Phytoestrogens and healthy aging: gaps in knowledge. A workshop report.

There is an increasing public interest in foods and dietary supplements containing phytoestrogens for the maintenance of health. A workshop was convened to assess evidence for the potential benefits of phytoestrogen-containing foods or supplements on diseases or conditions affecting older populations. Preclinical, clinical, and epidemiologic data on the cardiovascular system, various cancers, bone diseases, and menopausal symptoms were the focus of the discussions. Research on the basis of consumer food choices as well as a presentation from the FDA regarding approval of the use of soy foods to reduce the risk of cardiovascular disease were also presented. Based on the information presented, isoflavone-containing soy foods may have favorable effects on the cardiovascular system, but major knowledge gaps still exist regarding effects ofphytoestrogen supplements on bone diseases, various cancers, menopausal symptoms, and cognitive function.

Nonprimate animal models of menopause: workshop report.

OBJECTIVE: Menopause, an understudied, normal biological process in middle-aged women, is associated with loss of fertility and increased risk for osteoporosis and cardiovascular disease. Appropriate animal models allow in-depth investigation of biological mechanisms that underlie the increased risk for adverse health events in menopausal women. Although some species of older female nonhuman primates experience a menopause-like condition, with cessation of reproductive cycles, decreased bone density, and perhaps an increased risk for atherosclerosis, several factors restrict their usefulness for research (e.g., expense of purchase and care, relatively small numbers of animals available, risk for disease transmission to humans, limited facilities for experimentation). Thus, it may be useful to consider nonprimate animal species as potential models for pathophysiological changes associated with loss of reproductive function. DESIGN: A workshop was convened in June 1998 at the National Institutes of Health to explore the suitability of nonprimate animal species in this context. The focus of this workshop was on middle-aged, ovariectomized females of various laboratory animal species and the ability of exogenous estrogen to reverse pathophysiological changes in the skeleton, cardiovascular system, and thermoregulatory control mechanisms in these species. CONCLUSIONS: Of the species considered (mice, rats, dogs, rabbits, pigs, and sheep) and because of the limitations of relatively small amounts of research in ovariectomized, middle-aged animals for most of these species, mice (largely because of transgenic technology) have the potential to be good models for the effect of ovariectomy and estrogen replacement on associated bone and cardiovascular changes. Rats are an excellent model for bone but a poor model for the cardiovascular system changes associated with loss of reproductive function. Usefulness of the pig, which is usually considered to be a good model for the human cardiovascular system, is limited by the dearth of information available on ovariectomized mature pigs in cardiovascular and bone studies, sensitivity of bone density to dietary calcium, the difficult-to-manage size of regular pigs, and the relatively high cost of minipigs. Rabbits show good potential as a cardiovascular model despite the limited numbers of studies and the difference from primates in coronary artery structure. Although rabbits are the smallest species known to have Haversian bone remodeling processes, the limited number of bone studies in ovariectomized rabbits is confounded by effects of dietary calcium. Although there are virtually no studies on the cardiovascular system of the ovariectomized dog, bone studies that have been conducted suggest that it is a poor model for the menopausal human. Furthermore, the role of estrogen in bone and cardiovascular physiology is difficult to interpret because of the limitation of two estrus cycles per year in the dog. The sheep seems to be a promising large animal model for the bone and cardiovascular systems, but more research is needed. Of the species examined for estrogen effects on vasomotor symptoms (guinea pig, mouse, rat, and monkey), only rats and monkeys show evidence of hot flashes associated with loss of reproductive function.

Estrogen metabolism, not biosynthesis, in rabbit articular cartilage and isolated chondrocytes: a preliminary study.

Because serum estrogen levels are associated with the presence of osteoarthritis, and cartilage tissue is known to contain estrogen receptors, it is of interest to determine the extent to which estrogen is biosynthesized and/or metabolized in cartilage tissue or isolated chondrocytes. In this preliminary study, using a sensitive assay method, estrogen synthetase (aromatase) was undetectable in articular cartilage or isolated chondrocytes in culture from immature female rabbits. However, estrogen metabolism, specifically estrogen 17 beta-hydroxysteroid dehydrogenase activity, was detected in homogenized cartilage tissue, and at substantially higher specific activities in freshly isolated chondrocytes. These fresh chondrocytes, assayed in culture without any exogenous cofactor, demonstrated a significantly higher activity for converting the weak estrogen, estrone, to the more potent estrogen, estradiol. Chondrocytes grown to confluence in culture had very low estrogen 17 beta-hydroxysteroid dehydrogenase specific activity. Homogenized cartilage tissue, tested only with added NADPH as cofactor, also showed a preference for estradiol as the principal product, but this may have been primarily due to the use of reduced cofactor. If subsequent experiments confirm the presence of estrogen 17 beta-hydroxysteroid dehydrogenase activity, and its preference for converting estrone into estradiol, in human cartilage tissue and chondrocytes, this could have substantial implications in the estrogen dependency of osteoarthritis.

