Adverse effects of an aldosterone synthase (CYP11B2) inhibitor, fadrozole (FAD286), on inflamed rat colon.

Recently, we described local aldosterone production in the murine large intestine. Upregulated local aldosterone synthesis in different tissues has been linked with inflammatory conditions, which have been attenuated by the aldosterone synthase (CYP11B2) inhibitor, fadrozole (FAD286). Therefore, we investigated the effect of inhibition of intestinal aldosterone synthesis on the development of intestinal inflammation. Sprague-Dawley rats were administered 5% (v/w) dextran sodium sulphate (DSS) for 7 days with or without daily FAD286 (30 mg/kg/d) subcutaneous injections on 3 days before, during and one day after DSS. Tissue aldosterone concentrations were evaluated by ELISA, CYP11B2 by Western blot and RT-qPCR. FAD286 halved adrenal aldosterone production but, intriguingly, increased the colonic aldosterone concentration. The lack of inhibitory effect of FAD286 in the colon might have been affected by the smaller size of colonic vs. adrenal CYP11B2, as seen in Western blot. When combined with DSS, FAD286 aggravated the macroscopic and histological signs of intestinal inflammation, lowered the animals' body weight gain and increased the incidence of gastrointestinal bleeding and the permeability to iohexol in comparison to DSS-animals. To conclude, FAD286 exerted harmful effects during intestinal inflammation. Local intestinal aldosterone did not seem to play any role in the inflammatory pathogenesis occurring in the intestine.

Case Study in 21st-Century Ecotoxicology: Using In Vitro Aromatase Inhibition Data to Predict Reproductive Outcomes in Fish In Vivo.

To reduce the use of intact animals for chemical safety testing, while ensuring protection of ecosystems and human health, there is a demand for new approach methodologies (NAMs) that provide relevant scientific information at a quality equivalent to or better than traditional approaches. The present case study examined whether bioactivity and associated potency measured in an in vitro screening assay for aromatase inhibition could be used together with an adverse outcome pathway (AOP) and mechanistically based computational models to predict previously uncharacterized in vivo effects. Model simulations were used to inform designs of 60-h and 10-21-day in vivo exposures of adult fathead minnows (Pimephales promelas) to three or four test concentrations of the in vitro aromatase inhibitor imazalil ranging from 0.12 to 260 µg/L water. Consistent with an AOP linking aromatase inhibition to reproductive impairment in fish, exposure to the fungicide resulted in significant reductions in ex vivo production of 17ß-estradiol (E2) by ovary tissue (≥165 µg imazalil/L), plasma E2 concentrations (≥74 µg imazalil/L), vitellogenin (Vtg) messenger RNA expression (≥165 µg imazalil/L), Vtg plasma concentrations (≥74 µg imazalil/L), uptake of Vtg into oocytes (≥260 µg imazalil/L), and overall reproductive output in terms of cumulative fecundity, number of spawning events, and eggs per spawning event (≥24 µg imazalil/L). Despite many potential sources of uncertainty in potency and efficacy estimates based on model simulations, observed magnitudes of apical effects were quite consistent with model predictions, and in vivo potency was within an order of magnitude of that predicted based on in vitro relative potency. Overall, our study suggests that NAMs and AOP-based approaches can support meaningful reduction and refinement of animal testing. Environ Toxicol Chem 2023;42:100-116. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Case Study in 21st Century Ecotoxicology: Using In Vitro Aromatase Inhibition Data to Predict Short-Term In Vivo Responses in Adult Female Fish.

