Aromatase Assay Research Articles

Evaluation of a high-throughput H295R homogenous time resolved fluorescence assay for androgen and estrogen steroidogenesis screening

The H295R test guideline assay evaluates the effect of test substances on synthesis of 17β-estradiol (E2) and testosterone (T). The objective of this study was to leverage commercial immunoassay technology to develop a more efficient H295R assay to measure E2 and T levels in 384-well format. The resulting Homogenous Time Resolved Fluorescence assay platform (H295R-HTRF) was evaluated against a training set of 36 chemicals derived from the OECD inter-laboratory validation study, EPA guideline 890.1200 aromatase assay, and azole fungicides active in the HT-H295R assay. Quality control performance criteria were met for all conditions except E2 synthesis inhibition where low basal hormone synthesis was observed. Five proficiency chemicals were active for both the E2 and T endpoints, consistent with guideline classifications. Of the nine OECD core reference chemicals, 9/9 were concordant with outcomes for E2 and 7/9 for T. Likewise, 9/13 and 11/13 OECD supplemental chemicals were concordant with anticipated effects for E2 and T, respectively. Of the 10 azole fungicides screened, 7/10 for E2 and 8/10 for T exhibited concordant outcomes for inhibition. Generally, all active chemicals in the training set demonstrated equivalent or greater potency in the H295R-HTRF assay, supporting the sensitivity of the platform. The adaptation of HTRF technology to the H295R model provides an efficient way to evaluate E2 and T modulators in accordance with guideline specifications.

An in vitro assay approach to investigate the potential impact of different doping agents on the steroid profile.

The steroid profile, that is, the urinary concentrations and concentration ratios of selected steroids, is used in sports drug testing to detect the misuse of endogenous steroids such as testosterone. Since several years, not only population-based thresholds are applied but also the steroid profile is monitored via the Athlete Biological Passport whereby the individual reference ranges derived from multiple test results of the same athlete are compared to population-based thresholds. In order to maintain a high probative force of the passport, samples collected or analyzed under suboptimal conditions should not be included in the longitudinal review. This applies to biologically affected or degraded samples and to samples excluded owing to the presence of other substances potentially (or evidently) altering the steroid profile. Nineteen different doping agents comprising anabolic steroids, selective androgen receptor modulators, selective estrogen receptor modulators, ibutamoren, and tibolone were investigated for their effect on the steroid profile using an androgen receptor activation test, an androgen receptor binding assay, an aromatase assay, and a steroidogenesis assay. The in vitro tests were coupled with well-established liquid chromatography/mass spectrometry-based analytical approaches and for a subset of steroidal analytes by gas chromatography/mass spectrometry. The variety of tests employed should produce a comprehensive data set to better understand how a compound under investigation may impact the steroid profile. Although our data set may allow an estimate of whether or not a substance will have an impact on the overall steroid metabolism, predicting which parameter in particular may be influenced remains difficult.

High-Throughput Analysis of Ovarian Cycle Disruption by Mixtures of Aromatase Inhibitors.

Background:Combining computational toxicology with ExpoCast exposure estimates and ToxCast™ assay data gives us access to predictions of human health risks stemming from exposures to chemical mixtures.Objectives:We explored, through mathematical modeling and simulations, the size of potential effects of random mixtures of aromatase inhibitors on the dynamics of women's menstrual cycles.Methods:We simulated random exposures to millions of potential mixtures of 86 aromatase inhibitors. A pharmacokinetic model of intake and disposition of the chemicals predicted their internal concentration as a function of time (up to 2 y). A ToxCast™ aromatase assay provided concentration–inhibition relationships for each chemical. The resulting total aromatase inhibition was input to a mathematical model of the hormonal hypothalamus–pituitary–ovarian control of ovulation in women.Results:Above 10% inhibition of estradiol synthesis by aromatase inhibitors, noticeable (eventually reversible) effects on ovulation were predicted. Exposures to individual chemicals never led to such effects. In our best estimate, of the combined exposures simulated had mild to catastrophic impacts on ovulation. A lower bound on that figure, obtained using an optimistic exposure scenario, was 0.3%.Conclusions:These results demonstrate the possibility to predict large-scale mixture effects for endocrine disrupters with a predictive toxicology approach that is suitable for high-throughput ranking and risk assessment. The size of the effects predicted is consistent with an increased risk of infertility in women from everyday exposures to our chemical environment. https://doi.org/10.1289/EHP742