Human placental estrogen synthetase (aromatase). Effect of environment on the kinetics of protein-protein and substrate-protein interactions and the production of 19-oxygenated androgen intermediates in the purified reconstituted cytochrome P450 enzyme system.

Estrogen synthetase (aromatase) catalyzes the conversion of androgen into estrogen via two hydroxylations at C19 and a subsequent C19-10 lyase reaction. We report here the results of a reconstitution study using a highly purified aromatase cytochrome P450 monooxygenase enzyme system, with both protein components (cytochrome P450 and NADPH-cytochrome P450 reductase) obtained from human term placental microsomes. By varying one of the components (amounts of cytochrome P450, NADPH-cytochrome P450 reductase, or androgen substrate) as the other two were held constant in four different environments (phospholipid, non-ionic detergent, mixture of phospholipid and non-ionic detergent and buffer alone), we obtained evidence supporting the following conclusions. The reconstituted enzyme is more active and the protein components exhibit much lower apparent Km values in the detergent and/or lipid environment compared with buffer alone. Although the apparent Km and Vmax values for each aromatase protein component differ significantly in most cases with the particular limiting component and environment, the catalytic efficiency (Kcat/Km) was independent of the limiting protein component and varied with the environment only (highest in the lipid-detergent mixture and lowest in lipid alone). When the concentration of androgen substrate (androstenedione or testosterone) was varied at constant amounts of the aromatase protein components (NADPH-cytochrome P450 reductase saturating), the Km was lower and the Vmax was higher for adrostenedione. The specificity constant (Vmax/Km) was a function of the reconstitution environment (highest in lipid alone and lowest in detergent alone) and was, on average, about 4-fold higher for androstenedione in a particular environment. The extent of production of 19-oxygenated androgen intermediates (19-hydroxy and 19-oxo androstenedione) was examined at three different levels of aromatase cytochrome P450 (subsaturating, saturating, super-saturating) relative to the NADPH-cytochrome P450 reductase component in the three different hydrophobic environments using androstenedione as substrate. Both 19-oxygenated androgens, each made in comparable amounts relative to control, were isolatable in greatest amounts under cytochrome P450 super-saturating conditions in the detergent-lipid mixed environment, and in least amounts under cytochrome P450 subsaturating conditions in the lipid-only environment. Based on these data, we propose that 19-oxygenated androgen intermediates are biosynthesized sequentially in a step-wise fashion as the cytochrome P450 and NADPH-cytochrome P450 reductase form transient complexes, and that the amount of isolatable 19-oxygenated androgen is proportional to the amount of excess cytochrome P450 component.

Estrogen synthetase (aromatase). The cytochrome P-450 component of the human placental enzyme is a glycoprotein.

Estrogen synthetase (aromatase) is a cytochrome P-450 enzyme system which converts androgens to estrogens. Although this enzyme has been purified and the cDNA-derived amino acid sequence elucidated, very little is known regarding post-translational modifications of this physiologically crucial enzyme. We show here that the cytochrome P-450 component, P-450ES, purified from human term placental microsomes, is a glycoprotein based on the following evidence: its molecular weight is decreased following treatment with endoglycosidase F, concanavalin A-biotin specifically binds to this protein immobilized on nitrocellulose, and its oligosaccharide composition is consistent with a single N-linked fucosylated complex type carbohydrate chain. In a reconstitution system, the aromatase activity using the endoglycosidase F-treated P-450ES was reduced by about 35-40% relative to the native form, regardless of whether androstenedione or testosterone was used as substrate.

Estrogen synthetase (aromatase) Affinity purification of antibody against the cytochrome P450 component.

