Androgen Screen Research Articles

Cytotoxicity of water supply in a Palestinian refugee camp and a Syrian informal tented settlement in Lebanon.

Deficient water, sanitation, and hygiene (WASH) significantly account for a high burden of disease across the globe. Lebanon, an Eastern Mediterranean lower-middle-income country with a polluted environment, a fragmented healthcare system, and an ongoing severe economic crisis, faces serious challenges in sustaining safe water supplies, especially in vulnerable communities, while also hosting the world highest refugee population per capita. This study aimed to examine the mutagenicity, and the estrogenic and androgenic activities of water supplies, across both a Palestinian refugee camp and a Syrian informal settlement. Water samples were collected from two targeted camps in Dbayeh and Choueifat, North and South of the Capital City Beirut, respectively, between the months of September and October 2022. Microbial and physicochemical properties of samples were determined, including fecal contamination, total dissolved solids, and various minerals and salts. Organic pollutants were extracted using pre-packed solid phase extraction (SPE) columns, and then mutagenicity of extracts was examined using the Ames test in two Salmonella typhi bacterial strains. The estrogenic and androgenic activities of extracts were assessed using the yeast estrogen and androgen screen tests assays (YES/YAS). Results show excessive levels of total coliforms and total dissolved solids (TDS) in samples from both sites. In addition, the water supply from the Dbayeh Palestinian refugee camp is mutagenic, while the water supply from the Choueifat Syrian informal settlement shows anti-androgen activity. Our findings provide valuable WASH baseline data in two major vulnerable communities in Lebanon, and highlight the importance of a water toxicity testing approach concomitant with a water safety plan, based on a holistic strategy that covers all stages of the water supply chain.

Evaluation of new alternative methods for the identification of estrogenic, androgenic and steroidogenic effects: a comparative in vitro/in silico study

A suite of in vitro assays and in silico models were evaluated to identify which best detected the endocrine-disrupting (ED) potential of 10 test chemicals according to their estrogenic, androgenic and steroidogenic (EAS) potential compared to the outcomes from ToxCast. In vitro methods included receptor-binding, CALUX transactivation, H295R steroidogenesis, aromatase activity inhibition and the Yeast oestrogen (YES) and Yeast androgen screen (YAS) assays. The impact of metabolism was also evaluated. The YES/YAS assays exhibited a high sensitivity for ER effects and, despite some challenges in predicting AR effects, is a good initial screening assay. Results from receptor-binding and CALUX assays generally correlated and were in accordance with classifications based on ToxCast assays. ER agonism and AR antagonism of benzyl butyl phthalate were abolished when CALUX assays included liver S9. In silico final calls were mostly in agreement with the in vitro assays, and predicted ER and AR effects well. The efficiency of the in silico models (reflecting applicability domains or inconclusive results) was 43–100%. The percentage of correct calls for ER (50–100%), AR (57–100%) and aromatase (33–100%) effects when compared to the final ToxCast call covered a wide range from highly reliable to less reliable models. In conclusion, Danish (Q)SAR, Opera, ADMET Lab LBD and ProToxII models demonstrated the best overall performance for ER and AR effects. These can be combined with the YES/YAS assays in an initial screen of chemicals in the early tiers of an NGRA to inform on the MoA and the design of mechanistic in vitro assays used later in the assessment. Inhibition of aromatase was best predicted by the Vega, AdmetLab and ProToxII models. Other mechanisms and exposure should be considered when making a conclusion with respect to ED effects.

