Aromatase Inhibitor 4-hydroxyandrostenedione Research Articles

Acute developmental exposure to 4-hydroxyandrostenedione has a long-term effect on visually-guided behaviors

Estrogenic and anti-estrogenic endocrine disrupting compounds (EDCs) are recognized as critical modulators of neural development, including sensory system development. Using the zebrafish model, we tested the effect of transient developmental exposure to a known anti-estrogenic EDC on adult visually-guided behavior. In particular, we exposed zebrafish aged 24-hour post-fertilization (hpf), 72 hpf, or 7-days post-fertilization (dpf) to the aromatase inhibitor 4-hydroxyandrostenedione (4-OH-A) for 24h. After this time, the fish were removed from treatment, placed into control conditions, and reared until adulthood (3–4months) when visually-guided optomotor responses (OMR) were assessed. Our results show significant decreases in positive OMR in adults exposed to 4-OH-A at 72 hpf and 7 dpf. These deficits were not accompanied by changes in overall swimming behaviors and startle responses, suggesting 4-OH-A specifically effected the visual system. Overall, this study identified long-term, quantifiable effects in visually-guided adult behaviors resulting from transient developmental exposure to the anti-estrogenic EDC, 4-OH-A. Further, these effects were noted when 4-OH-A exposure occurred after hatching, suggesting estrogen signaling is important for visual system maturation.

Induction and inhibition of aromatase (CYP19) activity by natural and synthetic flavonoid compounds in H295R human adrenocortical carcinoma cells.

Flavonoids and related structures (e.g., flavones, isoflavones, flavanones, catechins) exert various biological effects, including anticarcinogenic, antioxidant and (anti-)estrogenic effects, and modulation of sex hormone homeostasis. A key enzyme in the synthesis of estrogens from androgens is aromatase (cytochrome P450 19; CYP19). We investigated the effects of various natural and synthetic flavonoids on the catalytic activity and promoter-specific expression of aromatase in H295R human adrenocortical carcinoma cells. Natural flavones were consistently more potent inhibitors than flavanones. IC(50) values for 7-hydroxyflavone, chrysin, and apigenin were 4, 7, and 20 microM, respectively; for the flavanones 7-hydroxyflavanone and naringenin the IC(50) values were 65 and 85 microM, respectively. The steroidal aromatase inhibitor (positive control) 4-hydroxyandrostenedione had an IC(50) of 20 nM. The inhibition by apigenin and naringenin coincided with some degree of cytotoxicity at 100 microM. The natural flavonoid derivative rotenone (IC(50) 0.3 microM) was the most potent aromatase inhibitor tested. Several synthetic flavonoid and structurally related quinolin-4-one analogs inhibited aromatase activity. The most potent inhibitor was 4'-tert-butyl-quinolin-4-one (IC(50) 2 microM), followed by two 2-pyridinyl-substituted alpha-naphthoflavones (IC(50)s 5 and >30 microM). The two 2-pyridinyl-substituted gamma-naphthoflavones consistently produced biphasic concentration-response curves, causing about 1.5-fold aromatase induction at concentrations below 1 microM and inhibition above that level (IC(50)s 7 and >30 microM). The natural flavone quercetin and isoflavone genistein induced aromatase activity 4- and 2.5-fold induction, respectively, at 10 microM. This coincided with increased intracellular cAMP concentrations and increased levels of the cAMP-dependent pII and to a lesser extent 1.3 promoter-specific aromatase transcripts. These results shed light on the structure-activity relationships for aromatase inhibition as well as mechanisms of induction in human H295R cells.

Dehydroepiandrosterone stimulates proliferation and gene expression in MCF-7 cells after conversion to estradiol

Dehydroepiandrosterone (DHEA) is a mitogen for estrogen-dependent MCF-7 breast cancer cells. Our aims were to determine whether DHEA required conversion to estrogens in order to stimulate cell proliferation and estrogen-dependent gene expression. After incubation of cells with 100 nM DHEA for 4 days, estradiol was present in the medium at a concentration of ∼200 pM. Other compounds identified were testosterone (∼300 pM) and estrone. Significant stimulation of cell proliferation by 1 nM estradiol and 100 nM DHEA was observed after 38 h and 4 days of incubation, respectively, indicating the necessity of DHEA conversion. DHEA doses ≥10 nM induced estrogen-dependent reporter gene expression in MCF-7 cells transfected with a luciferase reporter gene under the control of the estrogen response element. DHEA-dependent stimulation of proliferation and luciferase induction could be inhibited by the anti-estrogens ICI182,780 and tamoxifen, respectively, and by the aromatase inhibitor 4-hydroxyandrostenedione. An androgenic effect of DHEA on proliferation and gene expression of MCF-7 cells was not observed. We conclude that conversion of DHEA to estrogens, particularly estradiol, is required to exert a mitogenic response.

