Low dose of propyl-pyrazole-triol, an agonist of estrogen receptor alpha, administration stimulates the Coolidge effect in fadrozole-treated male rats.

Estrogen receptor (ER) α is involved in male sexual function. Here, we aim to investigate how ERα activation influences sexual satiety and the Coolidge effect (i.e., when a rat, that has reached sexual satiety, experiences an increased arousal after exposure to a novel sexual partner) in estrogen-deprived male rats. Male rats (8 per group) were treated daily for 29 days with either saline (Control group) or fadrozole dissolved in saline (1 mg/kg/day) 1 h before mating. On Days 13 and 29, rats treated with fadrozole received either no additional treatment (fadrozole group) or a single injection of propyl-pyrazole-triol (ERα-agonist group, dissolved in sesame oil, 1 mg/kg). Rats mated until reaching sexual satiety on Days 13 and 29. In these sessions, the Control group displayed higher frequency of intromission and ejaculation than the other groups. The ERα-agonist group mounted more frequently but reached sexual satiety sooner than the Control group. On Day 29, when exposed to a new sexual partner, the fadrozole-treated rats were less likely to display intromission than the other groups, or ejaculation than the Control group, or mounting than the ERα-agonist group. The Control group showed more ejaculatory behavior and shorter ejaculation latency than the other groups. Body weights, testosterone levels, estradiol levels, and ERα-immunoreactive cell counts in brain regions for sexual behavior were comparable between groups after 29 days of treatments. Our data suggest that estrogen helps regulate sexual satiety and the Coolidge effect in male rats.

Structure of human cortisol-producing cytochrome P450 11B1 bound to the breast cancer drug fadrozole provides insights for drug design.

Human cytochrome P450 11B1 (CYP11B1) is responsible for the final step generating the steroid hormone cortisol, which controls stress and immune responses and glucose homeostasis. CYP11B1 is a promising drug target to manage Cushing's disease, a disorder arising from excessive cortisol production. However, the design of selective inhibitors has been hampered because structural information for CYP11B1 is unavailable and the enzyme has high amino acid sequence identity (93%) to a closely related enzyme, the aldosterone-producing CYP11B2. Here we report the X-ray crystal structure of human CYP11B1 (at 2.1 Å resolution) in complex with fadrozole, a racemic compound normally used to treat breast cancer by inhibiting estrogen-producing CYP19A1. Comparison of fadrozole-bound CYP11B1 with fadrozole-bound CYP11B2 revealed that despite conservation of the active-site residues, the overall structures and active sites had structural rearrangements consistent with distinct protein functions and inhibition. Whereas fadrozole binds to both CYP11B enzymes by coordinating the heme iron, CYP11B2 binds to the R enantiomer of fadrozole, and CYP11B1 binds to the S enantiomer, each with distinct orientations and interactions. These results provide insights into the cross-reactivity of drugs across multiple steroidogenic cytochrome P450 enzymes, provide a structural basis for understanding human steroidogenesis, and pave the way for the design of more selective inhibitors of each human CYP11B enzyme.

Acute neuroestrogen blockade attenuates song-induced immediate early gene expression in auditory regions of male and female zebra finches.

Neuron-derived estrogens are synthesized by aromatase and act through membrane receptors to modulate neuronal physiology. In many systems, long-lasting hormone treatments can alter sensory-evoked neuronal activation. However, the significance of acute neuroestrogen production is less understood. Both sexes of zebra finches can synthesize estrogens rapidly in the auditory cortex, yet it is unclear how this modulates neuronal cell signaling. We examined whether acute estrogen synthesis blockade attenuates auditory-induced expression of early growth response 1 (Egr-1) in the auditory cortex of both sexes. cAMP response element-binding protein phosphorylation (pCREB) induction by song stimuli and acute estrogen synthesis was also examined. We administered the aromatase inhibitor fadrozole prior to song exposure and measured Egr-1 across several auditory regions. Fadrozole attenuated Egr-1 in the auditory cortex greater in males than females. Females had greater expression and clustering of aromatase cells than males in high vocal center (HVC) shelf. Auditory-induced Egr-1 expression exhibited a large sex difference following fadrozole treatment. We did not observe changes in pCREB expression with song presentation or aromatase blockade. These findings are consistent with the hypothesis that acute neuroestrogen synthesis can drive downstream transcriptional responses in several cortical auditory regions, and that this mechanism is more prominent in males.