Analysis of (Anti-)Oestrogenic and (Anti-)Androgenic Activities in Wastewater from the Lodz Sewer System

This article presents the results of a study on the oestrogenicity and androgenicity of urban wastewater in Lodz, and the possibility of their removal by the Group Wastewater Treatment Plant (GWWTP). Wastewater samples were taken at five points of the sewer system in the city and at the inlet and outlet of the GWWTP. The study was conducted using Yeast Oestrogen Screen (YES)/Yeast Androgen Screen (YAS) tests, which allow a general assessment of the content of compounds with (anti-)oestrogenic and (anti-)androgenic effects in wastewater, without identifying specific substances. Wastewater samples taken from the sewage network did not show (anti-)oestrogenic activity, while oestrogenic and antagonistic properties to androgens were detected in most of them. In the influent of the treatment plant, oestrogen agonistic activity was detected only in one sample (oestrogen equivalent—EEQ equal to 1.31 × 105 ng 17 β–oestradiol/L) and was 100% removed. The purification efficiencies in GWWTP for oestrogen and androgen antagonistic activity were 51.5–99.2% and 39.4–47.1%, respectively. Although no oestrogenic activity was detected in general wastewater in Lodz, observed high-antagonistic–androgenic activities may adversely affect the water body and cause, among others, the feminization of fish, especially in the case of discharge of untreated wastewater by combined sewer overflows.

Bioactivity Profiles on 15 Different Effect Mechanisms for 15 Golden Root Products via High-Performance Thin-Layer Chromatography, Planar Assays, and High-Resolution Mass Spectrometry

Planar chromatography has recently been combined with six different effect-directed assays for three golden root (Rhodiola rosea L.) samples. However, the profiles obtained showed an intense tailing, making zone differentiation impossible. The profiling was therefore improved to allow for the detection of individual bioactive compounds, and the range of samples was extended to 15 commercial golden root products. Further effect-directed assays were studied providing information on 15 different effect mechanisms, i.e., (1) tyrosinase, (2) acetylcholinesterase, (3) butyrylcholinesterase, (4) β-glucuronidase, and (5) α-amylase inhibition, as well as endocrine activity via the triplex planar yeast antagonist-verified (6-8) estrogen or (9-11) androgen screen, (12) genotoxicity via the planar SOS-Umu-C bioassay, antimicrobial activity against (13) Gram-negative Aliivibrio fischeri and (14) Gram-positive Bacillus subtilis bacteria, and (15) antioxidative activity (DPPH• radical scavengers). Most of the golden root profiles obtained were characteristic, but some samples differed substantially. The United States Pharmacopeia reference product showed medium activity in most of the assays. The six most active compound zones were further characterized using high-resolution mass spectrometry, and the mass signals obtained were tentatively assigned to molecular formulae. In addition to confirming the known activities, this study is the first to report that golden root constituents inhibit butyrylcholinesterase (rosin was tentatively assigned), β-glucuronidase (rosavin, rosarin, rosiridin, viridoside, and salidroside were tentatively assigned), and α-amylase (stearic acid and palmitic acid were tentatively assigned) and that they are genotoxic (hydroquinone was tentatively assigned) and are both agonistic and antagonistic endocrine active.

Multiplex planar bioassay with reduced diffusion on normal phase, identifying androgens, verified antiandrogens and synergists in botanicals via 12D hyphenation

Hormonal active compounds affecting health by altering the hormonal system are present in food. The planar yeast antagonist androgen screen (pYAAS) bioassay is a powerful tool to detect individual hormonal active compounds in complex samples separated by high-performance thin-layer chromatography (HPTLC). Previous methods lacked either detection sensitivity or zone sharpness. To overcome diffusion caused by long bioassay incubation on the normal–phase (NP) plate, zone fixation (fix) was achieved with a new polyisobutyl methacrylate coating, leading to enhanced zone sharpness. The exclusion of false-positive antagonists was integrated in the workflow, which allowed the verification (V) of true antagonists, apart from the detection of synergists. With the new multiplex bioassay providing information on 4 activities, 68 different botanicals were screened and hormonal active zones were identified by elution from the bioautogram to orthogonal reversed-phase high performance liquid chromatography with diode array detection and high-resolution mass spectrometry including fragmentation, resulting in the 12D hyphenation NP-HPTLCfix–UV/Vis/FLD–pYAVAS–FLD–heart cut–RP-HPLC–DAD–HRMS/MS.