2-Chloro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity?

There is increasing concern that certain chemicals in the environment can cause endocrine disruption in exposed humans and wildlife. Investigations of potential effects on endocrine function have been limited mainly to interactions with hormone receptors. A need exists for the development of alternate in vitro methods to evaluate chemicals for their potential to disturb various endocrine functions via other mechanisms. Our laboratory is using the human H295R adrenocortical carcinoma cell line to examine chemicals for their potential to interfere with the activity and/or expression of several key cytochrome P450 (CYP) enzymes involved in the biosynthesis of steroid hormones. In this report we demonstrated that the commonly used 2-chloro-s-triazine herbicides atrazine, simazine, and propazine dose-dependently (0-30 microM) induced aromatase (CYP19) activity to an apparent maximum of about 2.5-fold in H295R cells. Basal- and triazine-induced aromatase activity was completely inhibited by the irreversible aromatase inhibitor 4-hydroxyandrostenedione (100 microM). The triazines increased levels of CYP19 messenger ribonucleic acid (mRNA) between 1.5- and 2-fold. The time-response profile of the induction of aromatase activity and CYP19 mRNA by the triazines was similar to that by 8-bromo-cyclic adenosine monophosphate, a known stimulant of the protein kinase-A pathway that mediates the induction of aromatase in these cells. The observed induction of aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens, may be an underlying explanation for some of the reported hormonal disrupting and tumor promoting properties of these herbicides in vivo.

Evidence for sexual dimorphism of estrogen receptors in hypothalamus and thymus of neonatal and immature Wistar rats

It is hypothesized that the rat thymus is sexually differentiated. To begin testing this, we used the 5-day-old rat, whose hypothalamus is sexually differentiated during this period. Cytosolic estrogen receptors (ER) were measured in cytosols prepared from the brain, thymus and uterus of Wistar rats at 5, 18 and 30 days post-partum. ER concentrations were significantly higher in hypothalamus in cytosols of female 5-day-old rats, and this difference had disappeared by day 18. The pattern in thymus was identical to that observed in hypothalamus, suggesting the presence in the thymus of the aromatase system that converts androgen to estrogen, and that estrogen-mediated sexual differentiation of thymus might be proceeding at 5 days. Unlike the case for hypothalamus, no experimental model exists at present for testing functional sexual differentiation in thymus. Therefore we tested the effects of aromatase inhibitors on estrogen receptor activity in thymus well after the five-day period, and before atrophy of the thymus has commenced. Male and female rats were implanted at 15 days of age with SILASTIC implants containing 5 mg of estradiol or with 25 mg of the aromatase inhibitor 4-hydroxyandrostenedione (4-OHA) and cytosolic ER prepared at 30 days and activity measured. Administration of estradiol resulted in failure to detect available receptor, suggesting that the binding components measured were ER. After 4-OHA administration, ER concentrations were significantly increased in cytosols from male but not female hypothalamus and thymus. There is therefore a basis for exploring further the hypothesis that rat thymus is sexually differentiated.

Regulation of epidermal growth factor receptor by androgens in human endometrial cells in culture.

Women with polycystic ovaries (PCO) have a thicker endometrium than women with normal ovaries. This cannot be due to unopposed oestrogen, as it occurs in ovulatory cycles. Androgens may be involved, as these are raised in women with PCO. The effects of steroids are partly mediated by growth factors and their receptors. The aim of this study was to investigate the effect of androgens on epidermal growth factor (EGF) receptor in human endometrium. Endometrium was enzymatically dispersed and glands and stromal cells separated. Cells were incubated in Ham's F10 medium supplemented with 5% charcoal-stripped fetal calf serum and either androgens or vehicle. Specific binding of [125I]-labelled EGF was measured. Testosterone and dihydrotestosterone (DHT) (10 micromol/l) increased EGF receptor concentration (control 100 +/- 9%, testosterone 196 +/- 23% control; control 100 +/- 1%, DHT 244 +/- 6% control) but did not alter receptor affinity. The effect of testosterone was inhibited by the anti-androgen hydroxyflutamide, but not by the antioestrogen ICI182780 nor the aromatase inhibitor 4-hydroxyandrostenedione. EGF receptor levels were increased by androstenediol (control 100 +/- 2%, androstenediol 120 +/- 10% control) but not by androstanediol, dehydroepiandrosterone (DHA), DHA sulphate or androstenedione. Testosterone and DHT increased EGF receptor concentrations in glandular epithelium (control 100 +/- 24%, testosterone 147 +/- 5%, DHT 185 +/- 30% control). These data suggest that androgens may have an effect on the endometrium via an increase in EGF receptor concentrations.

