Chronic Exposure to Climbazole Induces Oxidative Stress and Sex Hormone Imbalance in the Testes of Male Zebrafish.

As the main active ingredient for the treatment of fungal infections, climbazole (CBZ) is commonly used in a variety of personal care products. After its use, CBZ enters the receiving environment directly or indirectly through domestic sewage. Its concentration can be up to several nanograms per liter in surface water. So far, the effects of CBZ on the reproductive system of female zebrafish have been systematically studied, but the potential toxicity mechanism of CBZ on male zebrafish still needs to be further explored. In this study, adult male zebrafish were exposed to CBZ at concentrations of 0.1, 10, and 1000 µg·L-1 for 28 days, and their testes were collected for histological, mass-spectrometry-based metabolomics, and biochemical analyses. We found that CBZ caused a significantly abnormal metabolism of purine and glutathione and triggered oxidative stress in zebrafish testes, thereby inducing testicular cell apoptosis. In addition, CBZ could inhibit the synthesis of essential sex hormones in the testis and thus reduce the sperm production. The conclusions of this study fill the data gap on the reproductive toxicity of CBZ to male zebrafish and highlight the ecotoxicological application of untargeted metabolomics in the biomarker discovery.

Sex Disparities in COVID-19 Severity and Outcome: Are Men Weaker or Women Stronger?

The coronavirus disease 2019 (COVID-19) outbreak, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health issue which has profound effects on most aspects of societal well-being, including physical and mental health. A plethora of studies globally have suggested the existence of a sex disparity in the severity and outcome of COVID-19 patients, mainly due to mechanisms of virus infection, immune response to the virus, development of systemic inflammation, and consequent systemic complications, particularly thromboembolism. Epidemiological data report a sex difference in the severity of COVID-19, with a more favorable course of the disease in women compared to men regardless of age, although the rate of SARS-CoV-2 infection seems to be similar in both sexes. Sex hormones, including androgens and estrogens, may not only impact virus entry and load, but also shape the clinical manifestations, complications, and ultimately the outcome of the disease. The current review comprehensively summarizes the current literature on sex disparities in susceptibility and outcome of COVID-19 as well as the literature underpinning the pathophysiological and molecular mechanisms, which may provide a rationale to a sex disparity. These mechanisms include sex hormone influence on factors that facilitate virus entry and priming, immune and inflammatory response, as well as coagulation and thrombosis diathesis. Based on present evidence, women appear to be relatively protected from COVID-19 because of a more effective immune response and a less pronounced systemic inflammation, with consequent moderate clinical manifestations of the disease, together with a lesser predisposition to thromboembolism. Conversely, men appear to be particularly susceptible to COVID-19 because of a less effective immune response with consequent severe clinical manifestations of the disease, together with a greater predisposition to thromboembolism. In the elderly, generally characterized by the phenomenon of inflammaging, sex disparities in overall mortality following SARS-CoV-2 infection are even more palpable as elderly men appear to be more prone to severe COVID-19 because of a greater predisposition to infections, a weaker immune defense, and an enhanced thrombotic state compared to women. The information revealed from the review highlights potential novel therapeutic approaches employing the administration of hormonal or antihormonal therapy in combination with antiviral drugs in COVID-19 patients.

Sex-tailored pharmacology and COVID-19: Next steps towards appropriateness and health equity.

Making gender bias visible allows to fill the gaps in knowledge and understand health records and risks of women and men. The coronavirus disease 2019 (COVID-19) pandemic has shown a clear gender difference in health outcomes. The more severe symptoms and higher mortality in men as compared to women are likely due to sex and age differences in immune responses. Age-associated decline in sex steroid hormone levels may mediate proinflammatory reactions in older adults, thereby increasing their risk of adverse outcomes, whereas sex hormones and/or sex hormone receptor modulators may attenuate the inflammatory response and provide benefit to COVID-19 patients. While multiple pharmacological options including anticoagulants, glucocorticoids, antivirals, anti-inflammatory agents and traditional Chinese medicine preparations have been tested to treat COVID-19 patients with varied levels of evidence in terms of efficacy and safety, information on sex-targeted treatment strategies is currently limited. Women may have more benefit from COVID-19 vaccines than men, despite the occurrence of more frequent adverse effects, and long-term safety data with newly developed vectors are eagerly awaited. The prevalent inclusion of men in randomized clinical trials (RCTs) with subsequent extrapolation of results to women needs to be addressed, as reinforcing sex-neutral claims into COVID-19 research may insidiously lead to increased inequities in health care. The huge worldwide effort with over 3000 ongoing RCTs of pharmacological agents should focus on improving knowledge on sex, gender and age as pillars of individual variation in drug responses and enforce appropriateness.