To procure an affinity gel capable of purifying antibody against the cytochrome P450 component of estrogen synthetase (P450ES), we coupled purified P450ES to agarose supports. OUr purpose was to compare two differently-activated agarose gels (Affi-Gel 15 and Tresyl-activated Sepharose) with the same P450ES preparation to compare the efficiency of coupling and the yield of purified antibody. Using supplier-directed protocols to covalently attach P450ES to each of the supports, and identical procedures to bind and elute anti-P450ES, we reached the following conclusions. Tresyl-activated Sepharose bound greater amounts of antigen than Affi-Gel 15 based on the amount of residual antigen after the coupling procedure and the amount of bound antigen detected by an ELISA-type method. However, both ligand-coupled supports yielded comparable amounts of purified anti-P450ES at a 48-fold purification relative to the starting IgG preparation.

Immunologic identification of the aromatase enzyme system in human endometrium.

Confluent human endometrial stromal cells were cultured in medium with no hormone or supplemented with medroxyprogesterone acetate (MPA), estradiol (E2), and porcine relaxin (RLX) for 5 days. These stromal cells were then labeled with [35S]methionine for 3 h. The radioactive proteins in the particulate fraction of cell homogenate were extracted by detergent and incubated with antisera to purified placental aromatase cytochrome P-450 (P-450arom) and NADPH-cytochrome P-450 reductase to isolate the radio-labeled aromatase enzyme components. Analysis of the radio-labeled protein, isolated by antibody to the cytochrome P-450arom from different preparations (P45FBIII or R-8-2) showed a major band at molecular weight 54k on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The intensity of 54k band was stronger in hormone treated stromal cells than that of control in parallel with the increase of aromatase activity. The radio-labeled protein isolated by anti-NADPH cytochrome P-450 reductase, REDFBIV, showed a major band at the molecular weight 73k on SDS-PAGE with comparable intensity in control and hormone treated samples. Thus, the apparent molecular weights of endometrial cytochrome P-450arom and cytochrome P-450 reductase were identical to placental aromatase enzyme system. When a secretory endometrium and a decidua were labeled with [35S]methionine, the cytochrome P-450arom was detected only in the decidua. NADPH cytochrome P-450 reductase was detected both in the endometrium and the decidua. These results show that antisera to placental aromatase enzyme system cross reacts with the endometrial aromatase enzyme components. The synthesis of cytochrome P-450arom was stimulated by MPA, E2 and RLX while the synthesis of the NADPH-cytochrome P-450 reductase aromatase component was not affected by the hormone.

Estrogen synthetase (aromatase) activity in primary culture of human term placental cells: effects of cell preparation, growth medium, and serum on adenosine 3',5'-monophosphate response.

The establishment of human term trophoblast cells in culture is dependent on the method of cell preparation, growth medium used, and presence of serum. Using freshly isolated term placental cells, we investigated 1) the effects of two different methods of removal of nontrophoblast cells and two culture media on trophoblast aromatase specific activity, cellular morphology, and hCG secretion over 72 h; and 2) the sensitivity of these hormonal parameters to cAMP added 24 h after the initiation of culture. Under conditions in which aromatase is responsive to cAMP, we studied the effect of removing serum after 24 h in culture with serum. After trypsin digestion of placental villi, isolated trophoblast cells were either treated with ammonium chloride (A) or purified on Percoll density gradients (P) and then grown in monolayer culture with medium 199 plus 10% fetal bovine serum (M) or Dulbecco's Modified Eagle's Medium (DMEM) plus 20% fetal bovine serum (D). Regardless of the method of treatment or growth medium used, aromatase specific activity increased 10- to 15-fold 24 h after plating, continued to increase from 24-48 h, and then decreased (AM) or remained constant (AD, PM, and PD) from 48-72 h. Addition of cAMP significantly increased activity in DMEM-grown cells (PD or AD) at both 48 and 72 h. Aromatase activity in PM cells grown with cAMP increased at 48 h, then decreased to near-control levels by 72 h; however, in AM cells, no response to cAMP was observed relative to control cells. Secretion of hCG was suppressed for the first 48 h in all cultures, but increased by 72 h (greatest increase in AM cultures). cAMP significantly increased hCG secretion 48 h after its addition under all conditions. We further evaluated the response of the Percoll-purified DMEM-grown cells (PD) to cAMP after serum removal at 24 h. Cells deprived of serum showed significantly higher aromatase specific activity over the entire culture period compared with serum-grown cells. Secretion of hCG increased 2- or 3-fold in the presence or absence of serum, respectively, after 72 h in culture. cAMP increased aromatase specific activity by 1.8- and 1.4-fold at 48 h and by 2.5- and 2.4-fold at 72 h in serum-containing and serum-free cells, respectively. The secretion of hCG increased 11- to 14-fold at 72 h under both serum-containing and serum-free conditions, respectively. The results show that cAMP can act as an intracellular messenger in the regulation of both aromatase activity and hCG secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Placental estrogen synthetase (aromatase): evidence for phosphatase-dependent inactivation.