The present study evaluated whether in vitro measures of aromatase inhibition as inputs into a quantitative adverse outcome pathway (qAOP) construct could effectively predict in vivo effects on 17ß-estradiol (E2) and vitellogenin (VTG) concentrations in female fathead minnows. Five chemicals identified as aromatase inhibitors in mammalian-based ToxCast assays were screened for their ability to inhibit fathead minnow aromatase in vitro. Female fathead minnows were then exposed to 3 of those chemicals: letrozole, epoxiconazole, and imazalil in concentration-response (5 concentrations plus control) for 24 h. Consistent with AOP-based expectations, all 3 chemicals caused significant reductions in plasma E2 and hepatic VTG transcription. Characteristic compensatory upregulation of aromatase and follicle-stimulating hormone receptor (fshr) transcripts in the ovary were observed for letrozole but not for the other 2 compounds. Considering the overall patterns of concentration-response and temporal concordance among endpoints, data from the in vivo experiments strengthen confidence in the qualitative relationships outlined by the AOP. Quantitatively, the qAOP model provided predictions that fell within the standard error of measured data for letrozole but not for imazalil and epoxiconazole. However, the inclusion of measured plasma concentrations of the test chemicals as inputs improved model predictions, with all predictions falling within the range of measured values. Results highlight both the utility and limitations of the qAOP and its potential use in 21st century ecotoxicology. Environ Toxicol Chem 2021;40:1155-1170. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

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.

Detection of biomarkers to differentiate endocrine disruption from hepatotoxicity in zebrafish (Danio rerio) using proteomics.

Measurement of specific biomarkers identified by proteomics provides a potential alternative method for risk assessment, which is required to discriminate between hepatotoxicity and endocrine disruption. In this study, adult zebrafish (Danio rerio) were exposed to the hepatotoxic substance acetaminophen (APAP) for 21 days, in a fish short-term reproduction assay (FSTRA). The molecular changes induced by APAP exposure were studied in liver and gonads by applying a previously developed combined FSTRA and proteomics approach. We observed a significant decrease in egg numbers, an increase in plasma hyaluronic acid, and the presence of single cell necrosis in liver tissue. Furthermore, nine common biomarkers (atp5f1b, etfa, uqcrc2a, cahz, c3a.1, rab11ba, mettl7a, khdrbs1a and si:dkey-108k21.24) for assessing hepatotoxicity were detected in both male and female liver, indicating hepatic damage. In comparison with exposure to fadrozole, an endocrine disrupting chemical (EDC), three potential biomarkers for liver injury, i.e. cahz, c3a.1 and atp5f1b, were differentially expressed. The zebrafish proteome response to fadrozole exposure indicated a significant regulation in estrogen synthesis and perturbed binding of sperm to zona pellucida in the ovary. This study demonstrates that biomarkers identified and quantified by proteomics can serve as additional weight-of-evidence for the discrimination of hepatotoxicity and endocrine disruption, which is necessary for hazard identification in EU legislation and to decide upon the option for risk assessment.

A combined FSTRA-shotgun proteomics approach to identify molecular changes in zebrafish upon chemical exposure.

The fish short-term reproduction assay (FSTRA) is a common in vivo screening assay for assessing endocrine effects of chemicals on reproduction in fish. However, the current reliance on measures such as egg number, plasma vitellogenin concentration and morphological changes to determine endocrine effects can lead to false labelling of chemicals with non-endocrine modes- of-action. Here, we integrated quantitative liver and gonad shotgun proteomics into the FSTRA in order to investigate the causal link between an endocrine mode-of-action and adverse effects assigned to the endocrine axis. Therefore, we analyzed the molecular effects of fadrozole-induced aromatase inhibition in zebrafish (Danio rerio). We observed a concentration-dependent decrease in fecundity, a reduction in plasma vitellogenin concentrations and a mild oocyte atresia with oocyte membrane folding in females. Consistent with these apical measures, proteomics revealed a significant dysregulation of proteins involved in steroid hormone secretion and estrogen stimulus in the female liver. In the ovary, the deregulation of estrogen synthesis and binding of sperm to zona pellucida were among the most significantly perturbed pathways. A significant deregulation of proteins targeting the transcriptional activity of estrogen receptor (esr1) was observed in male liver and testis. Our results support that organ- and sex-specific quantitative proteomics represent a promising tool for identifying early gene expression changes preceding chemical-induced adverse outcomes. These data can help to establish consistency in chemical classification and labelling.