Local paracrine effects of estradiol are central to parturition in the rhesus monkey.

The central biochemical mechanisms involved in primate parturition are still unclear. Studies in both humans and nonhuman primates such as the baboon and rhesus monkey indicate that many factors play a part in the cascade of interactive positive feedforward loops that progressively promote parturition: changes in maternal endocrinology, a nocturnal switch in myometrial activity from low amplitude, infrequent contractures to high amplitude, high frequency contractions (see Fig. 1), dilation of the cervix and biochemical changes in the fetal membranes that lead to rupture. Here we demonstrate that infusion of the aromatase inhibitor 4-hydroxyandrostenedione (4OHA) inhibits conversion of androgen to estrogen and prevents premature delivery caused by administration of androgen to pregnant rhesus monkeys at 0.8 of pregnancy term. 4OHA also inhibited the androstenedione induced maternal endocrine and fetal membrane biochemical changes, and alteration of myometrial activity patterns. Secondly, peripheral estrogen infusions increased myometrial activity but did not produce preterm delivery or fetal membrane changes. We conclude that paracrine functions of estrogen at its site of production play critical and central roles in delivery in the non-human primate.

Mechanisms of the actions of aromatase inhibitors 4-hydroxyandrostenedione, fadrozole, and aminoglutethimide on aromatase in JEG-3 cell culture

Selective inhibition of estrogen production with aromatase inhibitors has been found to be an effective strategy for breast cancer treatment. Most studies have focused on inhibitor screening and in vitro kinetic analysis of aromatase inhibition using placental microsomes. In order to determine the effects of different inhibitors on aromatase in the whole cell, we have utilized the human choriocarcinoma cell line, JEG-3 in culture to compare and study three classes of aromatase inhibitors, 4-hydroxyandrostenedione, fadrozole (CGS 16949A), and aminoglutethimide. Fadrozole is the most potent competitive inhibitor and aminoglutethimide is the least potent among the three. However, stimulation of aromatase activity was found to occur when JEG-3 cells were preincubated with aminoglutethimide. In contrast, 4-OHA and fadrozole caused sustained inhibition of aromatase activity in both JEG-3 cells and placental microsomes, which was not reversed even after the removal of the inhibitors. 4-OHA bound irreversibly to the active site of aromatase and caused inactivation of the enzyme which followed pseudo-first order kinetics. However, 4-OHA appears to be metabolized rapidly in JEG-3 cells. Sustained inhibition of aromatase induced by fadrozole occurs by a different mechanism. Although fadrozole bound tightly to aromatase at a site distinct from the steroid binding site, the inhibition of aromatase activity by fadrozole does not involve a reactive process. None of the inhibitors stimulated aromatase mRNA synthesis in JEG-3 cells during 8 h treatment. The stimulation of aromatase activity by AG appeared to be due to stabilization of aromatase protein. According to these results, 4-OHA and fadrozole would be expected to be more beneficial in the treatment of breast cancer patients than AG. The increase in aromatase activity by AG may counteract its therapeutic effect and might be partially responsible for relapse of breast cancer patients from this treatment.

Estrogen regulates vascular endothelial growth/permeability factor expression in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors.

