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.

Pathway-based approaches for assessment of real-time exposure to an estrogenic wastewater treatment plant effluent on fathead minnow reproduction.

Wastewater treatment plant (WWTP) effluents are known contributors of chemical mixtures into the environment. Of particular concern are endocrine-disrupting compounds, such as estrogens, which can affect the hypothalamic-pituitary-gonadal axis function in exposed organisms. The present study examined reproductive effects in fathead minnows exposed for 21 d to a historically estrogenic WWTP effluent. Fathead minnow breeding pairs were held in control water or 1 of 3 effluent concentrations (5%, 20%, and 100%) in a novel onsite, flow-through system providing real-time exposure. The authors examined molecular and biochemical endpoints representing key events along adverse outcome pathways linking estrogen receptor activation and other molecular initiating events to reproductive impairment. In addition, the authors used chemical analysis of the effluent to construct a chemical-gene interaction network to aid in targeted gene expression analyses and identifying potentially impacted biological pathways. Cumulative fecundity was significantly reduced in fish exposed to 100% effluent but increased in those exposed to 20% effluent, the approximate dilution factor in the receiving waters. Plasma vitellogenin concentrations in males increased in a dose-dependent manner with effluent concentration; however, male fertility was not impacted. Although in vitro analyses, analytical chemistry, and biomarker responses confirmed the effluent was estrogenic, estrogen receptor agonists were unlikely the primary driver of impaired reproduction. The results provide insights into the significance of pathway-based effects with regard to predicting adverse reproductive outcomes.

Integrated assessment of runoff from livestock farming operations: Analytical chemistry, in vitro bioassays, and in vivo fish exposures.

Animal waste from livestock farming operations can contain varying levels of natural and synthetic androgens and/or estrogens, which can contaminate surrounding waterways. In the present study, surface stream water was collected from 6 basins containing livestock farming operations. Aqueous concentrations of 12 hormones were determined via chemical analyses. Relative androgenic and estrogenic activity was measured using in vitro cell assays (MDA-kb2 and T47D-Kbluc assays, respectively). In parallel, 48-h static-renewal in vivo exposures were conducted to examine potential endocrine-disrupting effects in fathead minnows. Mature fish were exposed to surface water dilutions (0%, 25%, 50%, and 100%) and 10-ng/L of 17α-ethynylestradiol or 50-ng/L of 17ß-trenbolone as positive controls. Hepatic expression of vitellogenin and estrogen receptor α mRNA, gonadal ex vivo testosterone and 17ß-estradiol production, and plasma vitellogenin concentrations were examined. Potentially estrogenic and androgenic steroids were detected at low nanogram per liter concentrations. In vitro estrogenic activity was detected in all samples, whereas androgenic activity was detected in only 1 sample. In vivo exposures to the surface water had no significant dose-dependent effect on any of the biological endpoints, with the exception of increased male testosterone production in 1 exposure. The present study, which combines analytical chemistry measurements, in vitro bioassays, and in vivo fish exposures, highlights the integrated value and future use of a combination of techniques to obtain a comprehensive characterization of an environmental chemical mixture.

An inexpensive, temporally integrated system for monitoring occurrence and biological effects of aquatic contaminants in the field.

Assessment of potential risks of complex contaminant mixtures in the environment requires integrated chemical and biological approaches. In support of the US Great Lakes Restoration Initiative, the US Environmental Protection Agency lab in Duluth, MN, is developing these types of methods for assessing possible risks of aquatic contaminants in near-shore Great Lakes (USA) sites. One component involves an exposure system for caged fathead minnow (Pimephales promelas) adults suitable for the wide range of habitat and deployment situations encountered in and around the Great Lakes. To complement the fish exposure system, the authors developed an automated device for collection of composite water samples that could be simultaneously deployed with the cages and reflect a temporally integrated exposure of the animals. The present study describes methodological details of the design, construction, and deployment of a flexible yet comparatively inexpensive (<600 USD) caged-fish/autosampler system. The utility and performance of the system were demonstrated with data collected from deployments at several Great Lakes sites. For example, over 3 field seasons, only 2 of 130 deployed cages were lost, and approximately 99% of successfully deployed adult fish were recovered after exposures of 4 d or longer. A number of molecular, biochemical, and apical endpoints were successfully measured in recovered animals, changes in which reflected known characteristics of the study sites (e.g., upregulation of hepatic genes involved in xenobiotic metabolism in fish held in the vicinity of wastewater treatment plants). The automated composite samplers proved robust with regard to successful water collection (>95% of deployed units in the latest field season), and low within- and among-unit variations were found relative to programmed collection volumes. Overall, the test system has excellent potential for integrated chemical-biological monitoring of contaminants in a variety of field settings.