Passive sampling and in vitro assays to monitor antiandrogens in a river affected by wastewater discharge

Pharmaceuticals and personal care products, antibiotics, estrogens, and antiandrogens are found widely in aquatic environments. Monitoring studies by sampling surface water and effluents of wastewater treatment plants (WWTPs) have been conducted recently to monitor antiandrogens, which, along with estrogens, cause endocrine disruption. However, few studies have investigated antiandrogenic activity (AA) combined with a chemical analyses of emerging antiandrogens. Therefore, we analyzed the presence and persistence of 12 types of antiandrogens, atrazine, and carbamazepine using grab sampling and polar organic chemical integrative sampler (POCIS) along a river affected by WWTP discharges. Water and sediment samples were collected from the WWTP effluent (WW), as well as upstream (US) and downstream (DS) of the WWTP. We detected only tebuconazole, triclosan, propiconazole, and fluconazole during the two sampling campaigns in 2016 and 2017. Grab sampling of the site WW detected tebuconazole (7–77 ng/L), propiconazole (5–47 ng/L), and fluconazole (6–45 ng/L). However, the concentrations in the river water were below the detection limits. Nevertheless, fluconazole and triclosan were detected by POCIS in the site WW (45.7 and 26.8 ng/L, respectively) and all river samples ranges of 0.3–9.3 and 2.4–3.7, respectively. This detection was attributed to the limit of quantification of POCIS being lower than that of grab sampling. Nilutamide and triclosan were detected in the river sediment, suggesting that their concentrations in the water column were at least partly attenuated through sediment sorption. We also observed AA by analyzing POCIS extracts with the yeast androgen screen assay. The highest AA was found in the site WW and it was still observable several kilometers downstream of the point of discharge despite decreasing. Therefore, the WWTP effluent was most likely contributor to the persistent AA in the river.

Biological evaluation of antiproliferative and anti-invasive properties of an androstadiene derivative on human cervical cancer cell lines

Gynaecological cancers are leading cause of death: breast cancer is the most frequently diagnosed type of malignancies, and cervical neoplasms rank fourth for both incidence and mortality among women worldwide. In one of our previous studies, favourable antiproliferative and antimetastatic properties of a newly synthesized androstane derivative, 17APAD have been demonstrated on breast cancer cell lines with different expression patterns of hormone receptors. The aim of the current study was to investigate the antitumoral potential of this molecule in cervical cancer cell lines, including SiHa cells positive for human papilloma virus (HPV) type 16 and HPV-negative C33A cells.17APAD exerted pronounced growth-inhibition (with IC50 values ranging from 0.76 to 1.72 μM with considerable cancer selectivity), while cisplatin used as a reference agent yielded higher IC50 values (ranging from 3.69 to 12.43) and less selectivity, as evidenced by MTT assay. The proapoptotic effect and morphological changes induced by 17APAD were detected by Hoechst 33258–propidium iodide or Annexin V-Alexa488–propidium iodide fluorescent double staining methods, supplemented with a caspase-3 activity assay to identify the mechanism behind the programmed cell death induced by 17APAD. Additionally, significant and concentration-dependent elevation of the ratio of cells in the G2/M phase, on the expense of G0/G1 phase, was observed after 48 h of exposure to 17APAD. Besides its potent antiproliferative properties against both cervical cancer cell lines, 17APAD elicited a remarkable inhibition of cell migration and invasion as detected in wound-healing and Boyden chamber assays, respectively. The mechanisms of action underlying the effects of 17APAD on cell proliferation and motility were independent of androgenic activity, as demonstrated by the Yeast Androgen Screen method.Our results provide new evidence for the proapoptotic and anti-invasive properties of 17APAD, suggesting that it is worth of further research, as a promising prototype for designing novel anticancer agents.