Vascular endothelial growth/permeability factor (VEG/PF) is expressed in some normal tissues and at high levels in a wide range of tumors. This growth factor is believed to be a key mediator of angiogenesis. Recent reports have shown that VEG/PF mRNA in the normal rat uterus is stimulated by estradiol (E2). In this study, we investigated the expression of VEG/PF in the mammary gland and 7,12-dimethylbenz(a)anthracene (DMBA)-induced, hormone-dependent mammary tumor of the rat model, and also whether VEG/PF is regulated by E2. VEG/PF mRNA from tumor extracts was amplified by RT-PCR with VEG/PF primers and generated two main products which corresponded in size to those expected for VEG/PF 164 and 120. In some cases, a third product corresponding in size to that expected for VEG/PF 188 was also generated. No such PCR products were generated from equal amount of RNA from normal mammary tissue, rat brain, or liver. Using immunocytochemistry, VEG/PF expression was detected in the epithelial cells of the tumors. We developed an ELISA assay to measure VEG/PF protein concentrations and found a 4-fold difference between normal mammary glands (1.3 +/- 0.11 ng/mg protein) and tumors (4.44 +/- 0.66) (P < 0.01). E2 treatment (5 microg/rat, s.c.) of rats 24 h after ovariectomy, greatly enhanced the expression of RT-PCR products in tumors within 2 h, which reached a maximum at 6-8 h but declined by 48 h. VEG/PF concentrations were also increased 8-12 h after E2 injection. When rats were given two injections of aromatase inhibitor 4-hydroxyandrostenedione (4-OHA 10 mg/rat s.c.) 24 h apart, to reduce estrogen concentrations, a low level of RT-PCR products was maintained for at least 96 h. After a single injection of 4-OHA, RT-PCR products remained low until 36 h when an increase occurred corresponding with a rise in plasma E2 levels. Injection of E2 2 h after 4-OHA treatment, caused a rise in RT-PCR products in 6-8 h. However, there was no significant change in VEG/PF concentrations. An increase in VEG/PF protein concentrations followed the increase in mRNA levels by 4-6 h. Thus, it appears that E2 causes a rapid induction of VEG/PF expression in mammary tumors that is similar to that observed in the normal uterus. These findings suggest that one mechanism by which estrogen acts as a mammary tumor promotor is by stimulating VEG/PF, leading to increased tumor angiogenesis and/or permeability of the microvessels to allow tumor cell migration.

The aromatase inhibitor 4-hydroxyandrostenedione (4OHA) prevents androstenedione (A)-induced myometrial (MYO) contractions and increases in maternal plasma estradiol (E2) in pregnant monkeys.

The aromatase inhibitor 4-hydroxyandrostenedione (4OHA) prevents androstenedione (A)-induced myometrial (MYO) contractions and increases in maternal plasma estradiol (E2) in pregnant monkeys.

Characterization of oviductal aromatase in the northern leopard frog, Rana pipiens

Previous work has shown that the oviduct of the northern leopard frog, Rana pipiens, can convert testosterone to estradiol. The present paper examines the characteristics of the aromatase responsible for this reaction and compares it with human placental aromatase. Microsomes were isolated from the oviducts, and aromatase activity was assayed by a tritiated water release method. The K m and the V max for androstenedione were 188.1 ± 30.2 nM and 1.42 ± 0.11 fmol of estrogen produced/min/mg protein, respectively. Using the same method, human placental aromatase had a K m of 123.0 nM and a V max of 113.57 fmol of estrogen produced/min/mg protein. When tested at four temperatures between 15 and 45°C, the frog enzyme showed maximum activity at 37°C. The enzyme had a broad pH optimum between 7.4 and 10.4. The aromatase inhibitor 4-hydroxyandrostenedione inhibited activity by 20% at 0.3 μM and 40% at 0.5 μM. The present study provides additional evidence that an aromatase is present in the frog oviduct. Characterization of this enzyme revealed similarities to human placental aromatase, but the specific activity was much lower in the frog oviduct.

Dual regulation of luteal progesterone production by androstenedione during spontaneous and RU486-induced luteolysis in pregnant rats