Ecotoxicogenomics to support ecological risk assessment: a case study with bisphenol A in fish.

Effects of bisphenol A (BPA) on ovarian transcript profiles as well as targeted end points with endocrine/reproductive relevance were examined in two fish species, fathead minnow (Pimephales promelas) and zebrafish (Danio rerio), exposed in parallel using matched experimental designs. Four days of waterborne exposure to 10 µg BPA/L caused significant vitellogenin induction in both species. However, zebrafish were less sensitive to effects on hepatic gene expression and steroid production than fathead minnow and the magnitude of vitellogenin induction was more modest (i.e., 3-fold compared to 13,000-fold in fathead minnow). The concentration-response at the ovarian transcriptome level was nonmonotonic and violated assumptions that underlie proposed methods for estimating hazard thresholds from transcriptomic results. However, the nonmonotonic profile was consistent among species and there were nominal similarities in the functions associated with the differentially expressed genes, suggesting potential activation of common pathway perturbation motifs in both species. Overall, the results provide an effective case study for considering the potential application of ecotoxicogenomics to ecological risk assessments and provide novel comparative data regarding effects of BPA in fish.

Use of gene expression, biochemical and metabolite profiles to enhance exposure and effects assessment of the model androgen 17ß-trenbolone in fish.

The impact of exposure by water to a model androgen, 17ß-trenbolone (TRB), was assessed in fathead minnows using an integrated molecular approach. This included classical measures of endocrine exposure such as impacts on testosterone (T), 17ß-estradiol (E2), and vitellogenin (VTG) concentrations in plasma, as well as determination of effects on the hepatic metabolome using proton nuclear magnetic resonance spectroscopy. In addition, the rates of production of T and E2 in ovary explants were measured, as were changes in a number of ovarian gene transcripts hypothesized to be relevant to androgen exposure. A temporally intensive 16-d test design was used to assess responses both during and after the TRB exposure (i.e., depuration/recovery). This strategy revealed time-dependent responses in females (little impact was seen in the males), in which changes in T and E2 production in the ovary, as well as levels in plasma, declined rapidly (within 1 d), followed shortly by a return to control levels. Gene expression measurements revealed dynamic control of transcript levels in the ovary and suggested potential mechanisms for compensation during the exposure phase of the test. Proton nuclear magnetic resonance spectroscopy revealed a number of hepatic metabolite changes that exhibited strong time and dose dependence. Furthermore, TRB appeared to induce the hepatic metabolome of females to become more like that of males at both high test concentrations of TRB (472 ng/L) and more environmentally relevant levels (33 ng/L).

Assessment of the risk of solar ultraviolet radiation to amphibians. I. Dose-dependent induction of hindlimb malformations in the northern leopard frog (Rana pipiens).

A number of environmental stressors have been hypothesized as responsible for recent increases in limb malformations in several species of North American amphibians. The purpose of this study was to generate dose-response data suitable for assessing the potential role of solar ultraviolet (UV) radiation in causing limb malformations in a species in which this phenomenon seemingly is particularly prevalent, the northern leopard frog (Rana pipiens). Frogs were exposed from early embryonic stages through complete metamorphosis to varying natural sunlight regimes, including unaltered (100%) sunlight, sunlight subjected to neutral density filtration to achieve relative intensities of 85%, 75%, 65%, 50%, and 25% of unaltered sunlight, and sunlight filtered with glass or acrylamide to attenuate, respectively, the UVB (290-320 nm) and UVB plus UVA (290-380 nm) portions of the spectrum. The experiments were conducted in a controlled setting, with continual monitoring of UVB, UVA, and visible light to support a robust exposure assessment. Full sunlight caused approximately 50% mortality of the frogs during early larval development; no significant treatment-related mortality occurred under any of the other exposure regimes, including 100% sunlight with glass or acrylamide filtration. There was a dose-dependent (p < 0.0001) induction of hindlimb malformations in the frogs, with the percentage of affected animals ranging from about 97% under unaltered sunlight to 0% in the 25% neutral density treatment. Malformations were comprised mostly of missing or truncated digits, and generally were bilateral as well as symmetrical. Filtration of sunlight with either glass or acrylamide both significantly reduced the incidence of malformed limbs. The estimated sunlight dose resulting in a 50% limb malformation rate (ED50) was 63.5%. The limb ED50 values based on measured sunlight intensities corresponded to average daily doses of 4.5 and 100 Wh x m(-2) for UVB and UVA, respectively. Exposure to sunlight also resulted in increased eye malformations in R. pipiens, however, the dose-response relationship for this endpoint was not monotonic. The results of this study, in conjunction with measured or predicted exposure data from natural settings, provide a basis for quantitative prediction of the risk of solar UV radiation to amphibians.