Unexpected Observations: Probiotic Administration Greatly Aggravates the Reproductive Toxicity of Perfluorobutanesulfonate in Zebrafish.

The present study exposed adult zebrafish to 0, 10, and 100 µg/L perfluorobutanesulfonate (PFBS) with or without dietary supplement of probiotic Lactobacillus rhamnosus. Interaction between probiotic and PFBS on sex endocrine and reproduction was investigated. It was striking to find that PFBS and probiotic coexposures almost ceased the fecundity, which was accompanied by disturbances in sex hormones and oocyte maturation in females. In contrast, probiotic additive efficiently antagonized the estrogenic activity of PFBS in males. For the first time, this study reported that probiotic heavily depended on sex to modulate the endocrine disruption and reproductive toxicity of aquatic pollutants.

Curcumin: a phytochemical modulator of estrogens and androgens in tumors of the reproductive system.

Curcumin (Cur) is an active derivative extracted from turmeric which exerts a wide range of interactions with biomolecules through complex signaling pathways. Cur has been extensively shown to possess potential antitumor properties. In addition, there is growing body of evidence suggesting that Cur may exert potential anti-estrogen and anti-androgen activity. In vitro and in vivo studies suggest that anticancer properties of Cur against tumors affecting the reproductive system in females and males may be underlied by the Cur-mediated inhibition of androgen and estrogen signaling pathways. In this review we examine various studies assessing the crosstalk between Cur and both androgen and estrogen hormonal activity. Also, we discuss the potential chemopreventive and antitumor role of Cur in the most prevalent cancers affecting the reproductive system in females and males.

Migraine: navigating the hormonal minefield.

Migraine affects 959 million people worldwide,1 with the highest prevalence being in women of childbearing age. The interplay between female hormones and migraine can be a challenging area to navigate since issues relating to pregnancy, contraception and the menopause are often out of the neurology comfort zone. This review aims to help the neurologist to manage women with migraine, from menarche to menopause.

A Novel Action of Endocrine-Disrupting Chemicals on Wildlife; DDT and Its Derivatives Have Remained in the Environment.

Huge numbers of chemicals are released uncontrolled into the environment and some of these chemicals induce unwanted biological effects, both on wildlife and humans. One class of these chemicals are endocrine-disrupting chemicals (EDCs), which are released even though EDCs can affect not only the functions of steroid hormones but also of various signaling molecules, including any ligand-mediated signal transduction pathways. Dichlorodiphenyltrichloroethane (DDT), a pesticide that is already banned, is one of the best-publicized EDCs and its metabolites have been considered to cause adverse effects on wildlife, even though the exact molecular mechanisms of the abnormalities it causes still remain obscure. Recently, an industrial raw material, bisphenol A (BPA), has attracted worldwide attention as an EDC because it induces developmental abnormalities even at low-dose exposures. DDT and BPA derivatives have structural similarities in their chemical features. In this short review, unclear points on the molecular mechanisms of adverse effects of DDT found on alligators are summarized from data in the literature, and recent experimental and molecular research on BPA derivatives is investigated to introduce novel perspectives on BPA derivatives. Especially, a recently developed BPA derivative, bisphenol C (BPC), is structurally similar to a DDT derivative called dichlorodiphenyldichloroethylene (DDE).

"Not tonight zebrafish": the effects of Ruta graveolens on reproduction.