Endophytic fungi associated with Taxus fuana (West Himalayan Yew) of Pakistan: potential bio-resources for cancer chemopreventive agents

Context: Endophytic fungi, being a prolific source of bioactive secondary metabolites, are of great interest for natural product discovery.Objective: Isolation and partial characterization of endophytic fungi inhabiting the leaves and woody parts of Taxus fuana Nan Li & R.R. Mill. (Taxaceae) and evaluation of biological activity.Materials and methods: Endophytic fungal isolates were identified by molecular analysis of internal transcribed spacer (ITS) regions of 18S rDNA. Extracts of the endophytic fungi cultured on potato dextrose agar and modified medium were evaluated using cancer chemoprevention bioassays [inhibition of TNF-α-induced NFκB, aromatase and inducible nitric oxide synthase (iNOS); induction of quinone reductase 1 (QR1)] and growth inhibition with MCF-7 cells.Results: Nine of 15 fungal isolates were identified as belonging to Epicoccum, Mucor, Penicillium, Chaetomium, Paraconiothriym, Plectania or Trichoderma. Five of the 15 extracts inhibited NFκB activity (IC50 values ranging between 0.18 and 17 μg/mL) and five inhibited iNOS (IC50 values ranging between 0.32 and 12.9 μg/mL). In the aromatase assay, only two isolates mediated inhibition (IC50 values 12.2 and 10.5 μg/mL). With QR1 induction, three extracts exhibited significant activity (concentrations to double activity values ranging between 0.20 and 5.5 μg/mL), and five extracts inhibited the growth of MCF-7 cells (IC50 values ranging from 0.56 to 17.5 μg/mL). Six active cultures were derived from woody parts of the plant material.Conclusion: The endophytic fungi studied are capable of producing pharmacologically active natural compounds. In particular, isolates derived from the wood of Taxus fuana should be prioritized for the isolation and characterization of bioactive constituents.

Hedera nepalensis K. Koch: A Novel Source of Natural Cancer Chemopreventive and Anticancerous Compounds.

Traditional medicinal plants are often used for both the prevention and the treatment of local diseases. Taking into consideration the medicinal importance of Hedera nepalensis within local Pakistani traditions, the present study was undertaken to analyze the in vitro cancer chemopreventive and cytotoxic properties of the plant. The in vitro cancer chemopreventive testing was performed using nitrite assay, NFκB assay, aromatase assay, and quinone reductase 1 (QR1) assay. The cytotoxic potential was evaluated on three cancer-cell lines: MCF-7, MDA-MB-231, and HeLa using sulforhodamine B (SRB) assay. The results of cancer chemopreventive assays show that n-hexane and ethyl acetate fractions of tested plant have promising cancer chemopreventive potential. Lupeol isolated from n-hexane as well as ethyl acetate fraction showed lowest IC50 (0.20 ± 1.9 μM) in NFκB assay. Crude extract and its fractions inhibited the growth of three cancer cell lines by more than 60%, IC50 value of lupeol varied from 2.32 to 10.2 μM. HPLC-DAD-based quantification of lupeol in different plant tissues demonstrated that leaves of H. nepalensis are a rich source of lupeol (0.196 mg/100 mg dry weight). Our data have shown that H. nepalensis harbors cancer chemopreventive and cytotoxic agents.