Gonadosomatic index as a confounding variable in fish-based screening assays for the detection of anti-estrogens and nonaromatizable androgens.

The presence of reproductive endocrine-disrupting compounds (REDCs) in the environment poses a potential threat to fish and wildlife, because exposures are capable of altering sexual development, reproductive success, and behavior. Fish-based screening assays are often utilized to screen for the presence of REDCs in surface waters and to assess single chemicals for potential endocrine-disrupting activity. In an effort to improve such screening assays, the goal of the present study was to determine whether the gonadosomatic index (GSI) of female fathead minnows (Pimephales promelas), as assessed via external characteristics, influences their response to REDC exposure. Specifically, we sought to determine whether low-GSI females differed from high-GSI females in their responses to the model anti-estrogen fadrozole and the model androgen 17ß-trenbolone, and whether there was a preferable classification in the context of REDC screening. Low-GSI females were more sensitive to fadrozole at the lower concentration of fadrozole (5 µg/L) and to the higher concentration of trenbolone (50 ng/L), whereas high-GSI females were more sensitive at the lower concentration of trenbolone (5 ng/L). The differential response of low- and high-GSI females to REDCs indicates that GSI influences exposure outcome, and should subsequently be taken into consideration in the implementation of screening assays, as failure to utilize fish of the appropriate reproductive status may skew the test results. Environ Toxicol Chem 2019;38:603-615. © 2019 SETAC.

Quantitative Adverse Outcome Pathways and Their Application to Predictive Toxicology.

A quantitative adverse outcome pathway (qAOP) consists of one or more biologically based, computational models describing key event relationships linking a molecular initiating event (MIE) to an adverse outcome. A qAOP provides quantitative, dose-response, and time-course predictions that can support regulatory decision-making. Herein we describe several facets of qAOPs, including (a) motivation for development, (b) technical considerations, (c) evaluation of confidence, and (d) potential applications. The qAOP used as an illustrative example for these points describes the linkage between inhibition of cytochrome P450 19A aromatase (the MIE) and population-level decreases in the fathead minnow (FHM; Pimephales promelas). The qAOP consists of three linked computational models for the following: (a) the hypothalamic-pitutitary-gonadal axis in female FHMs, where aromatase inhibition decreases the conversion of testosterone to 17ß-estradiol (E2), thereby reducing E2-dependent vitellogenin (VTG; egg yolk protein precursor) synthesis, (b) VTG-dependent egg development and spawning (fecundity), and (c) fecundity-dependent population trajectory. While development of the example qAOP was based on experiments with FHMs exposed to the aromatase inhibitor fadrozole, we also show how a toxic equivalence (TEQ) calculation allows use of the qAOP to predict effects of another, untested aromatase inhibitor, iprodione. While qAOP development can be resource-intensive, the quantitative predictions obtained, and TEQ-based application to multiple chemicals, may be sufficient to justify the cost for some applications in regulatory decision-making.

Editor's Highlight: Computational Modeling of Plasma Vitellogenin Alterations in Response to Aromatase Inhibition in Fathead Minnows.

In vertebrates, conversion of testosterone into 17ß-estradiol (E2) is catalyzed by cytochrome P450 (CYP) 19A aromatase. An important role of E2 in oviparous vertebrates such as fish is stimulation of hepatic synthesis of the glycolipoprotein vitellogenin (VTG), an egg yolk precursor essential to oocyte development and larval survival. In fathead minnows (FHMs) (Pimephales promelas) exposed to the aromatase inhibitor fadrozole, plasma VTG levels do not change in concert with plasma E2 levels. Specifically, while plasma VTG and E2 levels both drop quickly when aromatase is first inhibited, the recovery of plasma VTG upon cessation of aromatase inhibition is substantially delayed relative to the recovery of plasma E2. We modified an existing computational model of the FHM hypothalamic-pituitary-gonadal axis to evaluate alternative hypotheses that might explain this delay. In the first hypothesis, a feedback loop involving active transport of VTG from the blood into the ovary is used. The activity of the transporter is negatively regulated by ovarian VTG. In the second hypothesis, a type 1 coherent feed-forward loop is implemented in the liver. This loop has 2 arms, both requiring E2 activation. The first arm describes direct, canonical E2-driven transcriptional induction of VTG, and the second describes an E2-driven intermediate transcriptional regulator that is also required for VTG synthesis. Both hypotheses accurately described the observed VTG dynamics. This result could be used to guide design of laboratory experiments intended to determine if either of the motifs, or perhaps even both of them, actually do control VTG dynamics in FHMs exposed to aromatase inhibitors.

