Antiestrogenic Potency Research Articles

Design and synthesis of phosphoryl-substituted steroidal pyridazines (Pho-STPYRs) as potent estrogen receptor alpha inhibitors: targeted treatment of hormone-dependent breast cancer cells.

Estrogen receptor alpha (ERα) is an important target for the discovery of new therapeutic drugs against hormone-dependent breast cancer. A series of phosphoryl-substituted steroidal pyridazines (Pho-STPYRs) were synthesized and biologically evaluated as potent ERα inhibitors. Pho-STPYRs showed cytotoxicity against breast cancer cells with IC50 values of 5.9 μM and higher. Pho-STPYRs 33 and 34 [IC50 (MCF7) = 6.5 and 5.9 μM, respectively] were found to block the expression of ERα, the main driver of breast cancer growth, and modulate the ERK, cyclin D1, and CDK4 pathways. Compound 34 showed selectivity, anti-estrogenic potency and high antiproliferative efficacy in combination with the AKT inhibitor. Molecular docking was used to more accurately define the binding mode of lead compounds 33 and 34 to ERα. The selectivity analysis showed that lead compounds 33 and 34 produce no effects on cytochromes P450, including CYP7A1, CYP7B1, CYP17A1, CYP19A1, and CYP21A2. In a word, Pho-STPYRs 33 and 34 are promising ERα inhibitors for the treatment of hormone-dependent breast cancer.

Synthesis and Biological Evaluation of Chalconesulfonamides: En Route to Proapoptotic Agents with Antiestrogenic Potency.

Breast and other estrogen receptor α-positive cancers tend to develop resistance to existing drugs. Chalcone derivatives possess anticancer activity based on their ability to form covalent bonds with targets acting as Michael acceptors. This study aimed to evaluate the anticancer properties of a series of chalcones (7a-l) with a sulfonamide group attached to the vinyl ketone moiety. Chalconesulfonamides showed a potent antiproliferative effect at low micromolar concentrations against several cancer cell lines, including ERα-positive 4-hydroxytamoxifen-resistant MCF7/HT2. Immunoblotting of samples treated with the lead compound 7e revealed its potent antiestrogenic activity (ERα/GREB1 axis) and induction of PARP cleavage (an apoptosis marker) in breast cancer cells. The obtained compounds represent a promising basis for further development of targeted drugs blocking hormone pathways in cancer cells.

Novel pentacyclic derivatives and benzylidenes of the progesterone series cause anti-estrogenic and antiproliferative effects and induce apoptosis in breast cancer cells.

The promising antitumor effects of progesterone derivatives have been identified in many studies. However, the specific mechanism of action of this class of compounds has not been fully described. Therefore, in this study, we investigated the antiproliferative and (anti)estrogenic activities of novel pentacyclic derivatives and benzylidenes of the progesterone series. The antiproliferative effects of the compounds were evaluated on hormone-dependent MCF7 breast cancer cells using the MTT test. Estrogen receptor α(ERα) activity was assessed by a luciferase-based reporter assay. Immunoblotting was used to evaluate the expression of signaling proteins. All benzylidenes demonstrated inhibitory effects with IC50 values below 10 µM, whereas pentacyclic derivatives were less active. These patterns may be associated with the lability of the geometry of benzylidene molecules, which contributes to an increase in the affinity of interaction with the receptor. The selected compounds showed significant anti-estrogenic potency. Benzylidene 1d ((8S,9S,10R,13S,14S,17S)-17-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15-decahydrocyclopenta[a]phenanthren-3-one) was the most active in antiproliferative and anti-estrogenic assays. Apoptosis induced by compound 1d was accompanied by decreases in CDK4, ERα, and Cyclin D1 expression. Compounds 1d and 3d were characterized by high inhibitory potency against resistant breast cancer cells. Apoptosis induced by the leader compounds was confirmed by PARP cleavage and flow cytometry analysis. Compound 3d caused cell arrest in the G2/M phase. Further analysis of novel derivatives of the progesterone series is of great importance for medicinal chemistry, drug design, and oncology.

