Adverse Outcome Pathway Network-Based Assessment of the Interactive Effects of an Androgen Receptor Agonist and an Aromatase Inhibitor on Fish Endocrine Function.

Predictive approaches to assessing the toxicity of contaminant mixtures have been largely limited to chemicals that exert effects through the same biological molecular initiating event. However, by understanding specific pathways through which chemicals exert effects, it may be possible to identify shared "downstream" nodes as the basis for forecasting interactive effects of chemicals with different molecular initiating events. Adverse outcome pathway (AOP) networks conceptually support this type of analysis. We assessed the utility of a simple AOP network for predicting the effects of mixtures of an aromatase inhibitor (fadrozole) and an androgen receptor agonist (17ß-trenbolone) on aspects of reproductive endocrine function in female fathead minnows. The fish were exposed to multiple concentrations of fadrozole and 17ß-trenbolone individually or in combination for 48 or 96 h. Effects on 2 shared nodes in the AOP network, plasma 17ß-estradiol (E2) concentration and vitellogenin (VTG) production (measured as hepatic vtg transcripts) responded as anticipated to fadrozole alone but were minimally impacted by 17ß-trenbolone alone. Overall, there were indications that 17ß-trenbolone enhanced decreases in E2 and vtg in fadrozole-exposed fish, as anticipated, but the results often were not statistically significant. Failure to consistently observe hypothesized interactions between fadrozole and 17ß-trenbolone could be due to several factors, including lack of impact of 17ß-trenbolone, inherent biological variability in the endpoints assessed, and/or an incomplete understanding of interactions (including feedback) between different pathways within the hypothalamic-pituitary-gonadal axis. Environ Toxicol Chem 2020;39:913-922. © 2020 SETAC.

Regarding the references for reference chemicals of alternative methods.

The selection of reference and proficiency chemicals is an important basis for method validation and proficiency evaluations. Reference chemicals are a set of test substances used by a method developer to evaluate the reliability and relevance of a new method, in comparison to reference data (usually to a validated reference method). Proficiency chemicals, as defined in OECD Guidance Document on Good In Vitro Method Practices, are defined post validation as a subset of the reference chemicals or other chemicals with sufficient supporting data that are used by naïve laboratories to demonstrate technical competence with a validated test method. Proficiency chemicals should cover different physical states, several chemical classes within the applicability domain of the method and yield the full range of responses (in the validated reference method and in vivo), they shall be commercially available (at non-prohibitive costs) and have high quality reference data. If reference and subsequent proficiency chemicals are chosen without sufficient evidence for their inclusion, both test method evaluation and demonstration of technical proficiency can be hampered. In this report we present cases in which the selection of reference chemicals led to problems in the reproduction of the reference results and demonstration of technical proficiency: The variability of results was not always taken into account in selection of several reference substances of the LLNA (OECD TG 429). Based on the available reference data one proficiency chemical for the Corrositex skin corrosion test (OECD TG 435) should be replaced. Likewise, the expected in vitro result for one of the proficiency chemicals for the BCOP (OECD TG 437) was difficult to reproduce in several labs. Furthermore, it was not possible to obtain one of the proficiency chemicals for the Steroidogenesis Assay (OECD TG 456) at non-prohibitive costs at a reasonable purity. Based on these, we recommend changes of current proficiency chemicals lists with established OECD Test Guidelines and provide recommendations for developing future sets of reference chemicals.

Time-Integrative Passive sampling combined with TOxicity Profiling (TIPTOP): an effect-based strategy for cost-effective chemical water quality assessment.

This study aimed at demonstrating that effect-based monitoring with passive sampling followed by toxicity profiling is more protective and cost-effective than the current chemical water quality assessment strategy consisting of compound-by-compound chemical analysis of selected substances in grab samples. Passive samplers were deployed in the Dutch river delta and in WWTP effluents. Their extracts were tested in a battery of bioassays and chemically analyzed to obtain toxicity and chemical profiles, respectively. Chemical concentrations in water were retrieved from publicly available databases. Seven different strategies were used to interpret the chemical and toxicity profiles in terms of ecological risk. They all indicated that the river sampling locations were relatively clean. Chemical-based monitoring resulted for many substances in measurements below detection limit and could only explain <20% of the observed in vitro toxicity. Effect-based monitoring yielded more informative conclusions as it allowed for ranking the sampling sites and for estimating a margin-of-exposure towards chronic effect ranges. Effect-based monitoring was also cheaper and more cost-effective (i.e. yielding more information per euro spent). Based on its identified strengths, weaknesses, opportunities, and threats (SWOT), a future strategy for effect-based monitoring has been proposed.

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.

Remediation efficiency of three treatments on water polluted with endocrine disruptors: Assessment by means of in vitro techniques.