Disruption of apoptosis pathways involved in zebrafish gonad differentiation by 17α-ethinylestradiol and fadrozole exposures.

Zebrafish (Danio rerio) sex determination seems to involve genetic factors (GSD) but also environmental factors (ESD), such as endocrine disrupting chemicals (EDCs) that are known to mimic endogenous hormones and disrupt gonad differentiation. Apoptosis has also been proposed to play a crucial role in zebrafish gonad differentiation. Nevertheless, the interactions between EDCs and apoptosis have received little attention. Thus, this study aimed to assess if and which apoptotic pathways are involved in zebrafish gonad differentiation and how EDCs may interfere with this process. With these purposes, zebrafish were exposed to 17α-ethinylestradiol (EE2, 4ng/L) and fadrozole (Fad, 50µg/L) from 2h to 35days post-fertilization (dpf). Afterwards, a gene expression analysis by qRT-PCR and a stereological analysis, based on systematic sampling and protein immunohistochemistry, were performed. The death receptors (FAS; TRADD), anti-apoptotic (BCL-2; MDM2), pro-apoptotic (CASP-2 and -6) and cell proliferation (BIRC5/survivin; JUN) genes and proteins were evaluated. In general, apoptosis was inhibited in females through the involvement of anti-apoptotic pathways, while in males apoptosis seemed to be crucial to the failure of the "juvenile ovary" development and the induction of testes transformation. The JUN protein was shown to be necessary in juvenile ovaries, while the BIRC5 protein seemed to be involved in zebrafish spermatogenesis. Both EDCs, EE2 and Fad, increased the apoptosis stimulus in zebrafish gonad. It was noticed that the few females that were resistant to Fad-induced sex reversal had increased anti-apoptotic factor levels, while males exposed to EE2 showed increased pro-apoptotic genes/proteins and were more advanced in gonad differentiation. Overall, our findings show that apoptosis pathways are involved in zebrafish gonad differentiation and that EDCs can disrupt this process.

Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test.

The Fish Sexual Development Test (FSDT) is a non-reproductive test to assess adverse effects of endocrine disrupting chemicals. With the present study it was intended to evaluate whether gene expression endpoints would serve as predictive markers of endocrine disruption in a FSDT. For proof-of-concept, a FSDT according to the OECD TG 234 was conducted with the non-steroidal aromatase inhibitor fadrozole (test concentrations: 10µg/L, 32µg/L, 100µg/L) using zebrafish (Danio rerio). Gene expression analyses using quantitative RT-PCR were included at 48h, 96h, 28days and 63days post fertilization (hpf, dpf). The selection of genes aimed at finding molecular endpoints which could be directly linked to the adverse apical effects of aromatase inhibition. The most prominent effects of fadrozole exposure on the sexual development of zebrafish were a complete sex ratio shift towards males and an acceleration of gonad maturation already at low fadrozole concentrations (10µg/L). Due to the specific inhibition of the aromatase enzyme (Cyp19) by fadrozole and thus, the conversion of C19-androgens to C18-estrogens, the steroid hormone balance controlling the sex ratio of zebrafish was altered. The resulting key event is the regulation of directly estrogen-responsive genes. Subsequently, gene expression of vitellogenin 1 (vtg1) and of the aromatase cyp19a1b isoform (cyp19a1b), were down-regulated upon fadrozole treatment compared to controls. For example, mRNA levels of vtg1 were down-regulated compared to the controls as early as 48 hpf and 96 hpf. Further regulated genes cumulated in pathways suggested to be controlled by endocrine mechanisms, like the steroid and terpenoid synthesis pathway (e.g. mevalonate (diphospho) decarboxylase (mvd), lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase; lss), methylsterol monooxygenase 1 (sc4mol)) and in lipid transport/metabolic processes (steroidogenic acute regulatory protein (star), apolipoprotein Eb (apoEb)). Taken together, this study demonstrated that the existing Adverse Outcome Pathway (AOP) for aromatase inhibition in fish can be translated to the life-stage of sexual differentiation. We were further able to identify MoA-specific marker gene expression which can be instrumental in defining new measurable key events (KE) of existing or new AOPs related to endocrine disruption.