Secosteroid–quinoline hybrids as new anticancer agents

An elegant approach to unknown secosteroid–quinoline hybrids is disclosed. A series of 13,17-secoestra-1,3,5(10)-trien-17-oic acid [N′-(iso)quinolylmethylene]hydrazides was prepared and these novel type of secosteroids was screened for antiproliferative activity against estrogen-responsive human breast cancer cell line MCF-7. Most of the synthesized compounds showed a cytotoxic effect superior to that of reference drug cisplatin; the lead compound exhibits the highest activity with the IC50 value of about 0.8 μM and is 7 times more active than cisplatin. A high selectivity index was observed for the hit 13,17-secoestra-1,3,5(10)-trien-17-oic acid [N′-quinolylmethylene]hydrazides 2a and 2c. Compounds 2a and 2c evaluated in luciferase reporter assays exhibited high antiestrogenic potency which was superior to that of tamoxifen. These hit compounds were characterized by high activity against MCF-7 cells that retained towards multidrug-resistant NCI/ADR-RES cells.

Design, Semisynthesis, and Estrogenic Activity of Lignan Derivatives from Natural Dibenzylbutyrolactones.

Based on molecular docking studies on the ERα, a series of lignan derivatives (3–16) were designed and semisynthesized from the natural dibenzylbutyrolactones bursehernin (1) and matairesinol dimethyl ether (2). To examine their estrogenic and antiestrogenic potencies, the effects of these compounds on estrogen receptor element (ERE)-driven reporter gene expression and viability in human ER+ breast cancer cells were evaluated. Lignan compounds induced ERE-driven reporter gene expression with very low potency as compared with the pure agonist E2. However, coincubation of 5 μM of lignan derivatives 1, 3, 4, 7, 8, 9, 11, 13, and 14 with increasing concentrations of E2 (from 0.01 pM to 1 nM) reduced both the potency and efficacy of pure agonists. The binding to the rhERα-LBD was validated by TR-FRET competitive binding assay and lignans bound to the rhERα with IC50 values from 0.16 μM (compound 14) to 6 μM (compound 4). Induced fit docking (IFD) and molecular dynamics (MD) simulations for compound 14 were carried out to further investigate the binding mode interactions. Finally, the in silico ADME predictions indicated that the most potent lignan derivatives exhibited good drug-likeness.

Glucose starvation greatly enhances antiproliferative and antiestrogenic potency of oligomycin A in MCF-7 breast cancer cells

Energy imbalance is one of the key properties of tumour cells, which in certain cases supports fast cancer progression and resistance to therapy.The simultaneous blocking of glycolytic processes and oxidative phosphorylation pathways seems to be a promising strategy for antitumor therapies. The study aimed to evaluate the effect of glucose starvation on the antiproliferative and antiestrogenic potency of oligomycin A against hormone-dependent breast cancer cells.Cell viability was assessed by the MTT test. Estrogen receptor alpha (ERα) activity was evaluated by reporter assay. mTOR, AMPK, Akt, and S6 kinase expression was assessed by immunoblotting.Glucose starvation caused multiple increases in the antiproliferative potency of oligomycin A in the hormone-dependent breast cancer MCF-7 cells, while its effect on the sensitivity of the second hormone-dependent cancer cell line, named T47D, was weak and limited. Glycolytic inhibitors, 3-bromopyruvate and 2-deoxyglucose, greatly enhanced the antiproliferative potency of oligomycin A in MCF-7 cells. Glucose starvation leads to remarkable activation of Akt in MCF-7 cells, whereas oligomycin A enhances its effect. The mTOR, S6 kinase, and AMPK signalling pathways are significantly modulated by oligomycin A under glucose starvation. Oligomycin A demonstrates more pronounced antiestrogenic effects under glucose starvation. Thus, glucose starvation and pharmacological inhibition of glycolysis are of interest for revealing the antitumor potential of macrolide oligomycin A against hormone-dependent breast cancers.

Discovery of derivatives of 6(7)-amino-3-phenylquinoxaline-2-carbonitrile 1,4-dioxides: novel, hypoxia-selective HIF-1α inhibitors with strong antiestrogenic potency