Chemical substances with potential to disrupt endocrine systems have been detected in aquatic environments worldwide, making necessary the investigation about water treatments able to inhibit such potential. The present work aimed to assess the efficiency for removing endocrine disruptors (with estrogenic and androgenic activity) of three simple and inexpensive substrates that could be potentially used in sectors or regions with limited resources: powdered activated carbon (PAC), powdered natural zeolite (ZEO) (both at a concentration of 500 mg L-1) and natural aquatic humic substances (AHS) (at 30 mg L-1). MilliQ-water and mature water from fish facilities (aquarium water, AW), were artificially spiked with 17ß-estradiol (E2), 17α-ethinylestradiol and dihydrotestosterone. Moreover, effluent samples from waste water treatment plants (WWTP) were also submitted to the remediation treatments. Estrogenic and androgenic activities were assessed with two cell lines permanently transfected with luciferase as reporter gene under the control of hormone receptors: AR-EcoScreen containing the human androgen receptor and HER-LUC transfected with the sea bass estrogen receptor. PAC was efficiently removing the estrogenic and androgenic compounds added to milliQ and AW. However, androgenic activity detected in WWTP effluents was only reduced after treatment with ZEO. The higher surface area of PAC could have facilitated the removal of spiked hormones in clean waters. However, it is possible that the substances responsible of the hormonal activity in WWTP have adsorbed to micro and nanoparticles present in suspension that would have been retained with higher efficiency by ZEO that show pores of several microns in size.

Assessment of toxic and endocrine potential of substances migrating from selected toys and baby products.

Analysis of literature data shows that there is limited information about the harmful biological effects of mixture of compounds from the EDC group that are released from the surface of toys and objects intended for children and infants. One of the tools that can be used to obtain such information is appropriate bioanalytical tests. The aim of this research involved determining whether tests that use living organisms as an active element (Vibrio fischeri-Microtox®, Heterocypris incongruens-Ostrocodtoxkit F™ and the XenoScreen YES/YAS™ test of oestrogenic/androgenic activity) can be a tool for estimating the combined toxic effects induced by xenobiotics released from objects intended for children. To reproduce the conditions to which objects are exposed during their use, liquids with a composition corresponding to that of human bodily fluids (artificial sweat and saliva) were used. This research focused on the main parameters influencing the intensification of the migration process (temperature, contact time and composition of the extraction mixture). The studies aimed to estimate the endocrine potential of the extracts showed that compounds released from the surface of studied objects exhibit antagonistic androgenic activity. While on the basis of the results of Microtox® test, one can state that the largest quantity of toxic compounds are released in the first 2 h of using the object. The FTIR spectra analyses confirmed that no degradation of polymeric material took place. On the basis of the results obtained, it was unanimously concluded that contact of the object with bodily fluids may result in the release of a large number of xenobiotics, which has disadvantageous effects on the metabolic processes of the indicator organisms.

High-dose testosterone and dehydroepiandrosterone induce cardiotoxicity in rats: Assessment of echocardiographic, morphologic, and oxidative stress parameters.

The aim of this study is to assess cardiotoxic effect of testosterone (TES) and dehydroepiandrosterone (DHEA) in Sprague Dawley rats. We compared the impact of subacute (14 days) and subchronic (90 days) administration of suprapharmacologic doses of TES and DHEA on body weight, locomotor activity, muscle strength, echocardiographic parameters, heart histopathology, and oxidative stress markers with the control group. Testosterone (10, 30, and 100 mg/100 g body weight) and DHEA (10 mg/100 g body weight) administration decreased the body weights and locomotor activity (p < 0.05), and the combination of both increased muscle strength (p < 0.05) in rats. In our histopathological evaluation, misshapen cell nuclei, disorganized myocardial fibers, and leukocytic infiltrates were observed in high-dose TES (100 mg/100 g)-treated rats, especially on day 14. On day 90, mild changes such as misshapen cell nuclei, disorganized myocardial fibers, and leukocytic infiltrates were observed in TES and DHEA-treated groups. According to our echocardiographic study on day 14 and day 90, TES, especially at high doses, induced increase in left ventricular posterior wall diameter and ejection fraction (p < 0.05). In this study, blood oxidative stress marker malondialdehyde was increased slightly but not significantly in TES and DHEA groups. On the other hand, antioxidant enzymes such as SOD and glutathione peroxidase (GSH-Px) levels were slightly but not significantly increased in TES and DHEA groups. These data demonstrate that the potential risk to cardiac health due to exogenous androgen use may be related to oxidative stress in rats.

Assessment of hormonal activities and genotoxicity of industrial effluents using in vitro bioassays combined with chemical analysis.