CONTEXT: There is growing interest in the pharmacological evaluation of Rue due to its potential to treat a variety of clinical diseases. The plant seems to present potent endocrine disrupting effects, and its excretion and disposal are not a concern. OBJECTIVE: The effects of Ruta graveolens L. (Rutaceae) ethanol extract (RE) on reproductive behaviour, fertility, and steroid and thyroid hormone levels in zebrafish were investigated. MATERIAL AND METHODS: We exposed subjects to varying concentrations of RE, and one-tenth the LC50 concentration (2.37 ppm) was established as the sublethal dose. After 2 weeks exposure, reproductive behaviour, cumulative number of eggs laid, percentage of fertilized eggs, and whole body steroid and thyroid hormones were measured. RESULTS: Reproductive association behaviour did not differ between control and RE-exposed animals, but spawning attempts were reduced in RE exposed animals. Cumulative egg production between days 9 to 14, RE exposed fish laid 672 eggs while control fish laid 1242 eggs. Also, percentage of fertilized eggs was higher for the control than for the RE exposed fish. Estradiol-17ß (E2) levels were reduced in females exposed to RE and testosterone (T) was statistically lower in both males and females treated with RE. Furthermore, thyroid hormones (T3 and T4) declined in fish treated with RE. CONCLUSION: RE has endocrine disrupting potential in fish, which has important implications for studying the effects of unintentional pharmaceutical exposure. Moreover, the results demonstrate that drug exposure may affect more than just the overall level of behaviour, emphasizing the relevance of examining the effects of individual exposure. We reinforce the use of zebrafish as a model organism in physiology and behaviour, and raise concerns about the toxic effects of RE in non-target organisms such as aquatic vertebrates, which may ultimately affect human health.

Postpartum inhibition of ovarian steroid action increases aspects of maternal caregiving and reduces medial preoptic area progesterone receptor expression in female rats.

The rapid peripartum onset of maternal caregiving involves progesterone synergizing with estradiol, but prolonging progesterone exposure past this time can prevent the emergence of mothering. Interestingly, there is a 7-10day-long rise in progesterone during mid-lactation, but its effects on mothering are unknown. Given progesterone's potential to inhibit mothering onset, this mid-lactational rise may contribute to the normal attenuation of caregiving behaviors across lactation. To evaluate this, recently-parturient rats were ovariectomized and caregiving observed from postpartum days (PPD) 7-18. Ovariectomized dams were found to lick, hover over, and nurse in kyphosis more frequently than controls. Ovariectomy also decreased medial preoptic area (mPOA) progesterone receptor (PR) mRNA, which was negatively correlated with pup licking and kyphosis, but it did not affect mPOA levels of oxytocin receptor or vasopressin V1a receptor mRNAs. In a second study, gonadally intact dams were given the PR antagonist, RU 486, and were found to display more kyphosis and less supine nursing compared to controls. Finally, progesterone sensitivity across lactation was examined by measuring numbers of PR immunoreactive (PR-ir) cells in the mPOA, ventral bed nucleus of the stria terminalis (BSTv) and periaqueductal gray (PAG). PR-ir was higher in the mPOA at parturition compared to virgins, while PR-ir in the mPOA and BSTv dropped from parturition to PPD 7 and remained low through PPD 18. The number of PR-ir cells in the PAG was constant. Thus, in addition to their well-known prepartum effects, ovarian hormones limit the display of some maternal behaviors during mid-to-late lactation and contribute to their decline as weaning approaches.

Neuroendocrine disruption of organizational and activational hormone programming in poikilothermic vertebrates.

In vertebrates, sexual differentiation of the reproductive system and brain is tightly orchestrated by organizational and activational effects of endogenous hormones. In mammals and birds, the organizational period is typified by a surge of sex hormones during differentiation of specific neural circuits; whereas activational effects are dependent upon later increases in these same hormones at sexual maturation. Depending on the reproductive organ or brain region, initial programming events may be modulated by androgens or require conversion of androgens to estrogens. The prevailing notion based upon findings in mammalian models is that male brain is sculpted to undergo masculinization and defeminization. In absence of these responses, the female brain develops. While timing of organizational and activational events vary across taxa, there are shared features. Further, exposure of different animal models to environmental chemicals such as xenoestrogens such as bisphenol A-BPA and ethinylestradiol-EE2, gestagens, and thyroid hormone disruptors, broadly classified as neuroendocrine disrupting chemicals (NED), during these critical periods may result in similar alterations in brain structure, function, and consequently, behaviors. Organizational effects of neuroendocrine systems in mammals and birds appear to be permanent, whereas teleost fish neuroendocrine systems exhibit plasticity. While there are fewer NED studies in amphibians and reptiles, data suggest that NED disrupt normal organizational-activational effects of endogenous hormones, although it remains to be determined if these disturbances are reversible. The aim of this review is to examine how various environmental chemicals may interrupt normal organizational and activational events in poikilothermic vertebrates. By altering such processes, these chemicals may affect reproductive health of an animal and result in compromised populations and ecosystem-level effects.

[On "2015 Guidelines for Prevention and Treatment of Osteoporosis". Osteoporosis associated with sex hormone depletion treatment].