In this article, we describe the synthesis of 3-phenylquinoxaline-2-carbonitrile 1,4-dioxides bearing cyclic diamine residues at positions 6 or 7; the synthesis is based on the nucleophilic substitution of halogens. All synthesized 6(7)-aminoquinoxaline-2-carbonitrile 1,4-dioxides 3–6 demonstrated higher cytotoxicity and hypoxia selectivity compared to the reference agent tirapazamine against breast adenocarcinoma cell lines (MCF7, MDA-MB-231). The structure and position of the diamine residue considerably affects the antiproliferative properties of the quinoxaline-2-carbonitrile 1,4-dioxides. The introduction of a halogen atom at position 7 in the quinoxaline ring of 4a considerably increases the cytotoxicity of compounds 5a and 6a under both normoxic and hypoxic conditions. However, the most hypoxia-selective derivatives were non-halogenated 7-aminosubstituted 3-phenylquinoxaline-2-carbonitrile 1,4-dioxides 3a–j. Of the 32 novel synthesized derivatives, approximately 20 of the 6(7)-amino-3-phenylquinoxaline-2-carbonitrile 1,4-dioxides demonstrated high antiproliferative potency against wild type leukemia cells K562 and drug-resistant subline K562/4 with the expression of p-glycoprotein (p-gp) compared to the reference agent doxorubicin, which exhibited one order of magnitude lower activity towards K562/4 cells than towards K562 cells. Lead compounds 5a and 3f inhibited HIF-1α expression and activity and induced apoptosis in hypoxic tumor cells, which was confirmed by poly(ADP-ribose)polymerase (PARP) cleavage. Moreover, 5a and 3f showed strong antiestrogenic potencies in MCF7 breast cancer cells. Thus, the described series of quinoxaline 1,4-dioxides has high anticancer potential and good aqueous solubility. Therefore, these compounds are promising for further drug development of hypoxia-targeted anticancer agents.

Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for CYP2D6 and Tamoxifen Therapy.

Tamoxifen is biotransformed by CYP2D6 to 4-hydroxytamoxifen and 4-hydroxy N-desmethyl tamoxifen (endoxifen), both with greater antiestrogenic potency than the parent drug. Patients with certain CYP2D6 genetic polymorphisms and patients who receive strong CYP2D6 inhibitors exhibit lower endoxifen concentrations and a higher risk of disease recurrence in some studies of tamoxifen adjuvant therapy of early breast cancer. We summarize evidence from the literature and provide therapeutic recommendations for tamoxifen based on CYP2D6 genotype.

Widely used pharmaceuticals present in the environment revealed as in vitro antagonists for human estrogen and androgen receptors

A considerable amount of scientific evidence indicates that a number of pharmaceuticals that could be detected in the environment can contribute towards the development of problems associated with human reproductive systems, as well as those of wildlife. We investigated the estrogenic and androgenic effects of select pharmaceuticals with high production volume and environmental relevance. We examined the receptor-binding activities of these pharmaceuticals in the T47D human cell line using altered secretion of cytokine CXCL12. Functional yeast-luciferase reporter gene assays were also employed to confirm the mechanism of receptor binding by estrogen and androgen. Non-steroidal anti-inflammatory drugs, namely ibuprofen, diclofenac and antiarrhythmic agent amiodarone showed strong anti-estrogenic effects in the T47D cell line.In the yeast-luciferase assay, these anti-inflammatory drugs also demonstrated anti-estrogenic potency and inhibited the E2 response in a concentration-dependent manner. Amiodarone did not exhibit any response in the yeast-luciferase assay; therefore, the endocrine disruption presumably occurred at a different level without directly involving the receptor. All the anti-inflammatory drugs considered in this study, including ketoprofen, naproxen and clofibrate, exhibited a dose-dependent antagonism towards the androgen receptor in the yeast-luciferase assays. Several other drugs, including the stimulant caffeine, did not show any response in the tests that were employed.A risk assessment analysis using ‘Hazard Quotient’ suggested a potential risk, especially in the cases of ibuprofen, ketoprofen, diclofenac and clofibrate.The results reveal the intrinsic endocrine disrupting nature of several pharmaceuticals and thus could contribute towards explaining a number of adverse health effects on humans and wildlife.

Effect-based assessment of passive air samples from four countries in Eastern Europe