Effects of selective serotonin reuptake inhibitors on three sex steroids in two versions of the aromatase enzyme inhibition assay and in the H295R cell assay

Selective serotonin reuptake inhibitors are known to have a range of disorders that are often linked to the endocrine system e.g. hormonal imbalances, breast enlargement, sexual dysfunction, and menstrual cycle disorders. The mechanisms behind most of these disorders are not known in details. In this study we investigated whether the endocrine effect due to SSRI exposure could be detected in well adopted in vitro steroidogenesis assays, two versions of the aromatase enzyme inhibition assay and the H295R cell assay. The five drugs citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline, were shown to inhibit the aromatase enzyme in both types of aromatase assays. The IC50 values ranged from 3 to 600μM. All five SSRIs, were further investigated in the H295R cell line. All compounds altered the steroid secretion from the cells, the lowest observed effect levels were 0.9μM and 3.1μM for sertraline and fluvoxamine, respectively. In general the H295R cell assay was more sensitive to SSRI exposure than the two aromatase assays, up to 20 times more sensitive. This indicates that the H295R cell line is a better tool for screening endocrine disrupting effects. Our findings show that the endocrine effects of SSRIs may, at least in part, be due to interference with the steroidogenesis.

Cell-Based High-Throughput Screening for Aromatase Inhibitors in the Tox21 10K Library.

Multiple mechanisms exist for endocrine disruption; one nonreceptor-mediated mechanism is via effects on aromatase, an enzyme critical for maintaining the normal invivo balance of androgens and estrogens. We adapted the AroER tri-screen 96-well assay to 1536-well format to identify potential aromatase inhibitors (AIs) in the U.S. Tox21 10K compound library. In this assay, screening with compound alone identifies estrogen receptor alpha (ERα) agonists, screening in the presence of testosterone (T) identifies AIs and/or ERα antagonists, and screening in the presence of 17β-estradiol (E2) identifies ERα antagonists. Screening the Tox-21 library in the presence of T resulted in finding 302 potential AIs. These compounds, along with 31 known AI actives and inactives, were rescreened using all 3 assay formats. Of the 333 compounds tested, 113 (34%; 63 actives, 50 marginal actives) were considered to be potential AIs independent of cytotoxicity and ER antagonism activity. Structure-activity analysis suggested the presence of both conventional (eg, 1, 2, 4, - triazole class) and novel AI structures. Due to their novel structures, 14 of the 63 potential AI actives, including both drugs and fungicides, were selected for confirmation in the biochemical tritiated water-release aromatase assay. Ten compounds were active in the assay; the remaining 4 were only active in high-throughput screen assay, but with low efficacy. To further characterize these 10 novel AIs, we investigated their binding characteristics. The AroER tri-screen, in high-throughput format, accurately and efficiently identified chemicals in a large and diverse chemical library that selectively interact with aromatase.

Discovery of novel aromatase inhibitors using a homogeneous time-resolved fluorescence assay.

Aromatase is an important target for drugs to treat hormone-dependent diseases, including breast cancer. The aim of this study was to develop a homogeneous time-resolved fluorescence (HTRF) aromatase assay suitable for high-throughput screening (HTS). A 384-well aromatase HTRF assay was established, and used to screen about 7000 compounds from a compound library. Anti-proliferation activity of the hit was evaluated using alamarBlue(R) assay in a hormone-dependent breast cancer cell line T47D. Molecular docking was conducted to elucidate the binding mode of the hit using the Discovery Studio program. The Z' value and signal to background (S/B) ratio were 0.74 and 5.4, respectively. Among the 7000 compounds, 4 hits (XHN22, XHN26, XHN27 and triptoquinone A) were found to inhibit aromatase with IC50 values of 1.60±0.07, 2.76±0.24, 0.81±0.08 and 45.8±11.3 μmol /L, respectively. The hits XHN22, XHN26 and XHN27 shared the same chemical scaffold of 4-imidazolyl quinoline. Moreover, the most potent hit XHN27 at 10 and 50 μmol/L inhibited the proliferation of T47D cells by 45.3% and 35.2%, respectively. The docking study revealed that XHN27 docked within the active site of aromatase and might form a hydrogen bond and had a π-cation interaction with amino acid residues of the protein. XHN27, an imidazolyl quinoline derivative of flavonoid, is a potent aromatase inhibitor with anti-proliferation activity against breast cancer in vitro. The established assay can be used in HTS for discovering novel aromatase inhibitor.