An Case of Poems Syndrome With Primary Adrenal Insufficiency

POEMS syndrome is rare multi-system disorder characterised by a paraneoplastic plasma cell disorder. The acronym POEMS stands for Polyneuropathy, Organomegaly, Endocrinopathy, M protein, and Skin changes. The underlying mechanisms are poorly understood, but chronic overproduction of pro-inflammatory cytokines have an integral role in the disorder. Diagnosis of POEMS is difficult owing to the variety of clinical manifestations. Mandatory diagnostic criteria are (a) one major criterion of either polyneuropathy or monoclonal gammopathy, in association with (b) one minor criterion including, but not limited to, organomegaly, endocrinopathy or skin changes. Endocrinopathies have been identified in 67–84% of patients with POEMS [1]. While hypogonadism and hypothyroidism are relatively common, primary adrenal failure is rarely reported. We present a 54-year-old woman who was found to have a raised hemoglobin, hematocrit and thrombocytosis on routine blood testing. She was concomitantly investigated for a rapidly-ascending, bilateral peripheral motor and sensory neuropathy. Diagnosis of POEMS was made and she underwent chemotherapy with Lenalidomide and high dose dexamethasone in 2015. Since then, she has not received endogenous glucocorticoids. She had an unplanned admission in April 2020 with a likely viral illness and was found to be glucocorticoid deplete. A 250-mcg short synacthen test demonstrated an inadequate response in cortisol, from 4.31 µg/dl to 6.13 µg/dl, with a raised adrenocorticotrophic hormone (ACTH) level of 66 mU/L. Adrenal antibodies were not detected. CT scan of the abdomen reported no adrenal gland abnormalities. The patient denied symptoms of mineralocorticoid deficiency, with no postural blood pressure (BP) drop - sitting BP 123/69 mmHg and standing BP 131/74 mmHg. Serum electrolytes were normal (renin and aldosterone levels are pending) and androgen screen was within normal limits. Thyroid stimulating hormone (TSH) was 8.07 mIU/L, free thyroxine (fT4) levels 0.93 ng/dL, and thyroid receptor and thyroid peroxidase antibodies were both negative. The patient is now established on Hydrocortisone therapy: 10mg (morning), 5mg (lunchtime). In summary, we present a 54-year-old woman with POEMS syndrome with subacute primary adrenal failure, characterised by glucocorticoid deficiency and ACTH excess. Although rare, it is important for all clinicians to be aware of POEMS syndrome as a potential diagnosis if the diagnostic criteria described above are filled, and for Endocrinologists to be aware that POEMS endocrinopathies can occur in any gland, including the adrenal glands.1.Gandhi, G.Y., et al., Endocrinopathy in POEMS syndrome: the Mayo Clinic experience. Mayo Clin Proc, 2007. 82(7): p. 836–42.

A cell-free bioassay for the detection of androgens.

Androgens remain abused performance-enhancing drugs in sports. Technologies based on mass spectrometry can detect all forms of androgens but fail if the androgen represents a novel structure. A bioassay detects androgens based on function rather than structure. To date, there has been limited adoption of cell-based in vitro bioassays as a screening tool for nontargeted androgen detection because they require expert personnel and specialized equipment to perform. We now describe the development of a cell-free version of an androgen in vitro bioassay. Stage 1 involved in vitro transcription/translation reactions (IVTT) using a DNA template encoding an enhancer/androgen response element (ARE) regulatory region upstream of a minimal promoter that drives expression of a reporter protein. The assay detected testosterone across the concentration range of 106.7 to 0.0144 ng/ml (3.7 × 10-7 to 5 × 10-11 M), with an EC50 of 6.63 ng/ml (23 nM). To reduce complexity, Stages 2-4 of development included just in vitro transcription (IVT) reactions, whereby the output was an RNA molecule. Stage 2 involved directly labelling the RNA molecule with fluorophore-labelled nucleotide triphosphates, Stage 3 involved reverse transcription-polymerase chain reaction (PCR) of the RNA molecule, and Stage 4 utilized an RNA aptamer, Mango II, as its RNA output. The Stage 4 product detected testosterone across the range of 106.7-0.0001 ng/ml (3.7 × 10-7 to 5 × 10-13 M), with an EC50 of 0.04 ng/ml (0.155 nM). Further to this, we show that the Stage 4 product can detect other androgenic molecules. Relative to cell-based bioassays, the Stage 4 product is easy to perform and could be developed into a routine, high-throughput, nontargeted androgen screen.