Breast cancer and prostate cancer are sex hormone-dependent cancers and suppression of estrogen or androgen function is the standard therapy for those cancers. One of its important adverse effects is bone loss or osteoporosis. Recent evidences are : 1) Endocrine therapy for breast cancer or prostate cancer is associated with significant bone loss. 2) Treatment with aromatase inhibitors (AI) for breast cancer is associated with significant increase of pathologic fractures. Androgen depletion treatment (ADT) for prostate cancer is also probably associated with increased risk of fracture. 3) Bisphosphonates and denosumab treatment increases bone mineral density of patients treated with endocrine therapy for breast cancer. Bisphosphonates, denosumab and SERMs (raloxifene and toremifene) increase bone mineral density of patients treated with ADT for prostate cancer. 4) Bisphosphonates and denosumab decrease fracture risk of AI-treated breast cancer patients. Toremifene and denosumab decrease fracture risk of ADT-treated prostate cancer patients.

Molecular mechanism(s) of endocrine-disrupting chemicals and their potent oestrogenicity in diverse cells and tissues that express oestrogen receptors.

Endocrine-disrupting chemicals (EDCs) are natural or synthetic compounds present in the environment which can interfere with hormone synthesis and normal physiological functions of male and female reproductive organs. Most EDCs tend to bind to steroid hormone receptors including the oestrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR). As EDCs disrupt the actions of endogenous hormones, they may induce abnormal reproduction, stimulation of cancer growth, dysfunction of neuronal and immune system. Although EDCs represent a significant public health concern, there are no standard methods to determine effect of EDCs on human beings. The mechanisms underlying adverse actions of EDC exposure are not clearly understood. In this review, we highlighted the toxicology of EDCs and its effect on human health, including reproductive development in males and females as shown in in vitro and in vivo models. In addition, this review brings attention to the toxicity of EDCs via interaction of genomic and non-genomic signalling pathways through hormone receptors.

Fluorochemicals used in food packaging inhibit male sex hormone synthesis.

Polyfluoroalkyl phosphate surfactants (PAPS) are widely used in food contact materials (FCMs) of paper and board and have recently been detected in 57% of investigated materials. Human exposure occurs as PAPS have been measured in blood; however knowledge is lacking on the toxicology of PAPS. The aim of this study was to elucidate the effects of six fluorochemicals on sex hormone synthesis and androgen receptor (AR) activation in vitro. Four PAPS and two metabolites, perfluorooctanoic acid (PFOA) and 8:2 fluorotelomer alcohol (8:2 FTOH) were tested. Hormone profiles, including eight steroid hormones, generally showed that 8:2 diPAPS, 8:2 monoPAPS and 8:2 FTOH led to decreases in androgens (testosterone, dehydroepiandrosterone, and androstenedione) in the H295R steroidogenesis assay. Decreases were observed for progesterone and 17-OH-progesterone as well. These observations indicated that a step prior to progestagen and androgen synthesis had been affected. Gene expression analysis of StAR, Bzrp, CYP11A, CYP17, CYP21 and CYP19 mRNA showed a decrease in Bzrp mRNA levels for 8:2 monoPAPS and 8:2 FTOH indicating interference with cholesterol transport to the inner mitochondria. Cortisol, estrone and 17ß-estradiol levels were in several cases increased with exposure. In accordance with these data CYP19 gene expression increased with 8:2 diPAPS, 8:2 monoPAPS and 8:2 FTOH exposures indicating that this is a contributing factor to the decreased androgen and the increased estrogen levels. Overall, these results demonstrate that fluorochemicals present in food packaging materials and their metabolites can affect steroidogenesis through decreased Bzrp and increased CYP19 gene expression leading to lower androgen and higher estrogen levels.

Thymic involution and immune reconstitution.

Chronic thymus involution associated with aging results in less efficient T-cell development and decreased emigration of naïve T cells to the periphery. Thymic decline in the aged is linked to increased morbidity and mortality in a wide range of clinical settings. Negative consequences of these effects on global health make it of paramount importance to understand the mechanisms driving thymic involution and homeostatic processes across the lifespan. There is growing evidence that thymus tissue is plastic and that the involution process might be therapeutically halted or reversed. We present here progress on the exploitation of thymosuppressive and thymostimulatory pathways using factors such as keratinocyte growth factor, interleukin 7 or sex steroid ablation for therapeutic thymus restoration and peripheral immune reconstitution in adults.