The acute regulation of estrogen synthetase (aromatase), the cytochrome P450 enzyme system responsible for estrogen production, is not well explored. We report here that aromatase, but not NADPH-cytochrome c (P450) reductase, activity from human term placental microsomes decreased when incubated in phosphate-free buffer at 37 degrees C. Aromatase activity was stabilized by phosphate buffer or by the phosphatase inhibitors tartaric acid or EDTA, but not NaF, in phosphate-free buffer. Alkaline phosphatase also inhibited aromatase in phosphate-free buffer relative to phosphate buffer, but the inactivation appears to be due primarily to proteolytic solubilization of NADPH-cytochrome c reductase from the microsomes by proteases within the alkaline phosphatase preparation. Based on these data, we suggest that the cytochrome P450 component of aromatase may be regulated acutely by phosphorylation-dependent processes.

Oestrogen synthetase (aromatase) and hormone secretion in primary cultures of human placental trophoblast cells. Effects of cyclic AMP addition at the start of culture in attached and unattached cell populations.

Term placental trophoblast cells, released by trypsin digestion of placental villi, purified on a Percoll gradient and grown in serum-containing medium, differentiate within 24 to 48 h in culture from mononucleated cytotrophoblast-like cells at the start of culture to highly multinucleated giant (syncytiotrophoblast-like) cells that are more active in hormonogenesis. To determine the changes in hormone biosynthesis and secretion that occur early in the trophoblast differentiation process in vitro, freshly isolated placental cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 20 per cent FBS, in the presence and absence of cAMP for up to 48 h. Cell attachment and growth, oestrogen synthetase (aromatase) activity in attached and unattached cells, and secretion of human chorionic gonadotropin (hCG) and progesterone were studied. The aromatase specific activity, low in freshly isolated cells, increased fourfold in attached cells by 3 h, and achieved a 10- to 15-fold increase by 40 to 48 h. In attached cells grown with cAMP, aromatase activity was further stimulated by about fourfold, relative to the control. The aromatase activity of the unattached cells removed from the culture dishes at various times up to 48 h showed a biphasic response: the activity decreased by 18 h and then increased back to the fresh cell levels. The effect of cAMP on aromatase in these unattached cells was manifested by a two-fold stimulation of activity by 18 h, relative to control unattached cells. Secretion of hCG from both attached and unattached cells remained at a low level (less than 200 ng/mg protein) in control cells; in the presence of cAMP, hCG secretion was stimulated by tenfold after 40 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen synthetase (aromatase) in cultured human term placental cells and neoplastic human trophoblast.

Estrogen synthetase (aromatase) is present in large amounts in human term placenta. However, the localization of aromatase within the cellular structure of the placental villus is obscure. By immunocytochemical techniques using antibodies that separately recognize each component of the aromatase cytochrome P-450 enzyme system, the fraction of term placental trophoblast cells in primary culture expressing each aromatase component antigen increased from 20% in fresh mononucleated cells to about 65% for multinucleated giant cells after 72 h. In contrast, about 80% of human choriocarcinoma cells in continuous culture (JAr line) expressed each aromatase component antigen. The fraction of trophoblast cells in primary culture containing human chorionic gonadotropin increased from about 14% in fresh mononucleated cells to about 45% after 72 h and was about 30% in the choriocarcinoma cells. Fibroblast cells in culture, derived from trypsin-treated placental villi, contained aromatase activity, albeit much lower than term placental trophoblast cells. Aromatase specific activity in these placental fibroblasts did not change following growth with dibutyryl cAMP plus theophylline for 72 h.

Antisera against estrogen synthetase from human placental microsomes. Antibody characterization and cross-reactivity studies in other organs.