Key Learnings from Performance of the U.S. EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 In Vitro Assays

Tier 1 of the U.S. EPA Endocrine Disruptor Screening Program comprises 11 studies: five in vitro assays, four in vivo mammalian assays, and two in vivo nonmammalian assays. The battery is designed to detect compounds with the potential to interact with the estrogen, androgen, or thyroid signaling pathways. This article examines the procedures, results, and data interpretation for the five Tier 1 in vitro assays: estrogen receptor (ER) and androgen receptor binding assays, an ER transactivation assay, an aromatase assay, and a steroidogenesis assay. Data are presented from two laboratories that have evaluated approximately 11 compounds in the Tier 1 in vitro assays. Generally, the ER and androgen receptor binding assays and the aromatase assay showed good specificity and reproducibility. As described in the guideline for the ER transactivation assay, a result is considered positive when the test compound induces a reporter gene signal that reaches 10% of the response seen with 1 nM 17β-estradiol (positive control). In the experience of these laboratories, this cutoff criterion may result in false-positive responses. For the steroidogenesis assay, there is variability in the basal and stimulated production of testosterone and estradiol by the H295R cells. This variability in responsiveness, coupled with potential cell stress at high concentrations of test compound, may make it difficult to discern whether hormone alterations are specific steroidogenesis alterations (i.e., endocrine active). Lastly, both laboratories had difficulty meeting some recommended performance criteria for each Tier 1 in vitro assay. Data with only minor deviations were deemed valid.

Abstract 520: Characterizing the AC1-exemestane resistant cell line to reveal the underlying mechanism of resistance to aromatase inhibitors in estrogen-dependent breast cancer .

Abstract Estrogen receptor positive (ER+) breast cancer depends on the hormone estrogen for growth and is one of the most common forms of breast cancer present among postmenopausal women. One type of therapy that has proven highly effective in treating this type of breast cancer is aromatase inhibitors. Aromatase is an enzyme that mediates the conversion of androgens (i.e. androstenedione) into estrogens. Aromatase inhibitors (AIs) letrozole, anastrazole, and exemestane, are used to treat estrogen receptor positive (ER+) patients by reducing the levels of estrogen produced via the Aromatase enzyme. Though AIs are dynamically effective at treating ER+ cancers, a significant amount of breast cancer patients become resistant to these drugs over time. The purpose of this study is to gain a better understanding of acquired AI resistance by 1) developing a breast cancer cell line (AC1-ExR) that mimics clinical acquired exemestane resistance; and 2) characterizing this cell line. The novel cell line was extracted from ER+ tumors of xenograft mouse models. The xenograft tumors initially regressed, but after long-term treatment, they became resistant to exemestane, a type of AI, and began to grow. We characterized the AC1-ExR cell line by analyzing their drug sensitivity. Aromatase Assays were performed to measure the aromatase activity in these cells following treatment with 10−6 M of exemestane. The aromatase activity in the AC1-ExR cell line, when treated with exemestane, was less than that of the AC1 parental cell line: average aromatase activity of 31.1 fmol/mg protein*hr compared to average aromatase activity of 72.1 fmol/mg protein*hr in the parental cell line. In addition, AC1-ExR cells exhibited more cancer stem cell-like properties than the AC1 cells. The average mammosphere count for AC1-ExR and AC1 cells was 54 and 33.3 mammospheres, respectively. In the ER+ pathway, interactions between several growth factor signaling pathways have shown to increase hypersensitivity of the ER-alpha receptor to estrogen stimuli. Specifically, inhibitors to the insulin-like growth factor receptor (IGF-1R) have shown to significantly suppress the growth of breast cancer cells in vitro. Identification of growth factor signaling pathways that may be relevant to the ER+ pathway and crosstalk between ER-alpha and IGF-1R, EGFR, and/or other active receptors may reveal their potential role in aromatase resistance in ACI-ExR cells. Characterization of the AC1-ExR cell line has revealed decrease in aromatase activity. A comprehensive understanding of this mechanism may serve as a means of treatment in patients with ER+ positive breast cancer. This work was funded in part by NIH/NIGMS MARC U*STAR T34 08663 National Research Service Award to UMBC and an RO1 CA-62483 Grant by the National Cancer Institute of the National Institutes of Health Citation Format: Joyce C. Ohiri, Armina Kazi, Amanda Schech, Angela M. Brodie. Characterizing the AC1-exemestane resistant cell line to reveal the underlying mechanism of resistance to aromatase inhibitors in estrogen-dependent breast cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 520. doi:10.1158/1538-7445.AM2013-520