The effect of androstenedione on luteal progesterone production was studied during luteolysis preceding parturition as well as that induced by the antiprogestin RU486 in late pregnant rats. Luteal cells from animals on days 19, 20 or 21 of pregnancy and incubated with 10 μM androstenedione increased progesterone production by 99, 136, and 277%, respectively. The animals receiving androstenedione (10 mg/rat s.c.) on day 19 of pregnancy showed an increase in serum progesterone levels, a decline in luteal 3β-hydroxysteroid dehydrogenase (3β-HSD) activity and an increase in corpus luteum weight without modifying 20α-hydroxysteroid dehydrogenase (20α-HSD) activity on day 21 of pregnancy. Androstenedione and testosterone but not dihydrotestosterone were able to prevent the decrease in serum progesterone concentration and corpus luteum weight observed 58 h after treatment with RU486 (2 mg/kg) on day 18 of pregnancy. However, the three androgens studied inhibited the luteal 3β-HSD activity but 20α-HSD activity was not affected, when compared with animals receiving RU486 alone. The co-administration of androstenedione with the aromatase inhibitor 4-hydroxyandrostenedione or with the specific antioestrogen ICI 164,384 did not modify the effects induced by androstenedione in RU486-treated rats, indicating that the action of androstenedione on progesterone production and secretion at the time of luteolysis seems to occur through an androgenic mechanism and is not mediated by previous conversion of the androgens to oestrogens. In all experiments the high luteal 20α-HSD activity, that characterizes a luteolytic process, was not modified by androgens. Androstenedione administered to adrenalectomized rats was also able to prevent the decrease in serum progesterone concentration observed in spontaneous or RU486-induced luteolysis. The administration of androstenedione to RU486-treated rats induced a decrease in luteal progesterone content concomitant with an increase in serum progesterone levels. These studies demonstrate that androgens during luteolysis, are able to stimulate luteal progesterone secretion, prevent the loss in corpora lutea weight and enhance the decrease in 3β-HSD activity, without affecting the increase in 20α-HSD activity.

The synthesis of 19-norandrostenedione from dehydroepiandrosterone in equine placenta is inhibited by aromatase inhibitors 4-hydroxyandrostenedione and fadrozole

19-Norandrostenedione was synthesized in vitro from dehydroepiandrosterone by explants of equine full-term placenta. The synthesis of 19-norandrostenedione was inhibited by two specific aromatase inhibitors, 4-hydroxyandrostenedione and fadrozole.

Angiotensin-II stimulates estradiol secretion from human placental explants through AT1 receptor activation.

A complete renin-angiotensin system has been shown to be present in human placenta, but its physiological role is poorly known. To investigate the implication of this system in the regulation of steroid hormone secretion, we studied the effect of angiotensin-II on the release of estradiol and progesterone from human placental explants. Our experiments showed that angiotensin-II stimulated estradiol secretion from term placental explants in a dose- and time-dependent fashion, although progesterone release was unaffected. Estradiol release induced by angiotensin-II (0.2 mumol/L) was blocked by angiotensin AT1 receptor antagonist losartan in a dose-dependent manner, suggesting the involvement of the AT1 receptor subtype in the process. On the contrary, the angiotensin AT2 receptor antagonist PD123319 (1 mumol/L) or the angiotensin AT2 receptor agonist CGP42112A (1 mumol/L) had no effect. Analysis of the amount of steroid hormones in the placental tissues incubated for 12 h showed that angiotensin-II increased estradiol production by 34% compared with the unstimulated explants, whereas the total levels of the estrogen precursor androstenedione and testosterone were decreased by 30-45% in the presence of the peptide, suggesting a stimulatory effect on the aromatization step. This hypothesis was reinforced by the absence of effect of angiotensin-II on both estradiol and testosterone concentrations in the placental explants pretreated with the aromatase inhibitor 4-hydroxyandrostenedione (25 mumol/L). Progesterone synthesis was not affected by angiotensin-II. The present study indicates that angiotensin-II induces the secretion of estradiol from human placenta through the angiotensin AT1 receptor subtype activation, and this effect seems to be linked to the stimulation of local androgen aromatization.

Regulation of c-fos expression and TGF-β production by gonadal and adrenal androgens in normal human osteoblastic cells