Prenatal aromatase inhibition alters postnatal immunity in domestic chickens (Gallus gallus).

In birds, exposure to exogenous testosterone during embryonic development can suppress measures of immune function; however, it is unclear whether these effects are due to direct or indirect action via aromatization. Estradiol (E2) is synthesized from testosterone by the enzyme aromatase, and this conversion is a necessary step in many signaling pathways that are ostensibly testosterone-dependent. Many lines of evidence in mammals indicate that E2 can affect immune function. We tested the hypothesis that some of the immunomodulatory effects observed in response to in ovo testosterone exposure in birds are mediated by conversion to E2 by aromatase, by using fadrozole to inhibit aromatization of endogenous testosterone during a crucial period of embryonic immune system development in domestic chickens (Gallus gallus). We then measured total IgY antibody count, response to PHA challenge, mass of thymus and bursa of Fabricius, and plasma testosterone post-hatch on days 3 and 18. Because testosterone has a reputation for immunosuppression, we predicted that if modulation of an immune measure by testosterone is dependent on aromatization, then inhibition of estrogen production by fadrozole treatment would lead to elevated measures of that parameter. Conversely, if testosterone inhibits an immune measure directly, then fadrozole treatment would likely not alter that parameter. Fadrozole treatment reduced circulating E2 in female embryos, but had no effect on males or on testosterone in either sex. Fadrozole-treated chicks had decreased day 3 plasma IgY antibody titers and a strong trend towards increased day 18 thymic mass. Furthermore, fadrozole treatment generated a positive relationship between testosterone and thymic mass in males, and tended to increase day 18 IgY levels for a given bursal mass in females. There was no effect on PHA response, bursal mass, or plasma testosterone at either age post-hatch. The alteration of several indicators of immune function in fadrozole-treated chicks implicates aromatization as a relevant pathway through which developmental exposure to testosterone can affect immunity in birds.

Quantitative Response-Response Relationships Linking Aromatase Inhibition to Decreased Fecundity are Conserved Across Three Fishes with Asynchronous Oocyte Development.

Quantitative adverse outcome pathways (qAOPs) describe quantitative response-response relationships that can predict the probability or severity of an adverse outcome for a given magnitude of chemical interaction with a molecular initiating event. However, the taxonomic domain of applicability for these predictions is largely untested. The present study began defining this applicability for a previously described qAOP for aromatase inhibition leading to decreased fecundity developed using data from fathead minnow (Pimephales promelas). This qAOP includes quantitative response-response relationships describing plasma 17ß-estradiol (E2) as a function of plasma fadrozole, plasma vitellogenin (VTG) as a function of plasma E2, and fecundity as a function of plasma VTG. These quantitative response-response relationships simulated plasma E2, plasma VTG, and fecundity measured in female zebrafish (Danio rerio) exposed to fadrozole for 21 days but not these responses measured in female Japanese medaka (Oryzias latipes). However, Japanese medaka had different basal levels of plasma E2, plasma VTG, and fecundity. Normalizing basal levels of each measurement to equal those of female fathead minnow enabled the relationships to accurately simulate plasma E2, plasma VTG, and fecundity measured in female Japanese medaka. This suggests that these quantitative response-response relationships are conserved across these three fishes when considering relative change rather than absolute measurements. The present study represents an early step toward defining the appropriate taxonomic domain of applicability and extending the regulatory applications of this qAOP.

17ß-Estradiol modulates cell proliferation of medullary cords during ovarian differentiation of the Lepidochelys olivacea sea turtle.

In turtles undergoing temperature sex determination (TSD), bipotential gonads express Sox9 in medullary cords at both female- (FPT) and male-producing temperatures (MPT). Subsequently, when the sex fate of medullary cords becomes dimorphic, at FPT, Sox9 is downregulated, whereas at MPT, its expression is maintained. Medullary cords in the ovary turn into ovarian lacuna, whereas in the testis they differentiate as seminiferous cords. When embryos of Lepidochelys olivacea sea turtle are incubated at MPT and treated with estradiol, Sox9 expression persists in the medullary cords in the form of tiny ovotestis-like formations. The perturbed development of the treated gonads is due to a significant decrease in the number of proliferating cells. This suggests that the disturbed effect caused by exogenous estradiol may be due to a conflict between the gene networks regulated by temperature and the increased level of endogenous estrogens, induced by the treatment. Here, we decided to use fadrozole and fulvestrant, an aromatase inhibitor and an estrogen-receptor antagonist, respectively, to provide insights into the role played by endogenous estrogens in regulating the cell proliferation of the two main gonadal compartments: the medullary cords and the cortex. Comparing cell proliferation patterns, our current results suggest that the endogenous estrogens are involved in determining the sex fate of medullary cords, by repressing proliferation. Interestingly, our results showed that endogenous estradiol levels are unnecessary for the thickening of the ovarian cortex.