Development and recovery of histopathological alterations in the gonads of zebrafish (Danio rerio) after single and combined exposure to endocrine disruptors (17α-ethinylestradiol and fadrozole).

Exposure of wildlife to endocrine disrupting chemicals (EDCs) is not necessarily continuous. Due to seasonal changes and variable industrial and agricultural activities it often occurs intermittently. Thus, it is possible that aquatic organisms may be more affected by periodic peak exposure than by chronic exposure. Therefore, an experimental scenario including an exposure from 2h to 90 days post-fertilization (dpf) and a subsequent recovery period until 150 dpf was chosen to assess the potential reversibility of the effects of sex steroids on sexual and gonad development of zebrafish (Danio rerio). The aim of this study was to investigate the persistence of the endocrine effects of an estrogen (EE2-17α-ethinylestradiol, 4ng/L), an inhibitor of estrogen synthesis (Fad-fadrozole, 50µg/L) or their binary mixture (Mix-EE2+ Fad, 4ng/L+50µg/L). Afterwards, a semi-quantitative histological assessment was used to investigate histopathological changes on gonad differentiation and development. The data showed that fadrozole, alone or in combination with EE2, permanently disrupts the sexual development, inducing masculinization and causing severe pathological alterations in testis, such as intersex associated to the enlargement of sperm ducts, interstitial changes, asynchronous development and detachment of basal membrane. After exposures to both EDCs and their mixture, the gonad histopathology revealed interstitial proteinaceous fluid deposits and, in ovaries, there were atretic oocytes, and presumably degenerative mineralization. On the other hand, the gonadal changes induced by EE2 alone seem to be partially reversible when the exposure regime changed to a recovery period. In addition, EE2 enhanced zebrafish growth in both genders, with male fish presenting signs of early obesity such as the presence of adipocytes in testis. Moreover, sex ratio was slightly skewed toward females, at 90 and 105 dpf, in zebrafish exposed to EE2. The data further indicate that long-term studies on impacts of single EDCs and their mixtures with recovery periods are crucial to reveal the possibility of sex reversal and pathological changes of gonads that can adversely affect breeding.

Computational model of the fathead minnow hypothalamic-pituitary-gonadal axis: Incorporating protein synthesis in improving predictability of responses to endocrine active chemicals.

There is international concern about chemicals that alter endocrine system function in humans and/or wildlife and subsequently cause adverse effects. We previously developed a mechanistic computational model of the hypothalamic-pituitary-gonadal (HPG) axis in female fathead minnows exposed to a model aromatase inhibitor, fadrozole (FAD), to predict dose-response and time-course behaviors for apical reproductive endpoints. Initial efforts to develop a computational model describing adaptive responses to endocrine stress providing good fits to empirical plasma 17ß-estradiol (E2) data in exposed fish were only partially successful, which suggests that additional regulatory biology processes need to be considered. In this study, we addressed short-comings of the previous model by incorporating additional details concerning CYP19A (aromatase) protein synthesis. Predictions based on the revised model were evaluated using plasma E2 concentrations and ovarian cytochrome P450 (CYP) 19A aromatase mRNA data from two fathead minnow time-course experiments with FAD, as well as from a third 4-day study. The extended model provides better fits to measured E2 time-course concentrations, and the model accurately predicts CYP19A mRNA fold changes and plasma E2 dose-response from the 4-d concentration-response study. This study suggests that aromatase protein synthesis is an important process in the biological system to model the effects of FAD exposure.