Effects of low dose treatment of tributyltin on the regulation of estrogen receptor functions in MCF-7 cells

Endocrine disrupting chemicals are the natural/synthetic compounds which mimic or inhibit the actions of endogenous hormones. Organotin compounds, such as tributyltin (TBT) are typical environmental contaminants and suspected endocrine-disrupting chemical. The present study evaluates the estrogenic potential of this compound in vitro in ER (+) breast adenocarcinoma, MCF-7 cell line. Our data showed that tributyltin chloride (TBTCl) had agonistic activities for estrogen receptor-α (ER-α). Its estrogenic potential was checked using cell proliferation assay, aromatase assay, transactivation assay, and protein expression analysis. Low dose treatment of TBTCl had a proliferative effect on MCF-7 cells and resulted in up-regulation of aromatase enzyme activity and enhanced estradiol production in MCF-7 cells. Immunofluorescence staining showed translocation of ER-α from cytoplasm to nucleus and increased expression of ER-α, 3β-HSD and aromatase on treatment with increasing doses of TBTCl. Further, to decipher the probable signaling pathways involved in its action, the MCF-7 cells were transfected with different pathway dependent luciferase reporter plasmids (CRE, SRE, NF-κB and AP1). A significant increase in CRE and SRE and decrease in NF-κB regulated pathway were observed (p<0.05). Our results thus showed that the activation of SRE by TBTCl may be due to ligand dependent ER-α activation of the MAPK pathway and increased phosphorylation of ERK. In summary, the present data suggests that low dose of tributyltin genomically and non-genomically augmented estrogen dependent signaling by targeting various pathways.

Characterization of Brain and Liver Aromatase of Gilthead Seabream (Sparus aurata L., 1758) using Dibenzylfluorescein

Aromatase catalyzes the conversion of androgens into estrogens. Data on native aromatase enzyme kinetics and thus actual catalytic activity are scarce in fish, impeding comparison of aromatase activity (AA) from different organs within and between species. In the present study, fluorescence aromatase assay was optimized to measure AA in the gilthead seabream (Sparus aurata L., 1758) using dibenzylfluorescein (DBF) as a fluorometric substrate in brain and liver microsomes. Brain and liver AA have showed linearity until 20 and 40 µg protein concentration throughout 30 minutes, respectively. In brain and liver optimum pH of the enzyme was found to be 6.50 and 8.25, respectively and optimum temperature was found to be 30°C for both tissues. It has been observed that brain and liver AA have saturated at and above 2 µM DBF concentrations. Determined Vmax and Km values of brain and liver aromatase using LineweaverBurk graph have calculated 8,054±0,550 and 8,389±0,543 pmol/min/mg protein and 0,840±0,161 µM and 0,959±0,152 µM, respectively. Testosterone appeared to competitively inhibit the Sparus aurata brain and liver aromatase. In conclusion, the parameters of this assay that are reported for brain and liver aromatase in gilthead seabream could be useful to measure AA in other species