Testosterone Differentially Affects T Cells and Neurons in Murine and Human Models of Neuroinflammation and Neurodegeneration.

The high female-to-male sex ratio of multiple sclerosis (MS) prevalence has continuously confounded researchers, especially in light of male patients' accelerated disease course at later stages of MS. Although multiple studies have concentrated on estrogenic mechanisms of disease modulation, fairly little attention has been paid to androgenic effects in a female system, and even fewer studies have attempted to dissociate hormonal effects on the neurodegenerative and neuroinflammatory processes of MS. Herein, we demonstrate the differential effects of hormone treatment on the acute inflammatory and chronic neurodegenerative phases of murine experimental autoimmune encephalomyelitis. Although s.c. treatment with testosterone and aromatase inhibitor applied beginning on the day of immunization ameliorated initial course of disease, similar treatment administered therapeutically exacerbated chronic disease course. Spinal cord analyses of axonal densities reflected the clinical scores of the chronic phase. In vitro, testosterone treatment not only decreased Th1 and Th17 differentiation in an aromatase-independent fashion, but also exacerbated cell death in induced pluripotent stem cell-derived primary human neurons under oxidative stress conditions in an aromatase inhibitor-dependent manner. Thus, through the alleviation of inflammatory processes and the exacerbation of neurodegenerative processes, androgens may contribute to the epidemiologic sex differentials observed in MS prevalence and course.

Crosstalk between Estrogen Withdrawal and NFκB Signaling following Penetrating Brain Injury.

OBJECTIVES: Characterized by neuroinflammation, traumatic brain injury (TBI) induces neuropathological changes and cognitive deficits. Estrogens are neuroprotective by increasing cell survival and this increase is mediated by a decrease in neuroinflammation. To further explore the relationship between estrogens, brain injury, and neuroinflammation, we examined the expression of the IKK/NFκB complex. The IKK/NFκB complex is a pleiotropic regulator of many cellular signaling pathways linked to inflammation, as well as three major cytokines (IL-1ß, IL-6, and TNF-α). We hypothesized that NFκB expression would be upregulated following injury and that this increase would be exacerbated when circulating estrogens were decreased with fadrozole (aromatase inhibitor). METHODS: Using adult zebra finches, we first determined the expression of major components of the NFκB complex (NFκB, IκB-α, and IκB-ß) following injury using qPCR. Next, male and female finches were collected at 2 time points (2 or 24 h after injury) and brain tissue was analyzed to determine whether NFκB expression was differentially expressed in males and females at either time point. Finally, we examined how the expression of NFκB changed when estrogen levels were decreased immediately after injury. RESULTS: Our study documented an increase in the expression of the major components of the NFκB complex (NFκB, IκB-α, and IκB-ß) following injury. Decreasing estrogen levels resulted in a surprising decrease in the NFκB complex studied here. DISCUSSION: These data further expand the model of how estrogens and other steroid hormones interact with the inflammatory pathways following injury and may prove beneficial when developing therapies for treatment of TBI.

The Aldosterone Synthase Inhibitor FAD286 is Suitable for Lowering Aldosterone Levels in ZDF Rats but not in db/db Mice.