Although passive sampling has been previously used for the monitoring of volatile and semi-volatile contaminants in air, there are limited data on the use of this technique coupled with bioassays based on specific biological responses. Biological responses including those mediated by the aryl hydrocarbon (AhR) receptor as well as (anti-)estrogenicity and (anti-)androgenicity of samples from four Eastern European countries (Lithuania, Slovakia, Romania, and Serbia) were determined. To address the potential differences of specific toxic potencies of pollutant mixtures in ambient air in Eastern Europe, each country was characterized by a single more remote location that served to determine regional background conditions and one location in more urbanized and industrialized locations, which were defined as "impacted" areas. Besides samples from Lithuania, a significant gradient in concentrations of AhR-mediated potency from background and impacted localities was observed. Greatest potencies were measured in samples from impacted locations in Romania and Slovakia. Concentrations of polycyclic aromatic hydrocarbons (PAHs) that were quantified accounted for 3-33 % of the 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents determined by use of the bioassay. No significant estrogenic potency was detected but anti-estrogenic effects were produced by air from two background locations (Lithuania, Slovakia) and three impacted locations (Lithuania, Romania, and Serbia). Anti-androgenic potency was observed in all samples. The greatest anti-estrogenic potency was observed at the background location in Slovakia. Anti-estrogenic and anti-androgenic potencies of studied air samples were probably associated with compounds that are not routinely monitored. The study documents suitability of passive air sampling for the assessment of specific toxic potencies of ambient air pollutants.

Cyclopamine is a novel Hedgehog signaling inhibitor with significant anti-proliferative, anti-invasive and anti-estrogenic potency in human breast cancer cells

Stimulation of Hedgehog (Hh) signaling induces carcinogenesis or promotes cell survival in cancers of multiple organs. In epithelial cancer with aberrant Hedgehog activation, abrogation of Hedgehog signaling by cyclopamine, a naturally occurring Hedgehog-specific small-molecule inhibitor, causes profound inhibition of tumor growth. In the present study, cyclopamine displayed a significant potency in suppressing the proliferation of both estrogen-responsive (MCF-7) and estrogen-independent (MDA-MB-231) human breast cancer cells. Cyclopamine induced a robust G1 cell cycle arrest and elicited notable effects on the expression of cyclin D1 through modulation of the MAPK/ERK signaling pathway. Cyclopamine also inhibited the invasive ability of both breast cancer cell lines by suppressing the expression levels of NF-κB, MMP2 and MMP9 protein. Furthermore, in estrogen-responsive MCF-7 cells, cyclopamine significantly downregulated the production of estrogen receptor-α protein. Our results implicate cyclopamine as a novel, potent inhibitor of human breast cancer proliferation and estrogen responsiveness that could potentially be developed into a promising therapeutic agent for the treatment of breast cancer.

Role and pharmacologic significance of cytochrome P‐450 2D6 in oxidative metabolism of toremifene and tamoxifen

We investigated the in vitro metabolism and estrogenic and antiestrogenic activity of toremifene (TOR), tamoxifen (TAM) and their metabolites to better understand the potential effects of cytochrome P-450 2D6 (CYP2D6) status on the activity of these drugs in women with breast cancer. The plasma concentrations of TOR and its N-desmethyl (NDM) and 4-hydroxy (4-OH) metabolites during steady-state dosing with TOR were also determined. Unlike TOR, TAM and its NDM metabolite were extensively oxidized to 4-OH TAM and 4-OH-NDM TAM by CYP2D6, and the rate of metabolism was affected by CYP2D6 status. 4-OH-NDM TOR concentrations were not measurable at steady state in plasma of subjects taking 80 mg of TOR. Molecular modeling provided insight into the lack of 4-hydroxylation of TOR by CYP2D6. The 4-OH and 4-OH-NDM metabolites of TOR and TAM bound to estrogen receptor (ER) subtypes with fourfold to 30-fold greater affinity were 35- to 187-fold more efficient at antagonizing ER transactivation and had antiestrogenic potency that was up to 360-fold greater than their parent drugs. Our findings suggest that variations in CYP2D6 metabolic capacity may cause significant differences in plasma concentrations of active TAM metabolites (i.e., 4-OH TAM and 4-OH-NDM TAM) and contribute to variable pharmacologic activity. Unlike TAM, the clinical benefits in subjects taking TOR to treat metastatic breast cancer would not likely be subject to allelic variation in CYP2D6 status or affected by coadministration of CYP2D6-inhibiting medications.