Development of a broad-spectrum monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for screening of androgens in animal edible tissues

Androgens efficiently promote skeletal muscle growth and protein synthesis but causes neurotoxicity, genotoxicity and carcinogenicity, which had been prohibited applying in livestock. In this study, a broad-spectrum monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been developed for rapidly screening of androgens in various animal tissues. Primarily, a group-specific mAb, 4D12, was produced and showed IC50 values for nortestosterone, methyltestosterone, testosterone and trenbolone of 2.2, 0.3, 1.6 and 4.4 μg L−1, respectively. The limits of detection and limits of quantification of this method for 4 androgens in 11 kinds of animal edible tissues ranged from 37.2 to 697.8 ng L−1 and 70.0 to 1524.0 ng L−1, respectively. The recoveries of the 11 kinds of samples spiked with 4 androgens were in the range from 65.0% to 106.6%, with coefficients of variation below 16.9%. A good correlation (R2 = 0.998) was shown between the ic-ELISA and HPLC-MS/MS results for beef.

Non-targeted detection and differentiation of agonists versus antagonists, directly in bioprofiles of everyday products

Xenoestrogens exert antiandrogenic effects on the human androgen receptor. In the analytical field, such antagonists block the detection of testosterone and falsify results obtained by sum parameter assays. Currently, such agonistic versus antagonistic effects are not differentiated in complex mixtures. Oppositely acting hormonal effects present in products of everyday use can only be differentiated after tedious fractionation and isolation of the individual compounds along with subjection of each fraction/compound to the status quo bioassay testing. However, such long-lasting procedures are not suited for routine. Hence, we developed a fast bioanalytical tool that figures out agonists versus antagonists directly in complex mixtures. Exemplarily, 8 cosmetics and 15 thermal papers were analyzed. The determined antagonistic potentials of active compounds found were comparable to the ones of known antagonists (in reference shown for bisphenol A, 4-n-nonylphenol and four parabens). Relevant biological/chromatographic parameters such as cell viability, culture conditions, dose response curves, limits of biological detection/quantification and working range (shown for testosterone, dihydrotestosterone, nandrolone and trenbolone) were investigated to obtain the best sensitivity of the biological detection. The developed and validated method was newly termed reversed phase high-performance thin-layer chromatography planar yeast ant-/agonistic androgen screen (RP-HPTLC-pYAAS bioassay). Results were also compared with the RP-HPTLC-Aliivibrio fischeri bioassay (applied on RP plates for the first time). As proof-of-concept, the transfer to another bioassay (RP-HPTLC-pYES) was successfully demonstrated, analogously termed RP-HPTLC-pYAES bioassay detecting anti-/estrogens (exemplarily shown for evaluation of 4 pharmaceuticals used in breast cancer treatment). The new imaging concept provides (1) detection and differentiation of individual agonistic versus antagonistic effects in the bioprofiles, (2) bioanalytical quantification of their activity potential by scanning densitometry and (3) characterization of unknown bioactive compound zones by hyphenation to high-resolution mass spectrometry. Depending on the hormonal bioassay, 15 samples were analyzed in parallel within 5 h or 6 h (calculated as 20 or 24 min per sample). For the first time, piezoelectric spraying of the yeast cells was successfully demonstrated for the planar yeast–based bioassays.

Андрогенный дефицит у пациентов с заболеваниями предстательной железы

Negative effects of androgen deficiency in males can be avoided by regular androgen screening in patients over 50 years of age. We measured testosterone levels in 521 men aged between 18 and 93 years (mean age 60.94 years). A total of 235 patients (45.2%) were found to have age-related hypogonadism. We have developed a diagnostic algorithm for androgen deficiency at outpatient facilities. We also assessed the prevalence of prostatic diseases and a number of somatic disorders in patients with age-related hypogonadism. We have proposed a variant of medical correction of androgen deficiency. Key words: age-related hypogonadism, testosterone, prostate, screening, androgen replacement therapy

Comparison of the three SARMs RAD-140, GLPG0492 and GSK-2881078 in two different in vitro bioassays, and in an in silico androgen receptor binding assay