Although the role of estrogens in bone formation is becoming clarified, the function of androgens in this process remains to be defined. Consequently, we have explored the mechanism of action for both gonadal and adrenal androgens in normal human osteoblastic (hOB) cells, which are responsible for the synthesis and mineralization of bone. Changes in the steady-state mRNA levels for two nuclear proto-oncogenes (c-fos and c-jun) and one cytokine (TGF-beta 1) were quantified in response to short (30 min) and long (24-48 h) treatments of these cells with physiologic concentrations of steroids. In addition, the levels of TGF-beta protein in the hOB cells conditioned-media were measured using a bioassay. The results indicated that neither 10 nM dihydrotestosterone, 10-20 nM testosterone, nor 10-100 nM androstenedione had a significant effect on the steady-state levels of c-fos, c-jun, or TGF-beta 1 mRNAs. Interestingly, 10-1000 nM dehydroepiandrosterone (DHEA) and 1-10 microM DHEA-sulfate rapidly reduced the steady-state level of c-fos mRNA by 60-80% in a dose-dependent manner within 30 min. In contrast, neither of these adrenal steroids had a significant effect on the message levels for c-jun or TGF-beta 1. Surprisingly, although TGF-beta 1 mRNA levels remained unchanged, the total amount of TGF-beta activity in the hOB cell conditioned-media increased 2-5-fold in response to 24-48 h treatments of the cells with gonadal or adrenal androgens. This increase in TGF-beta activity by DHEA-sulfate was both time- and dose-dependent, and was not blocked by cotreatment with the specific aromatase inhibitor 4-hydroxyandrostenedione (1 microM). Immunoprecipitations of the hOB cell conditioned-media with isoform-specific TGF-beta neutralizing-antibodies indicated that TGF-beta 2 was predominantly produced by the cells in response to DHEA-sulfate treatment. These results demonstrate that differences exist between the actions of estrogens and androgens on normal human osteoblasts with regard to the regulation of c-fos expression and TGF-beta production. Moreover, these data suggest that DHEA and DHEA-sulfate may play a distinct role in the regulation of human osteoblast function via the rapid repression of c-fos message levels and the slower increase in TGF-beta 2 protein levels.

The effect of aromatase inhibitor 4-hydroxyandrostenedione on steroid receptors in hormone-dependent tissues of the rat

In previous studies, we observed that aromatase inhibitor 4-hydroxy-androstenedione (4-OHA) treatment significantly decreased estrogen (ER) and progesterone receptor (PR) concentrations in the uterus of ovariectomized (OVX) rats. To investigate whether similar effects occur in mammary tumors, we have studied the hormone-dependent, carcinogen (DMBA)-induced tumors of the rat. After 2 weeks of 4-OHA treatment both ER and PR were reduced in mammary tumors, as well as in uteri of intact animals (P < 0.05). Following ovariectomy, receptor levels were also reduced. A further reduction in receptor concentration in mammary tumors occurred with 4-OHA treatment in OVX animals (P < 0.001). Treatment of OVX rats with estradiol (0.2 microgram/ml) restored tumor PR concentrations to the level of the control, whereas ER levels were increased to concentrations slightly higher than the control. 4-OHA treatment partially inhibited this increase in ER in mammary tumors of OVX rats treated with estradiol. In contrast to ER concentrations, mRNA ER levels in the uterus were not decreased significantly by ovariectomy although mRNA levels were reduced in the tumors. Ovariectomy was without effect on mRNA PR in either tissue. Treatment with 4-OHA reduced mRNA levels of ER and PR in uterus and tumors in intact and OVX animals. Levels of tumor mRNA of both ER and PR were inhibited by 4-OHA treatment in estradiol treated OVX rats. Thus, 4-OHA appears to inhibit ER and PR concentrations in mammary tumors of the rat by reducing transcription. Although aromatase inhibition which results in decreased estrogen production, is the major antitumor effect of 4-OHA, reduction in ER and PR could contribute to effective estrogen blockade and limit tumor growth by antagonizing estrogen action as well as production.

The effects of oral 4-hydroxyandrostenedione on peripheral aromatisation in post-menopausal breast cancer patients

This study investigated the influence of the aromatase inhibitor 4-hydroxyandrostenedione (4OHA, formestane), given orally, on peripheral aromatase activity and plasma oestradiol (E2) levels in post-menopausal women with advanced breast cancer. The aim was to establish whether an optimal dose could be identified that had a pharmacological effectiveness comparable with that of parenteral 4OHA. A total of 13 post-menopausal women were studied before treatment and after a minimum of 4 weeks on treatment with one or more of the following doses: 125 mg once daily (od), 125 mg b.i.d. (bd) and 250 mg od. In all, seven aromatase studied were performed at 125 mg od; four, at 125 mg bd; and ten, at 250 mg od. Three patients were studied at all doses. E2 was measured concurrently and was available at all dose increments for seven patients. Given at doses of 125 mg od, 125 mg bd and 250 mg od, treatment with formestane inhibited in vivo aromatisation by 62.3% +/- 9.5%, 70.0% +/- 5.1% and 57.3% +/- 5.3%, respectively (mean +/- SEM). Corresponding values for plasma E2 suppression were 30.7% +/- 6.5%, 43.4% +/- 4.5% and 42.9% +/- 6.7%, respectively. Thus, apart from a somewhat better suppression of plasma E2 levels by the two higher doses as compared with 125 mg od, no significant difference in the degree of aromatase inhibition or plasma E2 suppression was observed. The suppression of E2 by oral 4OHA at 125 mg bd or 250 mg od approaches that achieved by the recommended parenteral schedule of 250 mg fortnightly, but inhibition of aromatase at this dose was substantially inferior. The findings are consistent with a hypothesis that 4OHA given orally may cause substantial plasma oestrogen suppression during part of the day, but neither the od nor the bd regimens investigated in the present study were capable of producing optimal aromatase inhibition.