Endocrine-Disrupting Effects of Thioxanthone Photoinitiators

Photoinitiators used in food packaging ink, such as 2-isopropylthioxanthone (2-ITX), have been shown to migrate into food and beverages. Recently, several studies indicated that 2-ITX might be an endocrine-disrupting chemical. In this work, the effects of 2-ITX, 4-isopropylthioxanthone (4-ITX), 2,4-diethylthio xanthone (2,4-diethyl-TX), 2-chlorothioxanthone (2-chloro-TX), and 1-chloro-4-propoxythioxanthone (1-chloro-4-propoxy-TX) on steroidogenesis and androgen and estrogen receptor-mediated transcription activation have been studied using human H295R adrenocarcinoma cells and yeast hormone bioassays, respectively. None of the compounds showed androgenic or estrogenic activities, but clear antiandrogenic and antiestrogenic activities were observed for 2-ITX, 4-ITX, and 2,4-diethyl-TX, whereas 2-chloro-TX showed only antiandrogenic activity. In an adapted version of the H295R steroidogenesis assay, using gas chromatography-tandem mass spectrometry analysis of H295R media, all five compounds increased levels of 17ß-estradiol and estrone. H295R cells incubated with 2-ITX also showed significantly reduced androgen and increased pregnenolone and progesterone levels. Expression of particular steroidogenic genes, including the one encoding for aromatase (CYP19A1), was significantly upregulated after incubation of H295R cells with 2-ITX, 4-ITX, and 2,4-diethyl-TX. In line with the increased CYP19A1 mRNA expression, 2-ITX increased catalytic activity of aromatase in H295R cells as measured by cognate aromatase assays. The results indicate that thioxanthone derivatives can act as potential endocrine disruptors both at the level of nuclear receptor signaling and steroid hormone production.

Comment on “Glyphosate impairs male offspring reproductive development by disrupting gonadotropin expression” by Romano et al. 2012

Romano et al. (2012) recently published the results of a study in which pregnant rats were treated with a glyphosate-based herbicide formulation from gestational day 18 to postnatal day 5. Various parameters related to sexual behavior and reproductive development were assessed in the resulting male offspring. Based on their results, the study authors claim that glyphosate exposure in the perinatal period causes changes in males that are associated with hypersecretion of androgens. There are a number of issues with this study, however, that preclude such conclusions. First, as a premise for this study, the authors incorrectly assert that glyphosate is a ‘‘potential endocrine chemical disruptor.’’ As evidence, they cite three in vitro studies of altered aromatase activity conducted in a single laboratory (Benachour et al. 2007; Gasnier et al. 2009; Richard et al. 2005), and their own earlier work suggesting reduced serum testosterone concentrations in response to treatment (Romano et al. 2010). These studies, however, were conducted using glyphosate-based herbicide formulations rather than the active ingredient alone. Exposures were at high concentrations; for instance, in the study by Benachour et al. (2007), the concentration of glyphosate in the tissue culture medium was reported to be 1.27 mM (approximately 200,000 lg/L), which is not relevant to concentrations in target tissue concentrations or environmental media. Further, although sporadic positive responses were reported, aromatase activity was generally unaffected in the few experiments that tested glyphosate by itself. It should be additionally noted that the EPA OPPTS guideline for conducting the aromatase assay (U.S. Environmental Protection Agency 2009a) clearly warns that all glassware and apparatus used need to be free of detergent residue; this is because microsomes are extremely sensitive to the surfactants found in detergents. Surfactants are a major component of the various herbicide formulations tested in these studies and are the likely cause for the observed aromatase inhibition. Therefore, the claim that glyphosate is an endocrine disruptor is not substantiated. Second, it is not clear to what agent(s) the animals were exposed. The study authors indicate that Roundup Transorb (480 g/L glyphosate, 648 g/L isopropylamine salt, and 594 g/L inert ingredients) was the product administered. However, they report no additional details regarding the composition of the herbicide formulation used such as the identity and amount of surfactant(s) in the formulation. This is important because in other studies, Levine et al. (2007) have shown that steroidogenesis in Leydig cell cultures is inhibited by the presence of surfactants, which perturb membrane function of mitochondria. An appropriate control group that was exposed to the surfactant(s) only was not included in the study nor was there a group exposed to glyphosate only. As such, the study results are confounded. Third, the authors did not describe any methods to control for potential litter effects, and it is not clear whether this was done or not. Because the dams were the unit of exposure in these experiments, the exposed litter (the dam) is the appropriate unit of analysis. This is particularly important when assessing these experiments because dosing of the dams occurred from gestational day 18 through postnatal day 5; however, glyphosate does not cross well

ChemInform Abstract: Non‐Steroidal Dissociated Glucocorticoid Agonists: Indoles as A‐Ring Mimetics and Function‐Regulating Pharmacophores.