Selective androgen receptor modulators comprise compounds that bind as ligands to the androgen receptor and possess tissue-selective activities. Ideally, they show agonistic properties in anabolic target tissues, while inducing antagonistic or only weak agonistic effects in reproductive organs. Due to their myoanabolic effects, selective androgen receptor modulators are included in the list of prohibited substances and methods of the World Anti-Doping Agency.In the current investigation, the androgenic potential of RAD-140, GSK-2881078 and GLPG0492 was comparably investigated in two different in vitro bioassays. In the yeast androgen screen, the androgenic effects were lower than in the reporter gene assay in prostate carcinoma cells (e.g. for GSK-2881078, the EC50 values were 4.44 × 10−6M in the yeast screen and 3.99 × 10-9M in the prostate cells respectively). For future investigations, it is of importance whether the yeast androgen screen, which has been proven to detect androgenic compounds in urine, can detect an abuse of the selective androgen receptor modulators. Molecular modeling of the binding to the androgen receptor ligand binding domain suggests slight differences in the binding modes of RAD-140, GSK-2881078 and GLPG0492.In conclusion, androgenic activity of the three non-steroidal compounds in the two different in vitro test systems confirmed the results of the in silico modeling of the androgen receptor binding.

Androgen- and estrogen-receptor mediated activities of 4-hydroxytestosterone, 4-hydroxyandrostenedione and their human metabolites in yeast based assays

4-Hydroxyandrost-4-ene-3,17-dione, also named formestane, is an irreversible aromatase inhibitor and therapeutically used as anti-breast cancer medication in post-menopausal women. Currently, no therapeutical indication led to approval of its 17-hydroxylated analog 4-hydroxytestosterone, an anabolic steroid. However, it is currently investigated in a clinical trial for breast cancer. In context with sports doping, aromatase inhibitors are administered to reduce estrogenic side effects of misused anabolic substances or their metabolites. Therefore, both substances are prohibited in sports by the World Anti-Doping Agency (WADA). Analysis of urinary phase I and phase II metabolites showed similar results for both compounds. In the current investigation, 4-hydroxyandrost-4-ene-3,17-dione, 4-hydroxytestosterone and seven of their described urinary metabolites as well as 2α-hydroxyandrostenedione were tested in the yeast androgen screen and the yeast estrogen screen. Androgenic effects were observed for all tested substances, except for one, which showed anti-androgenic properties. With regard to the yeast estrogen screen, estrogenic effects were observed for only two metabolites at rather high concentrations, while six out of the ten substances tested showed anti-estrogenic properties. In terms of the strong androgenic effect observed for 4-hydroxytestosterone (10−8 M), 4-hydroxyandrost-4-ene-3,17-dione (10−8 M) and two more urinary metabolites, the yeast androgen assay may also be used to trace abuse in urine samples.

Assessment of the analgesic dipyrone as a possible (anti)androgenic endocrine disruptor

Mild analgesics have been associated with antiandrogenic effects, but there are no such studies on dipyrone, despite its high prevalence of use in many countries. We examined the production of steroid hormones in human H295R cells after exposure to dipyrone and two metabolites, 4-Methylaminoantipyrine (MAA) and 4-Aminoantipyrine (AA), as well as fetal testicular testosterone production in rats following maternal dipyrone exposure. Androgen agonistic/antagonistic effects were examined in vitro for dipyrone and its metabolites in the Yeast Androgen Screen (YAS) assay and in vivo for dipyrone through the Hershberger assay. In vitro we tested dipyrone, MAA, and AA (0.1–1000 μM) while in vivo we used dipyrone (50, 100, 200 mg/kg/day). In the H295R assay, dipyrone, MAA and AA reduced the production of androgens and corticosteroids. Testosterone was reduced at concentrations 4–13 times higher than the maximum plasma concentrations reported in humans for MAA and AA. No effects were observed in the fetal testosterone production assay. In the YAS and Hershberger assays, no androgen agonistic/antagonistic activities were observed. These results indicate that dipyrone and its metabolites do not interact with the androgen receptor, but have the potential to inhibit steroidogenesis, however only at concentrations that are not relevant under normal medical use.