Inhibition of aldosterone synthase is an alternative treatment option to mineralocorticoid receptor antagonism to prevent harmful aldosterone actions. FAD286 is one of the best characterized aldosterone synthase inhibitors to date. FAD286 improves glucose tolerance and increases glucose-stimulated insulin secretion in obese and diabetic ZDF rats. However, there is limited knowledge about the dose-dependent effects of FAD286 on plasma aldosterone, corticosterone, and 11-deoxycorticosterone in ZDF rats and in db/db mice, a second important rodent model of obesity and type 2 diabetes. In addition, effects of FAD286 on plasma steroids in mice and rats are controversial. Therefore, obese Zucker diabetic fatty (ZDF) rats and db/db mice were treated with FAD286 for up to 15 weeks and plasma steroids were evaluated using highly sensitive liquid chromatography-tandem mass spectrometry. In ZDF rats, FAD286 (10 mg/kg/d) treatment resulted in nearly complete disappearance of plasma aldosterone while corticosterone levels remained unaffected and those of 11-deoxycorticosterone were increased ~4-fold compared to vehicle control. A lower dose of FAD286 (3 mg/kg/d) showed no effect on plasma aldosterone or corticosterone, but 11-deoxycorticosterone was again increased ~4-fold compared to control. In contrast to ZDF rats, a high dose of FAD286 (40 mg/kg/d) did not affect plasma aldosterone levels in db/db mice although 11-deoxycorticosterone increased ~2.5-fold. A low dose of FAD286 (10 mg/kg/d) increased plasma aldosterone without affecting corticosterone or 11-deoxycorticosterone. In conclusion, the aldosterone synthase inhibitor, FAD286, lowers plasma aldosterone in obese ZDF rats, but not in obese db/db mice.

Impact of Aldosterone Synthase Inhibitor FAD286 on Steroid Hormone Profile in Human Adrenocortical Cells.

Inhibition of aldosterone synthase (CYP11B2) is an alternative treatment option to mineralocorticoid receptor antagonism to prevent harmful aldosterone effects. FAD286 is the best characterized aldosterone synthase inhibitor. However, to date, no study has used sensitive liquid chromatography-tandem mass spectrometry to characterize in detail the effect of FAD286 on the secreted steroid hormone profile of adrenocortical cells. Basal aldosterone production in NCI-H295R cells was detectable and 9-fold elevated after stimulation with angiotensin II. FAD286 inhibited this increase, showing a maximal effect at 10 nmol/l. Higher concentrations of FAD286 did not further reduce aldosterone concentrations, but showed a parallel reduction in corticosterone, cortisol and cortisone levels, reflecting additional inhibition of steroid-11ß-hydroxylase (CYP11B1). Pregnenolone, progesterone and 17-OH-progesterone levels remained unaffected. In conclusion, the aldosterone synthase inhibitor FAD286 lowers angiotensin II-induced aldosterone concentrations in adrenocortical cells but the relative lack of selectivity over CYP11B1 is evident at higher FAD286 concentrations.

Quantitative Adverse Outcome Pathways and Their Application to Predictive Toxicology.

A quantitative adverse outcome pathway (qAOP) consists of one or more biologically based, computational models describing key event relationships linking a molecular initiating event (MIE) to an adverse outcome. A qAOP provides quantitative, dose-response, and time-course predictions that can support regulatory decision-making. Herein we describe several facets of qAOPs, including (a) motivation for development, (b) technical considerations, (c) evaluation of confidence, and (d) potential applications. The qAOP used as an illustrative example for these points describes the linkage between inhibition of cytochrome P450 19A aromatase (the MIE) and population-level decreases in the fathead minnow (FHM; Pimephales promelas). The qAOP consists of three linked computational models for the following: (a) the hypothalamic-pitutitary-gonadal axis in female FHMs, where aromatase inhibition decreases the conversion of testosterone to 17ß-estradiol (E2), thereby reducing E2-dependent vitellogenin (VTG; egg yolk protein precursor) synthesis, (b) VTG-dependent egg development and spawning (fecundity), and (c) fecundity-dependent population trajectory. While development of the example qAOP was based on experiments with FHMs exposed to the aromatase inhibitor fadrozole, we also show how a toxic equivalence (TEQ) calculation allows use of the qAOP to predict effects of another, untested aromatase inhibitor, iprodione. While qAOP development can be resource-intensive, the quantitative predictions obtained, and TEQ-based application to multiple chemicals, may be sufficient to justify the cost for some applications in regulatory decision-making.

Cell-autonomous sex differences in gene expression in chicken bone marrow-derived macrophages.