Endoxifen Levels and Its Association With CYP2D6 Genotype and Phenotype

An association between CYP2D6 variation and clinical outcomes among women with breast cancer treated with tamoxifen (TAM) has been demonstrated, such that the presence of 2 functional CYP2D6 alleles was associated with better clinical outcomes. This association is mainly due to the CYP2D6-mediated hydroxylation of N-desmethyltamoxifen (NDT) to yield endoxifen (EDF), which because of its high antiestrogenic potency, is mainly responsible for the therapeutic efficacy of TAM. The aim of this study was to evaluate the relation of CYP2D6 genotyping and phenotyping with EDF levels and [NDT]/[EDF] metabolic ratio in breast cancer patients from South of Brazil under TAM therapy. Trough blood samples were collected from 97 patients. CYP2D6 genotyping was performed with a luminex assay and calculation of genotypic activity scores. Tamoxifen and metabolites EDF, NDT, and 4-hydroxy-TAM were measured in plasma by high performance liquid chromatography with photo diode array detector. CYP2D6 phenotyping was performed by the determination of dextromethorphan (DMT) and dextrorphan (DTF) by high-performance liquid chromatography with fluorescence detection at plasma collected 3 hours after oral administration of 33 mg of DMF. Phenotypes were given according to [DMT]/[DTF] metabolic ratio. CYP2D6 genotyping indicated a prevalence of 4.1% poor metabolizer, 4.1% intermediate metabolizer, 49.5% extensive metabolizer slow activity, 39.2% extensive metabolizer fast activity, and 3.1% ultrarapid metabolizer. Genotype (genotypic activity scores) was significantly correlated with phenotype ([DMT]/[DTF]), with a moderate association (rs = -0.463; P < 0.001). Median plasma concentrations (nanograms per milliliter; N = 97) were TAM 57.17; 4-hydroxy-TAM 1.01; EDF 6.21; NDT 125.50. EDF levels were lower in poor metabolizers than that in extensive metabolizers (P < 0.05). Phenotype showed stronger, but still moderate, association with EDF and [NDT]/[EDF] than genotype (r = -0.507, r = 0.625, P < 0.001 versus r = 0.356, r = 0.516, P < 0.01). Phenotype accounted for 26% of the variability in EDF levels and 38% of [NDT]/[EDF], whereas genotype accounted for 12% and 27%, respectively. CYP2D6 genotyping and/or phenotyping could not fully predict EDF concentrations. Monitoring EDF itself could be considered during TAM therapy.

Importance of CYP2D6 metabolism in the in vitro antiestrogenic activity of toremifene and tamoxifen.

e13067 Background: Toremifene (TOR) and tamoxifen (TAM) are approved treatments for advanced breast cancer. It has been suggested that poor metabolizers of TAM have worse clinical outcomes than patients who exhibit normal TAM metabolism. TOR and TAM are extensively metabolized by the human cytochrome P450 system (TAM - 3A4 and 2D6; TOR - 3A4). To gain a better understanding of CYP2D6 status and the potential impact on breast cancer activity, we investigated the estrogen and antiestrogen actions of TOR and TAM and their NDM, 4-OH, and 4-OH-NDM metabolites in Ishikawa endometrial and MCF-7 breast cancer cells in vitro. Methods: Estrogen receptor binding affinity was evaluated in vitro using purified ER-LBD and 3H-estradiol. Transactivation activity was determined in HEK-293 cells. TOR, TAM and their N-desmethyl (NDM), 4-OH, and 4-OH-NDM metabolites were evaluated at up to [10 μM] in Ishikawa endometrial and MCF-7 breast cancer cells. Results: TAM and metabolites had similar binding affinity to TOR analogs. The 4-OH and 4-OH-NDM metabolites bound to ER subtypes with 5-50-fold greater affinity and were 50-100 more efficient at ER transactivation than TOR and TAM. Endoxifen (a CYP2D6 product) exhibited the greatest potency of all compounds tested. Estrogen induced Ishikawa cell growth with maximal effects observed at 0.1 nM. TOR and TAM prevented estrogen-mediated agonist effects with similar activity. TOR and TAM had similar antiestrogen activity against Ishikawa cell growth, however, 4-OH metabolites of TAM and TOR are up to 377-fold more potent than their parent drugs. These data are consistent with in vivo studies in intact rats demonstrating TOR and TAM block estrogenic effects on the uterus. TOR and TAM and metabolites were also antagonists in MCF-7 cells, with the order of antiestrogenic potency being 4-OH and 4-OH-NDM metabolites > TOR/TAM and NDM metabolites. Conclusions: Our results provide mechanistic insight into the potential contributions of the 4-OH and 4-OH-NDM metabolites to TOR and TAM activity and perhaps an explanation for the importance of CYP2D6 metabolism to TAM but not TOR clinical outcomes.