Predicting Fecundity of Fathead Minnows (Pimephales promelas) Exposed to Endocrine-Disrupting Chemicals Using a MATLAB®-Based Model of Oocyte Growth Dynamics.

Fish spawning is often used as an integrated measure of reproductive toxicity, and an indicator of aquatic ecosystem health in the context of forecasting potential population-level effects considered important for ecological risk assessment. Consequently, there is a need for flexible, widely-applicable, biologically-based models that can predict changes in fecundity in response to chemical exposures, based on readily measured biochemical endpoints, such as plasma vitellogenin (VTG) concentrations, as input parameters. Herein we describe a MATLAB® version of an oocyte growth dynamics model for fathead minnows (Pimephales promelas) with a graphical user interface based upon a previously published model developed with MCSim software and evaluated with data from fathead minnows exposed to an androgenic chemical, 17ß-trenbolone. We extended the evaluation of our new model to include six chemicals that inhibit enzymes involved in steroid biosynthesis: fadrozole, ketoconazole, propiconazole, prochloraz, fenarimol, and trilostane. In addition, for unexposed fathead minnows from group spawning design studies, and those exposed to the six chemicals, we evaluated whether the model is capable of predicting the average number of eggs per spawn and the average number of spawns per female, which was not evaluated previously. The new model is significantly improved in terms of ease of use, platform independence, and utility for providing output in a format that can be used as input into a population dynamics model. Model-predicted minimum and maximum cumulative fecundity over time encompassed the observed data for fadrozole and most propiconazole, prochloraz, fenarimol and trilostane treatments, but did not consistently replicate results from ketoconazole treatments. For average fecundity (eggs•female(-1)•day(-1)), eggs per spawn, and the number of spawns per female, the range of model-predicted values generally encompassed the experimentally observed values. Overall, we found that the model predicts reproduction metrics robustly and its predictions capture the variability in the experimentally observed data.

Zebrafish sex differentiation and gonad development after exposure to 17α-ethinylestradiol, fadrozole and their binary mixture: A stereological study.

Current knowledge on zebrafish (Danio rerio) sex determination suggests that this trait has a polygenic genetic basis, although environmental factors, such as endocrine disrupting chemicals (EDC), may also be involved in modeling or disturbing the species sex differentiation and development. This study aimed to assess how sex steroids imbalance triggers impact on sex differentiation and gonad development in zebrafish. Fish where exposed to an estrogen (EE2, i.e. 17α-ethinylestradiol, 4ng/L), to an inhibitor of estrogen synthesis (Fad, i.e. fadrozole, 50µg/L) or to their binary mixture (Mix-EE2+ Fad, 4ng/L+50µg/L), from 2h to 60 days post-fertilization (dpf). Afterwards, a quantitative (stereological) analysis using light microscopy, based on systematic sampling, was made at 35 and 60dpf, to identify alterations on gonad differentiation and development. During the sex differentiation period, our histological data showed that not all zebrafish males develop a "juvenile ovary", contrarily to what is currently taken for granted. Furthermore, the stereological analysis suggests that EE2 alone enhanced both zebrafish growth and gonad development. On the other hand, exposure to Fad affected the sexual development in zebrafish, inducing masculinization of the specimens, with some degree of intersex observed in males. In addition, the binary mixture allowed identifying sex-dependent roles of steroid hormones in the general growth and gonad development of zebrafish, with estrogens acting as growth promoters in females and being essential for ovary development. Data further support that sex-specific and single EDC impact studies are important, but clearly not sufficient to understand what may occur in the environment.

Developmental toxicity of endocrine disruptors in early life stages of zebrafish, a genetic and embryogenesis study.