Antibodies were obtained against both protein components of the cytochrome P-450 enzyme system responsible for estrogen biosynthesis, estrogen synthetase (aromatase), from human placental microsomes. The antiserum against the NADPH-cytochrome P-450 reductase component (antiserum denoted RE-DFBIV) gave a single major band at the Mr of the authentic enzyme by immunoblotting after electrophoretic separation of SDS-solubilized microsomes and inhibited both the reductase and aromatase activities in human placental and endometrial microsomes (Tseng, L. and Bellino, F.L. (1985) J. Steroid Biochem. 22, 555-557) and in homogenates of cultured aromatase-stimulated human endometrial stromal cells and human ovarian microsomes. The antiserum against the cytochrome P-450 component of aromatase (antiserum denoted P45FBIII) also gave a single band at the Mr of the authentic protein by immunoblotting after electrophoresis, and inhibited aromatase activity in homogenates of human placental microsomes, ovarian and decidual particulate fractions and cultured aromatase-stimulated endometrial stromal cells. This antiserum had no effect on NADPH-cytochrome c reductase activity in any of the systems studied. We conclude that these antiserum preparations separately recognize the NADPH-cytochrome P-450 reductase and cytochrome P-450 components of aromatase in human placenta, ovary, decidua and endometrium. Epitopes on these aromatase component proteins involved in enzyme activity are shared among these various human tissue sources.

Inhibition of aromatase and NADPH cytochrome c reductase activities in human endometrium by the human placental NADPH cytochrome c reductase antiserum.

The cross-reactivity of human placental microsomal NADPH-cytochrome c reductase antiserum, REDFBIV, against the endometrial reductase alone and as a component of the endometrial aromatase was investigated. Human endometrial particulate fractions were incubated with various amounts of REDFBIV for 1 h at 4 degrees C and both enzyme activities were measured at the end of incubation. The extent of inhibition of these endometrial enzymes was compared with the ability of this antiserum to inhibit the placental microsomal reductase and aromatase activities. The antiserum effectively inhibited the activities of both enzymes in both tissues in a dose dependent manner with aromatase activity inhibited to a greater extent than reductase activity. These results indicate the antiserum to the placental microsomal NADPH-cytochrome c reductase component of aromatase recognizes the reductase component of the aromatase enzyme system in endometrium.

Estrogen synthetase activity in human term placental cells in monolayer culture.

Human term placental cells, isolated by trypsin treatment, were grown in culture with medium 199 and 10% fetal bovine serum for up to 1 week. Aromatase specific activity (+/-SE) of freshly isolated cells was low (0.63 +/- 0.04 pmol/min X mg protein; n = 15) compared to that of placental tissue before trypsin treatment (21.30 +/- 0.40 pmol/min X mg protein; n = 6). This activity in attached cells increased 10-fold 24 h after plating (6.32 +/- 0.75 pmol/min X mg protein; n = 19) and continued to increase up to 72-96 h (14.78 +/- 1.09 pmol/min X mg protein; n = 13) before declining to 6.50 +/- 1.40 pmol/min X mg protein (n = 4) after 120 h. The functional activity of the cells was assessed by daily measurements of hCG secretion into the medium. Secretion of hCG was maintained at about 0.3 micrograms/flask up to 48 h in culture, then rose rapidly to about 6.2 micrograms/flask from 96-168 h. The addition of 1 mM (Bu)2cAMP plus 1 mM theophylline to the culture medium 24 h after plating stimulated hCG secretion 7- to 8-fold relative to that by control cells, had no effect on aromatase specific activity 24 h after its addition, but decreased aromatase activity after 48 h. Freshly prepared cells were primarily (approximately 80%) mononucleated. With time in culture, the number and size of the multinucleated cells increased drastically until they accounted for virtually all of the cellular material in culture at 72 h. These morphological and functional changes in hCG secretion and aromatase activity suggest that trypsin-isolated cytotrophoblast cells differentiated in culture to form syncytiotrophoblast cells.

Estrogen synthetase stimulation by hemin in human choriocarcinoma cell culture.

The ability of hemin to stimulate estrogen synthetase (aromatase) in cultured human trophoblast cells and in cellular homogenates was investigated and compared with aromatase stimulation by dibutyryl cAMP [(Bu)2 cAMP]. Cells grown with hemin for 24 h, or homogenates incubated for 45 min with hemin, showed maximal aromatase stimulation (150 to 200% of activities in the absence of hemin) at 25 microM and 0.1 microM, respectively. Aromatase stimulation in culture by 25 microM hemin was observed within 4 h after hemin addition, while (Bu)2 cAMP required more than 6 h. Intracellular heme and porphyrin levels were higher (160 to 185%) in 96 h (Bu)2 cAMP-grown cells than control cells.