Wastewaters from various industries are a main source of the contaminants in aquatic environments. The authors evaluated the hormonal activities (estrogenic/anti-estrogenic activities, androgenic/anti-androgenic activities) and genotoxicity of various effluents from textile and dyeing plants, electronic and electroplate factories, pulp and paper mills, fine chemical factories, and municipal wastewater treatment plants in the Pearl River Delta region by using in vitro bioassays (yeast estrogen screen [YES]; yeast androgen screen [YAS]; and genotoxicity assay [umu/SOS]) combined with chemical analysis. The results demonstrated the presence of estrogenic, anti-estrogenic, and anti-androgenic activity in most industrial effluents, whereas no androgenic activities were detected in all of the effluents. The measured estrogenic activities expressed as estradiol equivalent concentrations (EEQs) ranged from below detection (3 of 26 samples) to 40.7 ng/L, with a mean of 7.33 ng/L in all effluents. A good linear relationship was found between the EEQs measured by YES bioassay and the EEQs calculated from chemical concentrations. These detected estrogenic compounds, such as 4-nonylphenol and estrone, were responsible for the estrogenic activities in the effluents. The genotoxic effects expressed as benzo[a]pyrene equivalent concentrations (BaP EQs) varied between below detection and 88.2 µg/L, with a mean of 8.76 µg/L in all effluents. The target polycyclic aromatic hydrocarbons were minor contributors to the genotoxicity in the effluents, and some nontarget compounds in the effluents were responsible for the measured genotoxicity. In terms of estrogenic activities and genotoxicity, discharge of these effluents could pose high risks to aquatic organisms in the receiving environments.

Doping and effects of anabolic androgenic steroids on the heart: histological, ultrastructural, and echocardiographic assessment in strength athletes.

Anabolic androgenic steroids (AAS) are used by some athletes to enhance performance despite the health risk they may pose in some persons. This work was carried out to evaluate the possible structural and functional alterations in the heart using two-dimensional, M-mode, tissue Doppler imaging (TDI) and strain rate imaging (SRI) in athletes using supraphysiological doses of AAS. Additionally, the histological and ultrastructural changes in cardiac muscles of adult albino rats after injection of sustanon, as an example of AAS, were studied. Fifteen male bodybuilders using anabolic steroids constituted group 1, five male bodybuilders who are not using anabolic steroids constituted group 2, and five nonathletic males constituted negative control group (group 3). They were investigated by two-dimensional, M-mode, TDI and SRI. This study was performed on 30 adult albino rats. They were divided into two groups. Group I (Control group) (10) was subdivided into negative control, subgroup 1a (5), and subgroup 1b (5), which received 0.8 ml olive oil intramuscular once a week for 8 weeks. Group II (Experimental group) (20) received sustanon 10 mg/kg intramuscularly once a week for 8 weeks. The heart specimens were prepared for light microscopy and transmission electron microscopy. Echocardiographic results showed that bodybuilders who use steroids have smaller left ventricular dimension with thicker walls, impaired diastolic function, as well as higher peak systolic strain rate in steroid-using bodybuilders as compared to the other two groups. Light microscopy examination of cardiac muscle fibers showed focal areas of degeneration with loss of striations and vacuolation in the experimental group. Ultrastructural examination showed disturbance of the banding pattern of the cardiac muscle fiber with disintegration, loss of striations, dehiscent intercalated disc, and interrupted Z-bands. Administration of supraphysiological doses of AAS caused severe deleterious effects in the myocardium both in athletes and in experimental animals. The SRI shows promise in the early detection of systolic dysfunction in those athletes who use steroids.

Stacking anabolic androgenic steroids (AAS) during puberty in rats: a neuroendocrine and behavioral assessment.

Anabolic androgenic steroid (AAS) abuse is increasing in teenagers. We examined the effects of stacked AAS in adolescent male rats. Stacking, in which multiple AAS are taken simultaneously, is commonly employed by humans. Beginning at puberty gonadally intact male rats received testosterone, nandrolone, or stanozolol. Additional groups received stacked AAS: testosterone + stanozolol, nandrolone + stanozolol, or nandrolone + testosterone. Injections continued during tests for sexual behavior, vocalizations, scent marking, partner preference, aggression and fertility. Body and reproductive tissue weights were taken. Sexual and aggressive behaviors were increased by testosterone yet inhibited by stanozolol; nandrolone had no effect. Stacking testosterone with stanozolol prevented the inhibitory effects of stanozolol. Body weight was decreased by testosterone and all stacked AAS. Cell nuclear androgen receptor binding in brain was significantly increased in nandrolone males and decreased in stanozolol males; testosterone males were slightly higher than controls. Androgen receptors in stacked groups were intermediate between individual AAS suggesting that stanozolol competed with other AAS for androgen receptors despite its low affinity. The results indicate that stacking AAS influences the effects of individual AAS on behavioral and endocrine measures, and levels of androgen receptor occupation are not directly correlated with AAS effects on behavior.