AbstractSAR analysis of non‐steroidal glycocorticoid mimetics lead to the discovery of new agonist (I), which shows high progesterone receptor and mineralocorticoid receptor selectivity, potent agonist activity, and reduced transactivation activity in the MMTV (mouse mammary tumor virus) and aromatase assays.

Non-steroidal dissociated glucocorticoid agonists: indoles as A-ring mimetics and function-regulating pharmacophores

We report a SAR of non-steroidal glucocorticoid mimetics that utilize indoles as A-ring mimetics. Detailed SAR is discussed with a focus on improving PR and MR selectivity, GR agonism, and in vitro dissociation profile. SAR analysis led to compound (R)-33 which showed high PR and MR selectivity, potent agonist activity, and reduced transactivation activity in the MMTV and aromatase assays. The compound is equipotent to prednisolone in the LPS-TNF model of inflammation. In mouse CIA, at 30 mg/kg compound (R)-33 inhibited disease progression with an efficacy similar to the 3 mg/kg dose of prednisolone.

Screening for Compounds with Aromatase Inhibiting Activities from Atractylodes macrocephala Koidz

Ten compounds were isolated from the dichloromethane extract of Atractylodes macrocephala and their aromatase inhibiting activities were tested using an in vitro fluorescent-based aromatase assay. The results indicated that atractylenolide I (1), atractylenolide II (2) and atractylenolide III (3) had inhibition ratios of 94.56 ± 0.70%, 90.93 ± 1.41% and 86.31 ± 8.46%, respectively, at a concentration of 10 μM. We conclude from our results that atractylenolide and its derivates may serve as potential aromatase inhibitors (AIs) and thus merit continued study in the future.

Assessment of chemical effects on aromatase activity using the H295R cell line

In response to concerns about chemical substances that can alter the function of endocrine systems and may result in adverse effects on human and ecosystem health, a number of in vitro tests have been developed to identify and assess the endocrine disrupting potential of chemicals and environmental samples. One endpoint that is frequently used in in vitro models for the assessment of chemical effects on the endocrine system is the alteration of aromatase activity (AA). Aromatase is the enzyme responsible for converting androgens to estrogens. Some commonly used aromatase assays, including the human microsomal assay that is a mandatory test in US-EPA's endocrine disruptor screening program (EDSP), detect only direct effects of chemicals on aromatase activity and not indirect effects, including changes in gene expression or transcription factors. This can be a problem for chemical screening initiatives such as the EDSP because chemicals can affect aromatase both indirectly and directly. Here we compare direct, indirect, and combined measurements of AA using the H295R cell line after exposure to seven model chemicals. Furthermore, we compare the predictability of the different types of AA measurements for 17beta-estradiol (E2) and testosterone (T) production in vitro. H295R cells were exposed to forskolin, atrazine, letrozole, prochloraz, ketoconazole, aminoglutethimide, and prometon for 48 h. Direct, indirect, and combined effects on aromatase activity were measured using a tritiated water-release assay. Direct effects on aromatase activity were assessed by exposing cells only during the conduct of the tritium-release assay. Indirect effects were measured after exposing cells for 48 h to test chemicals, and then measuring AA without further chemical addition. Combined AA was measured by exposing cells prior and during the conduction of the tritium-release assay. Estradiol and testosterone were measured by ELISA. Exposure to the aromatase inhibitors letrozole, prochloraz, ketoconazole, and aminoglutethimide resulted in greater indirect aromatase activity after a 48-h exposure due to presumed compensatory mechanisms involved in aromatase activity regulation. Forskolin and atrazine caused similar changes in hormone production and enzyme profiles, and both chemicals resulted in a dose-dependent increase in E2, T, and indirect AA. Neither of these two chemicals directly affected AA. For most of the chemicals, direct and combined AA and E2 were good predictors of the mechanism of action of the chemical, with regard to AA. Indirect aromatase activity was a less precise predictor of effects at the hormone level because of presumed feedback loops that made it difficult to predict the chemicals' true effects, mostly seen with the aromatase inhibitors. Further, it was found that direct and indirect AA measurements were not reliable predictors of effects on E2 for general inducers and inhibitors, respectively. Differential modulation of AA and hormone production was observed in H295R cells after exposure to seven model chemicals, illustrating the importance of measuring multiple endpoints when describing mechanisms of action in vitro. For future work with the H295R, it is recommended that a combination of direct and indirect aromatase measurements is used because it was best in predicting the effects of a chemical on E2 production and its mechanism of action. Further, it was shown that direct AA measurements, which are a common way to measure AA, must be used with caution in vitro.