We have identified differences in gene expression in macrophages grown from the bone marrow of male and female chickens in recombinant chicken M-CSF (CSF1). Cells were profiled with or without treatment with bacterial LPS for 24 h. Approximately 600 transcripts were induced by prolonged LPS stimulation to an equal extent in the male and female macrophages. Many transcripts encoded on the Z chromosome were expressed ∼1.6-fold higher in males, reflecting a lack of dosage compensation in the homogametic sex. A smaller set of W chromosome-specific genes was expressed only in females. LPS signaling in mammals is associated with induction of type 1 IFN-responsive genes. Unexpectedly, because IFNs are encoded on the Z chromosome of chickens, unstimulated macrophages from the female birds expressed a set of known IFN-inducible genes at much higher levels than male cells under the same conditions. To confirm that these differences were not the consequence of the actions of gonadal hormones, we induced gonadal sex reversal to alter the hormonal environment of the developing chick and analyzed macrophages cultured from male, female, and female sex-reversed embryos. Gonadal sex reversal did not alter the sexually dimorphic expression of either sex-linked or IFN-responsive genes. We suggest that female birds compensate for the reduced dose of inducible IFN with a higher basal set point of IFN-responsive genes.

Rapid effects of the aromatase inhibitor fadrozole on steroid production and gene expression in the ovary of female fathead minnows (Pimephales promelas).

Cytochrome P450 aromatase catalyzes conversion of C19 androgens to C18 estrogens and is critical for normal reproduction in female vertebrates. Fadrozole is a model aromatase inhibitor that has been shown to suppress estrogen production in the ovaries of fish. However, little is known about the early impacts of aromatase inhibition on steroid production and gene expression in fish. Adult female fathead minnows (Pimephales promelas) were exposed via water to 0, 5, or 50µg fadrozole/L for a time-course of 0.5, 1, 2, 4, and 6h, or 0 or 50µg fadrozole/L for a time-course of 6, 12, and 24h. We examined ex vivo ovarian 17ß-estradiol (E2) and testosterone (T) production, and plasma E2 concentrations from each study. Expression profiles of genes known or hypothesized to be impacted by fadrozole including aromatase (cytochrome P450 [cyp] 19a1a), steriodogenic acute regulatory protein (star), cytochrome P450 side-chain cleavage (cyp11a), cytochrome P450 17 alpha hydroxylase/17,20 lyase (cyp17), and follicle stimulating hormone receptor (fshr) were measured in the ovaries by quantitative real-time polymerase chain reaction (QPCR). In addition, broader ovarian gene expression was examined using a 15k fathead minnow microarray. The 5µg/L exposure significantly reduced ex vivo E2 production by 6h. In the 50µg/L treatment, ex vivo E2 production was significantly reduced after just 2h of exposure and remained depressed at all time-points examined through 24h. Plasma E2 concentrations were significantly reduced as early as 4h after initiation of exposure to either 5 or 50µg fadrozole/L and remained depressed throughout 24h in the 50µg/L exposure. Ex vivo T concentrations remained unchanged throughout the time-course. Expression of transcripts involved in steroidogenesis increased within the first 24h suggesting rapid induction of a mechanism to compensate for fadrozole inhibition of aromatase. Microarray results also showed fadrozole exposure caused concentration- and time-dependent changes in gene expression profiles in many HPG-axis pathways as early as 4h. This study provides insights into the very rapid effects of aromatase inhibition on steroidogenic processes in fish.

Estrogen-regulated expression of cyp19a1a and cyp19a1b genes in swim-up fry of Labeo rohita.

P450 aromatase is the terminal enzyme in the steroidogenic pathway and catalyzes the conversion of androgens to estrogens. The expression of cyp19a1 genes in brain and gonad of Indian major carp, Labeo rohita swim-up fry was measured by quantitative real-time polymerase chain-reaction. Results demonstrated that cyp19a1b and cyp19a1a predominate in brain and gonad respectively. Treatment of fry with an aromatase inhibitor fadrozole for 6days attenuated brain cyp19a1b expression, but not cyp19a1a of gonad. Fadrozole also attenuated brain aromatase activity. Treatment with 17ß-estradiol (E2) for 6days resulted in up-regulation of brain cyp19a1b transcripts in a dose- and time-dependent manner, but not cyp19a1a. Whole-body concentration of vitellogenin also increased in response to E2. Altogether, these results indicate L. rohita swim-up fry can be used to detect environmental estrogens either using vitellogenin induction or cyp19a1b gene expression.

Role of aromatase and radial glial cells in neurotoxin-induced dopamine neuron degeneration and regeneration.