Investigation of full-scale ozonation at a municipal wastewater treatment plant using a toxicity-based evaluation concept

ABSTRACT Effluents from municipal wastewater treatment plants (WWTPs) are known to be point sources of micropollutants for surface waters. The aim of this study was to examine a reconstructed full-scale ozonation equipped with a pump-injector system for ozone (O3) dosage and a fluidized moving-bed reactor as biological posttreatment at a municipal WWTP utilizing an effect-directed approach. This approach consists of chemical analysis in combination with toxicological tests for the assessment of treatment efficiency of the plant. Chemical analysis showed elimination rates > 80% for pharmaceuticals and industrial chemicals. Analysis of endocrine disruptors was limited due to substance concentrations below the limit of detection (LOD). Estrogenic activity was detected by the Arxula Adeninivorans yeast estrogen screen (A-YES) at low concentrations (pg to ng EEQ/l range). Estrogenic activity was reduced by more than 90% after ozonation. In contrast, androgenic activity (measured in the Adeninivorans yeast androgen screen, A-YAS) was still found after O3 treatment and after biological posttreatment, which is consistent with the data obtained by chemical analysis. Furthermore, no marked genotoxic or cytotoxic effects were observed after ozonation using the alkaline comet and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) assays, respectively. Results suggest that the applied specific O3 dose of 0.4 mgO3/mgDOC is a safe operation setup in terms of toxicologically relevant transformation products. In addition, no adverse effects on primary producers, as evidenced by algae growth inhibition tests, were detected. The monitored biofilm growth in the biological posttreatment exhibited a steady state after one month. Based on computational fluid dynamics (CFD) simulations and biomass, one might conclude that O3 did not apparently enter biological posttreatment to a great extent and that hydraulic retention time in the O3 reactor was sufficient. Our data demonstrate the effectiveness of a full-scale O3 treatment in combination with a fluidized moving-bed reactor as biological posttreatment for the reduction of a majority of micropollutants without the release of relevant toxic transformation products as assessed by a chemical and toxicity-based approach.

The ability of the YAS and AR CALUX assays to detect the additive effects of anti-androgenic fungicide mixtures.

Actual risk assessment only takes single pesticides into account, although about one third of the analyzed food samples in the European Union was contaminated with pesticide mixtures in 2013. A cumulative approach would group pesticides that share the same mechanism of toxicity. We evaluated the combination effects of low effect concentrations of binary and ternary mixtures of six anti-androgenic fungicides (procymidone, vinclozolin, tebuconazole, propiconazole, fenarimol and prochloraz) antagonizing the human androgen receptor (hAR) in the Yeast-based Androgen Screen assay (YAS) as well as in the AR Chemical-Activated LUciferase gene eXpression (AR CALUX) assay by means of concentration addition and nonlinear regression. The mixture effects were essentially additive when the fungicides were applied as iso-effective low inhibitory concentration combinations, independently from the used assay and as shown by the excellent agreement between experimental and predicted data. Both assays were successfully applied to evaluate the additive effects of fungicide mixtures at low concentrations. Since pesticide residues occur in/on foodstuffs in the EU at rather low concentrations and hormonally active environmental contaminants may concomitantly be present, more complex mixtures of anti-androgenic chemicals, not only pesticides, should be tested in the future when wanting to apply a target organ-based risk assessment approach.

Development of a novel cell based androgen screening model

The androgen receptor (AR) mediates the majority of androgen effects on target cells. The DNA cis-regulatory elements that respond to AR share sequence similarity with cis-regulatory elements for glucocorticoid, mineralocorticoid and progesterone receptors (GR, MR and PR, respectively). As a result, many of the current AR screening models are complicated by inaccurate activation of reporters by one of these receptor pathways. Identification of more selective androgen testing systems would be beneficial for clinical, pharmacological and toxicologic screening of AR activators. The present study describes the development of a selective androgen-responsive reporter cell line that expresses AR but does not express GR, MR and PR. CV1 cells were stably transduced to express human AR and an androgen-responsive gaussia luciferase gene. Clonal populations of AR expressing cells were isolated. Quantitative RT-PCR (qPCR) and western analysis confirmed stable integration of AR in the most responsive clonal line which was named ‘CV1-ARluc’. Stimulation of CV1AR-luc with androgenic ligands (testosterone and 5α-dihydrotestosterone) for 18h caused an increase in luciferase activity in a dose-dependent manner. Other steroid hormones including aldosterone, cortisol, and progesterone did not stimulate luciferase response. The CV1-ARluc also increased luciferase activity when treated with human serum extracts. In conclusion, the CV1-ARluc cells provide a novel model system for screening of new AR agonists and antagonists and can determine the androgenic activity of human serum samples.