In vitro profiling of endocrine disrupting potency of 2,2′,4,4′-tetrabromodiphenyl ether (BDE47) and related hydroxylated analogs (HO-PBDEs)

The potential of 2,2',4,4'-tetrabromodiphenyl ether (BDE47) and its related hydroxylated analogs (2'-HO-BDE28, 6-HO-BDE47, 4'-HO-BDE17, and 4'-HO-BDE49) to modulate estrogen/thyroid/androgen receptor-(ER, TR, AR), mediated responses were investigated by use of reporter gene assays. Exposure to 1 or 10 μM, 4'-HO-BDE17 significantly up-regulated expression of Luc, whereas other four chemicals did not induce Luc expression under control of the ER. Anti-estrogenic potency was observed for 4'-HO-BDE17 (IC50=1.14 μM)>6-HO-BDE47 (IC50=2.65 μM)>2'-HO-BDE28 (IC50=9.49 μM)>BDE47 (IC50=21.11 μM). No anti-estrogenic effect of 4'-HO-BDE49 was observed. Both 4'-HO-BDE17, 4'-HO-BDE49 resulted in greater responses of Luc expression induced by T3. BDE47, 2'-HO-BDE28, 6-HO-BDE47 did not show any effect on the expression of Luc induced by 5 nM T3. 6-HO-BDE47 (IC50=0.34 μM)>4'-HO-BDE17 (IC50=1.41 μM)>BDE47 (IC50=3.83 μM)>2'-HO-BDE28 (IC50=29.22 μM) exhibited anti-androgenic potency, while 4'-HO-BDE49 did not show androgenic transcriptional activity.

Endocrine disruption effects of 2,2′,4,4′,6-pentabromodiphenylether (BDE100) in reporter gene assays

Polybrominated diphenyl ethers (PBDEs) constitute an important group of flame retardants. 2,2',4,4',6-Pentabromodiphenylether (BDE100) is a prominent PBDE congener in some human populations. The potential of BDE100 to modulate responses mediated by the estrogen (ER), thyroid hormone (ThR) or androgen receptors (AR) were investigated by use of transactivation reporter gene assays. The African green monkey kidney CV-1 cell transiently transfected with the constructed reporter gene plasmid ERE-TATA-Luc and pUAS-tk-Luc with luciferase (Luc) under control of the estrogen response (ERE), or thyroid hormone response (ThRE) elements were used to evaluate (anti)estrogen and thyroid effects of BDE100. The (anti)androgenic potency of BDE100 was also evaluated by use of MDA-kb2 cells, which were stably transfected with MMTV-luciferase. The assays displayed appropriate responses to known natural estrogen 17β-estradiol (E2), ThR ligand triiodothyronine (T3), and the AR agonist 5α-dihydrotestosterone (DHT). 10 or 50 μM BDE100 significantly up-regulated expression of Luc under control of the ER. Antiestrogenic potency was observed for BDE100 (IC50 = 6.21 μM). Co-exposure to 50 μM BDE100 significantly enhanced expression of Luc caused by 5 nM T3. BDE100 was antiandrogenic at 10 and 50 μM with an IC50 of 28.60 μM BDE100. These results suggest that BDE100 can modulate the endocrine system in multiple ways by interfering with several hormonal signaling pathways simultaneously.

AhR-agonistic, anti-androgenic, and anti-estrogenic potencies of 2-isopropylthioxanthone (ITX) as determined by in vitro bioassays and gene expression profiling

2-Isopropylthioxanthone (2-ITX) has been widely used as a photoinitiator in printing ink of packaging materials. A few years ago, this compound got special attention since it was detected in milk and fruit drinks. Since little is known about possible effects of this compound on human health, studies were initiated to investigate its properties, starting with in vitro studies. Structural similarities between ITX and the AhR-agonists 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and furanocoumarins, prompted us to investigate whether ITX could have the potency to activate the AhR. ITX showed a clear response in the DR CALUX® bioassay and also induced EROD activity in H4IIE rat hepatoma cells. Microarray studies in these cells showed a gene expression profile similar to that of TCDD. Moreover, using yeast-based estrogen and androgen bioassays, it was demonstrated that ITX, but not TCDD, has potential anti-estrogenic and anti-androgenic properties. The observed in vitro effects warrant further in vivo studies for confirmation and hazard characterization.