Radial glial cells (RGCs) in teleost brain are progenitor cells that express aromatase B and produce estrogens. Controversial data suggest that estrogens are critical for brain repair and neurogenesis in teleosts. Using a goldfish model for neurotoxin-induced Parkinson-like syndrome, we investigated the possible roles of RGCs, especially estrogen synthetic function, in the processes underlying dopamine neuron regeneration. The data indicate that dopamine neuron degeneration and aromatase activity inhibition could be respectively achieved in vivo with treatments with the neurotoxin 1-methyl-1,2,3,6-tetrahydropyridine (MPTP) and fadrozole in female goldfish. The expression of genes in the telencephalon and hypothalamus related to RGC functions including gfap, gdnf and bdnf as well as genes related to mature dopamine neuron functions including th, slc6a3 and pitx3 are under modulation of estrogens. Together these results revealed that the activation of radial glial cells and dopamine neuron recovery after MPTP insult is aromatase-dependent. Findings in this study provide support for the hypothesis that endogenous estrogens are neuroprotective in goldfish. Future studies focus on the molecular pathways for enhancing protective functions of estrogens and understanding global effects of estrogens in the central nervous system.

Selectivity of BI 689648, a Novel, Highly Selective Aldosterone Synthase Inhibitor: Comparison with FAD286 and LCI699 in Nonhuman Primates.

The mineralocorticoid aldosterone is an important regulator of blood pressure, volume, and electrolyte balance. However, excess aldosterone can be deleterious as a driver of vascular remodeling and tissue fibrosis associated with cardiometabolic diseases. Aldosterone synthase (AS) inhibitors (ASI) attenuate the production of aldosterone directly and have been proposed as an alternative to mineralocorticoid receptor antagonists for blocking the pathologic effects of excess aldosterone. Discovery of selective ASIs has been challenging because of the high sequence identity (93%) AS shares with cortisol synthase (CS), and the low identity of rodent AS compared with human (63%). Using cynomolgus (cyno) monkey-based models, we identified BI 689648 [6-(5-methoxymethyl-pyridin-3-yl)-3,4-dihydro-2H-[1,8]naphthyridine-1-carboxylic acid amide], a novel, highly selective ASI that exhibits an in vitro IC50 of 2 nM against AS and 300 nm against CS (150-fold selectivity) compared with the recently described ASIs FAD286 [4-(5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-5-yl)benzonitrile] (3 nM AS; 90 nM CS; 40-fold) and LCI699 (4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluorobenzonitrile) (10 nM AS; 80 nM CS; 8-fold). After oral administration in cyno monkeys, BI 689648 (5 mg/kg) exhibits a peak plasma concentration of ∼500 nM. For in vivo profiling we used an adrenocorticotropin-challenge model in which BI 689648 was >20-fold more selective compared with FAD286 and LCI699. Because both FAD286 and LCI699 failed to provide adequate selectivity for CS when tested in patients, the desire for more selective molecules to test the ASI hypothesis remains high. Therefore, highly selective aldosterone synthase inhibitors such as BI 689648 represent an important step forward toward developing ASIs with greater potential for clinical success in cardiometabolic diseases.

Aromatized testosterone attenuates contextual generalization of fear in male rats.

Generalization is a common symptom of many anxiety disorders, and females are 60% more likely to suffer from an anxiety disorder than males. We have previously demonstrated that female rats display significantly accelerated rates of contextual fear generalization compared to male rats; a process driven, in part, by activation of ERß. The current study was designed to determine the impact of estrogens on contextual fear generalization in male rats. For experiment 1, adult male rats were gonadectomized (GDX) and implanted with a capsule containing testosterone proprionate, estradiol, dihydrotestosterone proprionate (DHT), or an empty capsule. Treatment with testosterone or estradiol maintained memory precision when rats were tested in a different (neutral) context 1day after training. However, male rats treated with DHT or empty capsules displayed significant levels of fear generalization, exhibiting high levels of fear in the neutral context. In Experiment 2, we used acute injections of gonadal hormones at a time known to elicit fear generalization in female rats (e.g. 24h before testing). Injection treatment followed the same pattern of results seen in Experiment 1. Finally, animals given daily injections of the aromatase inhibitor, Fadrozole, displayed significant fear generalization. These data suggest that testosterone attenuates fear generalization likely through the aromatization testosterone into estradiol as animals treated with the non-aromatizable androgen, DHT, or animals treated with Fadrozole, displayed significant generalized fear. Overall, these results demonstrate a sex-dependent effect of estradiol on the generalization of contextual fear.

Marketed Drugs Can Inhibit Cytochrome P450 27A1, a Potential New Target for Breast Cancer Adjuvant Therapy.