Epidermal growth factor receptor signalling mediates growth hormone-induced growth of chondrocytes from sex hormone-inhibited adolescent rats.

1. Growth hormone (GH) has been demonstrated to overcome the inappropriate deceleration of growth rate in children with central precocious puberty treated with gonadotropin-releasing hormone analogue (GnRHa). However, the underlying mechanisms remain largely unclear. In the present study, we investigated the potential involvement of the epidermal growth factor receptor (EGFR) pathway in the growth promotion by GH using in vitro cultured growth plate chondrocytes isolated from adolescent rats treated with GnRHa. 2. Chondrocytes were stimulated with GH in the presence or absence of the Janus tyrosine kinase (JAK) 2 inhibitor AG490 (1, 10 and 100 nmol/L), the EGFR kinase inhibitor AG1478 (0.1, 1 and 10 nmol/L), U0126 (an inhibitor of extracellular signal-regulated kinase (Erk) activation; 10 µmol/L) or a neutralizing antibody against epidermal growth factor (EGF Ab; 0.1, 1 and 10 µg/mL). The proliferation of chondrocytes was assessed by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and immunostaining for proliferating cell nuclear antigen (PCNA). Phosphorylation of Erk1/2 and EGFR was detected by western-blotting. Intracellular mRNA and extracellular protein levels of EGF were detected using reverse transcription-polymerase chain reaction and ELISA, respectively. 3. Growth hormone promoted the proliferation of chondrocytes, which was correlated with increased phosphorylation of Erk1/2 and EGFR and enhanced expression of EGF. Pretreatment with AG490, AG1478, U0126 or EGF Ab completely or partially inhibited the proliferation of chondrocytes and activation of Erk1/2 and EGFR. Pretreatment with AG490, AG1478, or U0126 partially inhibited the expression of EGF. 4. The findings indicate that GH promotes chondrocyte proliferation by activating EGFR signalling.

Mandibular and femoral growth alteration after sex hormone disruption in growing mice.

OBJECTIVES: To investigate how mandibular and femoral growth is affected when sex hormone- specific receptor antagonist is administered in growing mice. MATERIALS AND METHODS: Forty C57BL/6J mice were used in this experiment. At 5 days of age, the mice received daily injection of estrogen receptor alpha (ERα), beta (ERß), or androgen receptor (AR) antagonists, and their body weight was assessed every 4 days. One, four and eight weeks after the initial injection, radiographs of the mandible and femur were taken and measured. Analyses of variance and pairwise comparisons (Fisher) were performed to examine the differences in values measured among the groups. RESULTS: Mandibular growth was affected by ERß antagonist injection in male mice at 4 and 8 weeks. In female mice, the growth was affected during all the experimental period, when ERß was administered. Moreover, at 8 weeks, mandibular growth was also affected in male and female mice injected with ERα antagonist and in male mice injected with AR antagonist. Femoral growth was affected during all the experimental period in male and female mice injected with ERß antagonist. Moreover, at 8 weeks, the growth was affected in male and female mice injected with ERα antagonist and in male mice injected with AR antagonist. CONCLUSIONS: Growth of the mandible and femur in mice, in part, is induced in response to the stimulation of ERß in chondrocytes before and during early puberty. In late and after puberty, the growth is induced by the stimulation of ERα in male and female mice and that of AR in male mice.

Statin therapy and sex hormones.

Current guidelines recommend statin therapy for all adult patients with coronary artery disease irrespective of sex. Over recent years, some concerns have been raised concerning the effects of statins on endogenous steroid hormones synthesis. The aim of this review was to summarize the effects of statins on endogenous sex hormones in order to clarify their role and safety in different clinical settings. Results suggest that HMG-CoA inhibitors may slightly impair adrenal and/or gonadal steroid hormone production. In men, statins do not cause any clinically-relevant harmful effects on erectile function and spermatogenesis and, in women, statins have beneficial effects in treatment of polycystic ovary syndrome (PCOS). Additional research is needed to provide specific clinical recommendations concerning this topic.

The Effect of Metformin vs Placebo on Sex Hormones in Canadian Cancer Trials Group MA.32.