Development and Validation of Chemical and Biological Analyses to Determine the Antiestrogenic Potency of Resin Acids in Paper Mill Effluents

This study combined chemical analysis and bioassays of paper mill effluents and their components in order to determine their antiestrogenic activity. The bioassay comprised a yeast two-hybrid assay incorporating the estrogen receptor alpha (hERalpha) and an hERalpha competitive enzyme-linked immunosorbent assay (ELISA). Samples were fractionated by solid phase extraction (SPE) with a C18 disk and a Florisil cartridge to obtain four fractions. The final fraction, eluted with methanol from the Florisil cartridge after pre-extraction by the C18 disk, was the most active in the two-hybrid assay, and its antiestrogenic potency, expressed as the equivalent concentration to 4-hydroxytamoxifen (OHT), was 277 nM. Seven resin acids had antiestrogenic activity in the active fraction as determined by gas chromatography/mass spectrometry (GC/MS), and their concentration levels ranged from 0.11 to 12 microg/L. All the resin acids exhibited greater activity than OHT; their activity relative to OHT ranged from 2.8- to 4.0-fold in the yeast two-hybrid assay. Based on the chemical analysis data and relative potency of resin acids from the yeast two-hybrid assay, the contribution ratio of resin acids accounted for 72% of the observed antiestrogenic activity of the extract. Furthermore, no resin acid showed any affinity for the estrogen receptor in the ELISA. This study showed that analysis combining the SPE method and the yeast two-hybrid assay is likely to be effective for the comprehensive monitoring of resin acids in paper mill industrial discharge areas.

Some nonylphenol isomers show antiestrogenic potency in the MVLN cell assay

It has been shown that nonylphenol (NP) isomers vary in their estrogenic potency. These differences may be due to varieties in receptor affinity, receptor activation, or activation/deactivation of non-receptor mediated side paths of reporter gene translation. In the present study we investigated the underlying mechanism of the different estrogenic potency of seven nonylphenol isomers. An estrogen receptor binding assay was conducted with the human estrogen receptor alpha (hERα). Additionally we co-incubated the nonylphenol isomers with two concentrations of 17β-estradiol (E2) in the MVLN cell assay to measure the potency of the isomers to compete with E2. No significant differences were found between the nonylphenol isomer binding affinities for the hERα. The IC 50 were in the range of 2.1–8.1 × 10 −6 M and the binding affinity relative to estradiol (set to 1) were between 2.6 and 6.7 × 10 −3. Only two test items ( p353-NP and p-NP) were able to increase the estrogenic response of 100 pM estradiol. The response of the other isomers co-incubated with 100 pM E2 showed varying degrees of inhibition of the response in the MVLN reporter gene assay. Thus, it appears that all isomers bind to the ER but some are partial agonists while others are antagonists in the MVLN reporter gene assay.

Pollutants in particulate and gaseous fractions of ambient air interfere with multiple signaling pathways in vitro

Traditionally, contamination of air has been evaluated primarily by chemical analyses of indicator contaminants and these studies have focused mainly on compounds associated with particulates. Some reports have shown that air contaminants can produce specific biological effects such as toxicity mediated by the aryl hydrocarbon receptor (AhR) or modulation of the endocrine system. This study assessed the dioxin-like toxicity, anti-/estrogenicity, anti-/androgenicity and anti-/retinoic activity of both the particulate and gas phase fractions of air in two regions with different types of pollution sources and a background locality situated in an agricultural area of Central Europe. The first region (A) is known to be significantly contaminated by organochlorine pesticides and chemical industry. The other region (B) has been polluted by historical releases of PCBs, but the major current sources of contamination are probably combustion sources from local traffic and heating. Samples of both particle and gas fractions produced dioxin-like (AhR-mediated) activity, anti-estrogenic and antiandrogenic effects, but none had any effect on retinoid signaling. AhR-mediated activities were observed in all samples and the TEQ values were comparable in both fractions in region A, but significantly greater in the particulate fraction in region B. The greater AhR-mediated activity corresponded to a greater coincident antiestrogenicity of both phases in region B. Our study is the first report of antiestrogenicity and antiandrogenicity in ambient air. Anti-androgenicity was observed in the gas phase of all regions, while in the particulate phase only in one region due to the specific type of pollution in that area. Even though based on concentrations of individual compounds, except for the OCPs, the level of contamination of the two regions was similar, there were strong differences in responses in the bioassays between the two regions. Moreover, AhR-mediated activity and antiestrogenic potencies were greater in region B, where the pollution level according to the chemical analysis was similar or less than in the other region, which indicates the presence of other atmospheric pollutants with specific effects. The results document the advantage and utility of the simultaneous use of bioassays and chemical analysis in risk assessment of complex environmental samples.