Cytochrome P450 CYP27A1 is the only enzyme in humans converting cholesterol to 27-hydroxycholesterol, an oxysterol of multiple functions, including tissue-specific modulation of estrogen and liver X receptors. Both receptors seem to mediate adverse effects of 27-hydroxycholesterol in breast cancer when the levels of this oxysterol are elevated. The present work assessed druggability of CYP27A1 as a potential antibreast cancer target. We selected 26 anticancer and noncancer medications, most approved by the Food and Drug Administration, and evaluated them first in vitro for inhibition of purified recombinant CYP27A1 and binding to the enzyme active site. Six strong CYP27A1 inhibitors/binders were identified. These were the two antibreast cancer pharmaceuticals anastrozole and fadrozole, antiprostate cancer drug bicalutamide, sedative dexmedetomidine, and two antifungals ravuconazole and posaconazole. Anastrozole was then tested in vivo on mice, which received subcutaneous drug injections for 1 week. Mouse plasma and hepatic 27-hydroxycholesterol levels were decreased 2.6- and 1.6-fold, respectively, whereas plasma and hepatic cholesterol content remained unchanged. Thus, pharmacologic CYP27A1 inhibition is possible in the whole body and individual organs, but does not negatively affect cholesterol elimination. Our results enhance the potential of CYP27A1 as an antibreast cancer target, could be of importance for the interpretation of Femara versus Anastrozole Clinical Evaluation Trial, and bring attention to posaconazole as a potential complementary anti-breast cancer medication. More medications on the US market may have unanticipated off-target inhibition of CYP27A1, and we propose strategies for their identification.

Two aromatase inhibitors inhibit the ability of a third to promote mating in male rats.

Aromatase, the enzyme that aromatizes androstenedione (A) to estrone and testosterone (T) to estradiol (E), affects androgen control of male sex behavior in many vertebrates. In male monkeys, rats and quail, E mimics the ability of T to promote mating, and aromatase inhibitors block mating induced by T but not E. Aromatase inhibitors include androgens with different A-rings than T and A, e.g., 1,4,6-androstatriene-3,17-dione (ATD), azoles, e.g., fadrozole, and androgens α-halogenated at carbon 6, e.g., 6α-bromoA, 6α-fluoroA and 6α-fluoroT. 6α-FluoroT is the only 6α-halogenated androgen studied in regard to mating. It promotes mating in male rats and quail and was studied, before it was known to inhibit aromatase, because it cannot be aromatized yet has the same A-ring as T. 6α-FluoroT might promote mating by binding estrogen receptors (ER) directly, i.e., unassisted, or by metabolism to an androgen that binds ER. Since neither process would require aromatase, this study tested both hypotheses by determining how mating induced in castrated male rats by 6α-fluoroT is affected by ATD and fadrozole. Both aromatase inhibitors inhibited the effects of 6α-fluoroT on mating. Thus, 6α-fluoroT does not promote mating by direct ER binding or metabolism to another androgen. Since aromatase underlies a process in which 6α-fluoroT, unlike most nonaromatizable androgens, mimics T effects on male sex behavior, the process must involve a feature that 6α-fluoroT shares with T but not other nonaromatizable androgens. A-ring structure is a candidate. A hypothesis is also offered for how aromatase may participate without aromatizing the androgen.

Effects of estradiol on neural responses to social signals in female túngara frogs.

Estradiol plays an important role in mediating changes in female sexual behavior across reproductive cycles. In the túngara frog [Physalaemus (=Engystomops) pustulosus], the relationship between gonadal activity and female sexual behavior, as expressed by phonotaxis, is mediated primarily by estradiol. Estradiol receptors are expressed in auditory and motivational brain areas and the hormone could serve as an important modulator of neural responses to conspecific calls. To better understand how estradiol modifies neural responses to conspecific social signals, we manipulated estradiol levels and measured expression of the immediate early gene egr-1 in the auditory midbrain, thalamus and limbic forebrain in response to conspecific or heterospecific calls. We found that estradiol and conspecific calls increased egr-1 expression in the auditory midbrain and limbic forebrain, but in the thalamus, only conspecific calls were effective. In the preoptic area, estradiol enhanced the effect of the conspecific call on egr-1 expression, suggesting that the preoptic area could act as a hormonal gatekeeper to phonotaxis. Overall, the results suggest that estradiol has broad influences on the neural circuit involved in female reproduction, particularly those implicated in phonotaxis.