Endocrine disrupting compounds (EDCs) are capable of interfering with the endocrine system and are increasingly widespread in the aquatic environments. In the present study, zebrafish (Danio rerio) embryos and larvae were used to assess how EDCs may interfere with embryogenesis. Therefore, zebrafish embryos were exposed to 17α-ethinylestradiol (EE2: 0.4, 2, 4 and 20 ng/L), genistein (Gen: 2, 20, 200 and 2000 ng/L) and fadrozole (Fad: 2, 10, 50 and 250 µg/L), between 2 and 144 h post-fertilization (hpf). Somite development, heartbeat, malformations, mortality and hatching rates were evaluated. In parallel, the expression patterns of hormone receptors (esr1, esr2a, esr2b and ar) and apoptotic pathways related genes (p53 and c-jun) were determined using quantitative real-time PCR. Results showed that EE2, Gen and Fad caused a higher mortality and also malformations in larvae compared with control. A significant toxic effect was observed in the heartbeat rate, at 144 hpf, in larvae exposed to EE2 and Fad. QPCR revealed alterations in the expression levels of all the evaluated genes, at different time points. esr1 and c-jun genes were upregulated by EE2 and Gen exposure while the expression of esr2a, esr2b and ar genes was downregulated. Fad exposure decreased esr1, p53 and c-jun expression levels. This study shows a toxic effect of EE2, Gen and Fad to vertebrate embryogenesis and a relation between hormones action and apoptosis pathways.

Effects of bisphenol A and fadrozole exposures on cyp19a1 expression in the Murray rainbowfish, Melanotaenia fluviatilis.

Several endocrine-disrupting chemicals (EDCs) have been attributed to the alteration of reproduction in fish through disrupting endogenous sex steroidogenic pathways including aromatisation of androgens to oestrogen by CYP19 aromatase. Here we investigate this hypothesis in adult male and female Melanotaenia fluviatilis by examining the mRNA expression of cyp19a1 isoforms after exposure for ≤96 h to two EDCs with contrasting modes of action: one a weak oestrogen mimic, bisphenol A [BPA (100 or 500 µg/L)], and the other a nonsteroidal aromatase inhibitor, fadrozole [FAD (10 or 50 µg/L)]. The results suggest that BPA did not affect cyp19a1a expression significantly at both concentrations, whereas 50 µg/L of FAD significantly upregulated its expression in ovary. In contrast, BPA exposures increased expression of cyp19a1b in brain of both males and females, whilst FAD had contrasting effects in brain: It increased in males but decreased in females. Similar contrasting responses of cyp19a1b were induced by BPA in gonads: upregulation in ovary and downregulation in testis. FAD did not have a significant effect on gonadal expression of cyp19a1b. Collectively, the results suggest that BPA and FAD can disrupt cyp19a1b activity more readily than can cyp19a1a, albeit with contrasting effects in either a tissue- or sex-specific context that is conceivably consistent with their (BPA and FAD) opposing modes of action. Enhanced spatial and temporal sensitivity of cyp19a1b compared with cyp19a1a suggests that brain sex of fish is more susceptible to disruption by environmental pollutants such as BPA and FAD. Therefore, we propose that the response of cyp19a1b in brain tissue of M. fluviatilis is a more suitable indicator of oestrogenic pollution in the aquatic environment.

Developing predictive approaches to characterize adaptive responses of the reproductive endocrine axis to aromatase inhibition: II. Computational modeling.