BACKGROUND: Metformin has been associated with lower breast cancer (BC) risk and improved outcomes in observational studies. Multiple biologic mechanisms have been proposed, including a recent report of altered sex hormones. We evaluated the effect of metformin on sex hormones in MA.32, a phase III trial of nondiabetic BC subjects who were randomly assigned to metformin or placebo. METHODS: We studied the subgroup of postmenopausal hormone receptor-negative BC subjects not receiving endocrine treatment who provided fasting blood at baseline and at 6 months after being randomly assigned. Sex hormone-binding globulin, bioavailable testosterone, and estradiol levels were assayed using electrochemiluminescence immunoassay. Change from baseline to 6 months between study arms was compared using Wilcoxon sum rank tests and regression models. RESULTS: 312 women were eligible (141 metformin vs 171 placebo); the majority of subjects in each arm had T1/2, N0, HER2-negative BC and had received (neo)adjuvant chemotherapy. Mean age was 58.1 (SD=6.9) vs 57.5 (SD=7.9) years, mean body mass index (BMI) was 27.3 (SD=5.5) vs 28.9 (SD=6.4) kg/m2 for metformin vs placebo, respectively. Median estradiol decreased between baseline and 6 months on metformin vs placebo (-5.7 vs 0 pmol/L; P < .001) in univariable analysis and after controlling for baseline BMI and BMI change (P < .001). There was no change in sex hormone-binding globulin or bioavailable testosterone. CONCLUSION: Metformin lowered estradiol levels, independent of BMI. This observation suggests a new metformin effect that has potential relevance to estrogen sensitive cancers.

Effects of fluorotelomer alcohol 8:2 FTOH on steroidogenesis in H295R cells: targeting the cAMP signalling cascade.

Previous studies have demonstrated that perfluorinated chemicals (PFCs) can affect reproduction by disruption of steroidogenesis in experimental animals. However, the underlying mechanism(s) of this disruption remain unknown. Here we investigated the effects and mechanisms of action of 1H, 1H, 2H, 2H-perfluoro-decan-1-ol (8:2 FTOH) on steroidogenesis using a human adrenocortical carcinoma cell line (H295R) as a model. H295R cells were exposed to 0, 7.4, 22.2 or 66.6 microM 8:2 FTOH for 24h and productions of progesterone, 17alpha-OH-progesterone, androstenedione, testosterone, deoxycorticosterone, corticosterone and cortisol were quantified by HPLC-MS/MS. With the exception of progesterone, 8:2 FTOH treatment significantly decreased production of all hormones in the high dose group. Exposure to 8:2 FTOH significantly down-regulated cAMP-dependent mRNA expression and protein abundance of several key steroidogenic enzymes, including StAR, CYP11A, CYP11B1, CYP11B2, CYP17 and CYP21. Furthermore, a dose-dependent decrease of cellular cAMP levels was observed in H295R cells exposed to 8:2 FTOH. The observed responses are consistent with reduced cellular cAMP levels. Exposure to 8:2 FTOH resulted in significantly less basal (+GTP) and isoproterenol-stimulated adenylate cyclase activities, but affected neither total cellular ATP level nor basal (-GTP) or NaF-stimulated adenylate cyclase activities, suggesting that inhibition of steroidogenesis may be due to an alteration in membrane properties. Metabolites of 8:2 FTOH were not detected by HPLC-MS/MS, suggesting that 8:2 FTOH was not metabolized by H295R cells. Overall, the results show that 8:2 FTOH may inhibit steroidogenesis by disrupting the cAMP signalling cascade.

High resolution effect-directed analysis of steroid hormone (ant)agonists in surface and wastewater quality monitoring.

Monitoring of chemical water quality is extremely challenging due to the large variety of compounds and the presence of biologically active compounds with unknown chemical identity. Previously, we developed a high resolution Effect-Directed Analysis (EDA) platform that combines liquid chromatography with high resolution mass spectrometry and parallel bioassay detection. In this study, the platform is combined with CALUX bioassays for (anti)androgenic, estrogenic and glucocorticoid activities, and the performance of the platform is evaluated. It appeared to render very repeatable results, with high recoveries of spiked compounds and high consistency between the mass spectrometric and bioassay results. Application of the platform to wastewater treatment plant effluent and surface water samples led to the identification of several compounds contributing to the measured activities. Eventually, a workflow is proposed for the application of the platform in a routine monitoring context. The workflow divides the platform into four phases, of which one to all can be performed depending on the research question and the results obtained. This allows one to make a balance between the effort put into the platform and the certainty and depth by which active compounds will be identified. The EDA platform is a valuable tool to identify unknown bioactive compounds, both in an academic setting as in the context of legislative, governmental or routine monitoring.