Pomegranate Ellagitannin–Derived Compounds Exhibit Antiproliferative and Antiaromatase Activity in Breast Cancer Cells In vitro

Estrogen stimulates the proliferation of breast cancer cells and the growth of estrogen-responsive tumors. The aromatase enzyme, which converts androgen to estrogen, plays a key role in breast carcinogenesis. The pomegranate fruit, a rich source of ellagitannins (ET), has attracted recent attention due to its anticancer and antiatherosclerotic properties. On consumption, pomegranate ETs hydrolyze, releasing ellagic acid, which is then converted to 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one ("urolithin") derivatives by gut microflora. The purpose of this study was to investigate the antiaromatase activity and inhibition of testosterone-induced breast cancer cell proliferation by ET-derived compounds isolated from pomegranates. A panel of 10 ET-derived compounds including ellagic acid, gallagic acid, and urolithins A and B (and their acetylated, methylated, and sulfated analogues prepared in our laboratory) were examined for their ability to inhibit aromatase activity and testosterone-induced breast cancer cell proliferation. Using a microsomal aromatase assay, we screened the panel of ET-derived compounds and identified six with antiaromatase activity. Among these, urolithin B (UB) was shown to most effectively inhibit aromatase activity in a live cell assay. Kinetic analysis of UB showed mixed inhibition, suggesting more than one inhibitory mechanism. Proliferation assays also determined that UB significantly inhibited testosterone-induced MCF-7aro cell proliferation. The remaining test compounds also exhibited antiproliferative activity, but to a lesser degree than UB. These studies suggest that pomegranate ET-derived compounds have potential for the prevention of estrogen-responsive breast cancers.

Inhibition of placental aromatase activity in a cell free assay by ovarian protein.

Previously we identified a fraction of follicular fluid (follicle regulatory protein: FRP) which inhibits granulosa cell aromatase activity. During the course of these studies the question of FRP acting via autocrine as well as paracrine mechanisms arose in addition to the need for a more efficient method of screening for aromatase inhibitory activity during the purification of FRP. Accordingly, we assessed the effects of FRP on aromatase activity in a microsomal assay. Placental microsome preparations were preincubated for 20 minutes with or without FRP prior to a 20 minute incubation with testosterone. Significantly less conversion of testosterone to estrogen occurred with FRP compared to control preincubation. When follicular protein was added without pre-incubation, there was no apparent change in microsomal aromatase activity, whereas after a 20 minute pre-incubation with the follicular protein fraction, significantly less testosterone was converted into estrogen. When various concentrations of FRP were assayed in the placental aromatase assay, a dose-response curve demonstrated a 50% inhibitory dose (ID50) of approximately 400 micrograms/ml. To further purify the aromatase inhibitory activity, 5 mg of the crude follicular fluid preparation was eluted through an anion exchange column via HPLC using a sodium acetate gradient. The fractions in the central elution peak contained aromatase inhibitor activity with ID50 values of 25-160 micrograms/ml. Thus Fractions were further purified by elution through a gel exclusion column via HPLC which demonstrated inhibition of cell free placental aromatase activity in the 15,000-18,000 molecular weight range with an ID50 of 5 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)