Analysis of the aromatase cytochrome P450 gene in human breast cancers

Aromatase activity may be detected in most, but not all, breast cancers, and in certain tumours there appears to be decreased sensitivity to the aromatase inhibitor 4-hydroxyandrostenedione (4-OHA). The aims of the present study were to measure aromatase activity, and its sensitivity to 4-OHA, in breast tumours, and to examine the CYP19 gene encoding the aromatase cytochrome P450 (P450arom) for the presence of mutations. In vitro aromatase activity and sensitivity to 4-OHA were measured by determining the conversion of tritiated testosterone to tritiated oestradiol in breast tumour tissue in the absence and presence of 4-OHA (10 nM). Genomic DNA was extracted from five tumours: one showing no detectable aromatase activity and four displaying evidence of aromatase activity (two sensitive and two insensitive to 4-OHA). Subsequent PCR-single-strand conformation polymorphism analysis revealed a variation in the mobility of single-stranded DNA for exons III, VII and X, corresponding, as shown by direct sequencing of PCR products, to common polymorphism of the aromatase gene. This study does not provide evidence for mutation in the coding exons of the P450arom gene which would account for either the absence of aromatase activity or its changed sensitivity to 4-OHA in breast cancers.

Role of P‐450 aromatase in sex determination of the diamondback terrapin, Malaclemys terrapin

Sex determination in the diamondback terrapin, Malaclemys terrapin, is temperature-dependent. Eggs incubated at 31 degrees C, and above, hatch in approximately 45 days as females. Eggs incubated below 27 degrees C hatch in about 60 days as males. Sex is not reversible after hatching. Nest temperatures in the wild can be as low as 20 degrees C and as high as 37 degrees C with as much as a 10 degrees C diel cycle. The shortest incubation time measured in nature was 56 days and the longest approaching 120 days. Nests in our study site produced predominantly (> 95%) male hatchlings. Treatment of developing embryos with estrogen produces females at male producing temperatures while treatment with fadrozole (a nonsteroidal aromatase inhibitor) induces partial male-like gonads. Treatment with a steroidal aromatase inhibitor (4-hydroxyandrostenedione, 4-OHA) had no effect on sex determination. Both fadrozole and 4-OHA are potent competitive inhibitors (Ki approximately 40-50 nM) for terrapin in vitro aromatase activity. These findings are consistent with aromatase expression being a key step in sex determination of terrapins. We have cloned a partial single copy P-450 aromatase from the terrapin using a cDNA library constructed from ovarian mRNA. This partial clone is highly homologous to other vertebrate aromatases.

Treatment of advanced breast cancer with the aromatase inhibitor 4-hydroxyandrostenedione: a phase II trial

50 postmenopausal women with advanced breast cancer were treated with 4-hydroxy-androstenedione (4-OHA) by intramuscular injection of 500 mg given every 2 weeks for 24 weeks unless progression of disease or intolerable side-effects precluded further treatment. All patients had received prior chemotherapy or hormone therapy and 16 patients had responded to their most recent treatment. 46 patients were evaluable for efficacy; 1 died of progressive disease within 3 weeks and 3 were withdrawn for unrelated medical conditions and were ineligible for assessment. All 50 patients were evaluated for toxicity; systemic symptoms were uncommon but 10 patients reported buttock discomfort. Of the 46 patients assessable for response, 7 (15%) had a partial response and 12 (26%) stable disease with 27 (59%) showing disease progression. The median duration of response was 85 weeks (range 32–219 weeks) and the median time to progression was 12 weeks (range 2–219 weeks).