Endocrine-disrupting chemicals can affect reproduction and development in humans and wildlife. We developed a computational model of the hypothalamic-pituitary-gonadal (HPG) axis in female fathead minnows to predict dose-response and time-course (DRTC) behaviors for endocrine effects of the aromatase inhibitor, fadrozole (FAD). The model describes adaptive responses to endocrine stress involving regulated secretion of a generic gonadotropin (LH/FSH) from the hypothalamic-pituitary complex. For model development, we used plasma 17ß-estradiol (E2) concentrations and ovarian cytochrome P450 (CYP) 19A aromatase mRNA data from two time-course experiments, each of which included both an exposure and a depuration phase, and plasma E2 data from a third 4-day study. Model parameters were estimated using E2 concentrations for 0, 0.5, and 3 µg/l FAD exposure concentrations, and good fits to these data were obtained. The model accurately predicted CYP19A mRNA fold changes for controls and three FAD doses (0, 0.5, and 3 µg/l) and plasma E2 dose response from the 4-day study. Comparing the model-predicted DRTC with experimental data provided insight into how the feedback control mechanisms in the HPG axis mediate these changes: specifically, adaptive changes in plasma E2 levels occurring during exposure and "overshoot" occurring postexposure. This study demonstrates the value of mechanistic modeling to examine and predict dynamic behaviors in perturbed systems. As this work progresses, we will obtain a refined understanding of how adaptive responses within the vertebrate HPG axis affect DRTC behaviors for aromatase inhibitors and other types of endocrine-active chemicals and apply that knowledge in support of risk assessments.

Developing predictive approaches to characterize adaptive responses of the reproductive endocrine axis to aromatase inhibition: I. Data generation in a small fish model.

Adaptive or compensatory responses to chemical exposure can significantly influence in vivo concentration-duration-response relationships. This study provided data to support development of a computational dynamic model of the hypothalamic-pituitary-gonadal axis of a model vertebrate and its response to aromatase inhibitors as a class of endocrine active chemicals. Fathead minnows (Pimephales promelas) were either exposed to the aromatase inhibitor fadrozole (0.5 or 30 µg/l) continuously for 1, 8, 12, 16, 20, 24, or 28 days or exposed for 8 days and then held in control water (no fadrozole) for an additional 4, 8, 12, 16, or 20 days. The time course of effects on ovarian steroid production, circulating 17ß-estradiol (E2) and vitellogenin (VTG) concentrations, and expression of steroidogenesis-related genes in the ovary was measured. Exposure to 30 µg fadrozole/l significantly reduced plasma E2 and VTG concentrations after just 1 day and those effects persisted throughout 28 days of exposure. In contrast, ex vivo E2 production was similar to that of controls on day 8-28 of exposure, whereas transcripts coding for aromatase and follicle-stimulating hormone receptor were elevated, suggesting a compensatory response. Following cessation of fadrozole exposure, ex vivo E2 and plasma E2 concentrations exceeded and then recovered to control levels, but plasma VTG concentrations did not, even after 20 days of depuration. Collectively these data provide several new insights into the nature and time course of adaptive responses to an aromatase inhibitor that support development of a computational model (see companion article).

Application of a label-free, gel-free quantitative proteomics method for ecotoxicological studies of small fish species.

Although two-dimensional electrophoresis (2D-GE) remains the basis for many ecotoxicoproteomic analyses, newer non-gel-based methods are beginning to be applied to overcome throughput and coverage limitations of 2D-GE. The overall objective of our research was to apply a comprehensive, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic approach to identify and quantify differentially expressed hepatic proteins from female fathead minnows exposed to fadrozole, a potent inhibitor of estrogen synthesis. Female fathead minnows were exposed to 0 (control), 0.04, and 1.0 µg of fadrozole/L of water for 4 days, and proteomic analysis was performed. Proteins were extracted and digested, and proteolytic peptides were separated via high-resolution one- or two-dimensional (1-D or 2-D) ultrapressure liquid chromatography (UPLC) and analyzed by tandem mass spectrometry. Mass spectra were searched against the National Center for Biotechnology Information (NCBI) ray-finned fish ( Actinopterygii ) database, resulting in identification of 782 unique proteins by single-dimension UPLC. When multidimensional LC analysis (2-D) was performed, an average increase of 1.9× in the number of identified proteins was observed. Differentially expressed proteins in fadrozole exposures were consistent with changes in liver function, including a decline in concentrations of vitellogenin as well as other proteins associated with endocrine function and cholesterol synthesis. Overall, these results demonstrate that a gel-free, label-free proteomic analysis method can successfully be utilized to determine differentially expressed proteins in small fish species after toxicant exposure.