Screening of multiple hormonal activities in water and sediment from the river Nile, Egypt, using in vitro bioassay and gonadal histology.

In Egypt, until yet no records are available regarding possible multiple hormonal activities in the aquatic systems and especially in the river Nile. In this paper, in vitro yeast estrogen screen (YES) and yeast androgen screen (YAS) were used to assess (for the first time) the multiple hormonal activities in surface waters and sediments of the river Nile. This study aimed to determine whether river Nile water can cause changes in gonadal histology of Nile tilapia (Oreochromis niloticus niloticus). All water samples exhibited extremely low levels of estrogenicity. Estrogenicity was nearly not detected in any of the sediment samples. Unlike the estrogenicity, significant androgenic activities were recorded in the water and sediment samples along the course of the river Nile. The present study reports for the first time quantification anti-estrogenic and anti-androgenic activities with high levels in both water and sediment of the river Nile. The greatest anti-estrogenic and anti-androgenic activities were observed in samples from downstream river Nile. These results indicated that the anti-estrogenic and anti-androgenic activities along the Nile course were great and the pollution of the sites at downstream was more serious than the upstream sites due to industrial and anthropogenic activities at these sites. Good correlations were observed among some hormonal activities, suggesting coexistence of these contaminants in the environmental matrices. There were no signs of sexual disruption in any of the gonads analyzed from either male or female Nile tilapia, demonstrating that no hormonal activity present along the Nile course was sufficient to induce adverse effects on reproductive development. Further investigation is necessary to identify the compounds responsible for the hormonal activities in the river Nile and to examine effects of very low levels of hormonally active compounds on gonadal histology, as well as in the development of more sensitive biomarkers.

In vitro assessment of endocrine disrupting potential of naphthenic Acid fractions derived from oil sands-influenced water.

Oil sands-influenced process waters have been observed to cause reproductive effects and to induced CYP1A activity in fishes; however, little progress has been made in determining causative agents. Naphthenic acids (NAs) are the predominant organic compounds in process-affected waters, but due to the complexity of the mixture, it has been difficult to examine causal linkages in fishes. The aim of this study was to use in vitro assays specific to reproductive and CYP1A mechanisms to determine if specific acid extractable fractions of NAs obtained from oil sands-influenced water are active toward reproductive processes or interact with the Ah receptor responsible for CYP1A activity. NAs were extracted from aged oil sands-influenced waters by use of acid precipitation, and the mixture was fractionated into three acidic and one neutral fraction. The four fractions were examined for Ah receptor-mediated potency by use of the H4IIE-luc bioassay, effects on production of steroid hormones by use of the H295R steroidogenesis assay, and sex steroid receptor binding activity using the yeast estrogen screen and yeast androgen screen. The mixtures were characterized by high resolution mass spectrometry, (1)H nuclear magnetic resonance, and attenuated total reflectance infrared spectroscopy. The neutral fraction elicited Ah-receptor mediated activity after 24 h but not after 48 or 72 h. None of the fractions contained measurable levels of estrogen or androgen receptor agonists nor did they cause reductions in steroidogenesis. A number of fractions showed antiestrogenic or antiandrogenicity potency, with the neutral and main acidic fractions being the most potent. Neutral aromatic compounds are likely responsible for the CYP1A activity observed. Direct estrogenic, androgenic, or steroidogenic mechanisms are unlikely for NAs based on these results, but NAs act as potent antiandrogen or antiestrogens.