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.

Weight of evidence evaluation of a network of adverse outcome pathways linking activation of the nicotinic acetylcholine receptor in honey bees to colony death.

Ongoing honey bee (Apis mellifera) colony losses are of significant international concern because of the essential role these insects play in pollinating crops. Both chemical and non-chemical stressors have been implicated as possible contributors to colony failure; however, the potential role(s) of commonly-used neonicotinoid insecticides has emerged as particularly concerning. Neonicotinoids act on the nicotinic acetylcholine receptors (nAChRs) in the central nervous system to eliminate pest insects. However, mounting evidence indicates that neonicotinoids also may adversely affect beneficial pollinators, such as the honey bee, via impairments on learning and memory, and ultimately foraging success. The specific mechanisms linking activation of the nAChR to adverse effects on learning and memory are uncertain. Additionally, clear connections between observed impacts on individual bees and colony level effects are lacking. The objective of this review was to develop adverse outcome pathways (AOPs) as a means to evaluate the biological plausibility and empirical evidence supporting (or refuting) the linkage between activation of the physiological target site, the nAChR, and colony level consequences. Potential for exposure was not a consideration in AOP development and therefore this effort should not be considered a risk assessment. Nonetheless, development of the AOPs described herein has led to the identification of research gaps which, for example, may be of high priority in understanding how perturbation of pathways involved in neurotransmission can adversely affect normal colony functions, causing colony instability and subsequent bee population failure. A putative AOP network was developed, laying the foundation for further insights as to the role of combined chemical and non-chemical stressors in impacting bee populations. Insights gained from the AOP network assembly, which more realistically represents multi-stressor impacts on honey bee colonies, are promising toward understanding common sensitive nodes in key biological pathways and identifying where mitigation strategies may be focused to reduce colony losses.

A study of temporal effects of the model anti-androgen flutamide on components of the hypothalamic-pituitary-gonadal axis in adult fathead minnows.

The aim of this study was to investigate temporal changes in the hypothalamic-pituitary-gonadal (HPG) axis of fathead minnows (Pimephales promelas) treated with the model androgen receptor (AR) antagonist flutamide. Reproductively-mature fish were exposed in a flow-through test to analytically-confirmed concentrations of either 50 or 500µg flutamide/L for 8 d, followed by an 8-d recovery period in clean water. Fish were sampled at 1, 2, 4 and 8days during each phase of the experiment. Flutamide (500µg/L) caused significant reductions in relative gonad size of the females on day 8 of the exposure and day 1 of the recovery, and reduced expression of secondary sex characteristics in males during the exposure phase of the experiment. Ex vivo gonadal synthesis of testosterone in both sexes (and 17ß-estradiol in females) was reduced in the 500µg/L treatment within 2 d of exposure; however, steroid synthesis returned to levels comparable to controls by the end of the exposure portion of the test. Ex vivo testosterone synthesis in males exposed to 50µg flutamide/L was greater than in controls on days 4 and 8 of the exposure. Both the enhanced steroid production in the low treatment males, and return to control levels in the high treatment males and females during chemical exposure are indicative of a compensatory HPG response. One contributor to this response could be increased expression of genes responsible for enzymes involved in steroid synthesis; for example, transcripts for both cytochrome P450 side- chain cleavage and 11ß-hydroxysteroid dehydrogenase were significantly elevated in flutamide-exposed males. Overall, responses of the HPG axis in adult male and female fathead minnows exposed to flutamide were both dynamic and comparatively rapid during exposure and recovery. These observations have ramifications both for the development of short-term fish assays to detect endocrine-active chemicals, and the derivation of robust adverse outcome pathways for AR antagonists in fish.

Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow.

In the present study, a hypothesized adverse outcome pathway linking inhibition of thyroid peroxidase (TPO) activity to impaired swim bladder inflation was investigated in two experiments in which fathead minnows (Pimephales promelas) were exposed to 2-mercaptobenzothiazole (MBT). Continuous exposure to 1mg MBT/L for up to 22 days had no effect on inflation of the posterior chamber of the swim bladder, which typically inflates around 6 days post fertilization (dpf), a period during which maternally-derived thyroid hormone is presumed to be present. In contrast, inflation of the anterior swim bladder, which occurs around 14dpf, was impacted. Specifically, at 14dpf, approximately 50% of fish exposed to 1mg MBT/L did not have an inflated anterior swim bladder. In fish exposed to MBT through 21 or 22dpf, the anterior swim bladder was able to inflate, but the ratio of the anterior/posterior chamber length was significantly reduced compared to controls. Both abundance of thyroid peroxidase mRNA and thyroid follicle histology suggest that fathead minnows mounted a compensatory response to the presumed inhibition of TPO activity by MBT. Time-course characterization showed that fish exposed to MBT for at least 4 days prior to normal anterior swim bladder inflation had significant reductions in anterior swim bladder size, relative to the posterior chamber, compared to controls. These results, along with similar results observed in zebrafish (see part II, this issue) are consistent with the hypothesis that thyroid hormone signaling plays a significant role in mediating anterior swim bladder inflation and development in cyprinids, and that role can be disrupted by exposure to thyroid hormone synthesis inhibitors. Nonetheless, possible thyroid-independent actions of MBT on anterior swim bladder inflation cannot be ruled out based on the present results. Overall, although anterior swim bladder inflation has not been directly linked to survival as posterior swim bladder inflation has, potential links to adverse ecological outcomes are plausible given involvement of the anterior chamber in sound production and detection.

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 chemical and toxicological investigation of UV-chlorine/chloramine drinking water treatment.

As the use of alternative drinking water treatment increases, it is important to understand potential public health implications associated with these processes. The objective of this study was to evaluate the formation of disinfection byproducts (DBPs) and cytotoxicity of natural organic matter (NOM) concentrates treated with chlorine, chloramine, and medium pressure ultraviolet (UV) irradiation followed by chlorine or chloramine, with and without nitrate or iodide spiking. The use of concentrated NOM conserved volatile DBPs and allowed for direct analysis of the treated water. Treatment with UV prior to chlorine in ambient (unspiked) samples did not affect cytotoxicity as measured using an in vitro normal human colon cell (NCM460) assay, compared to chlorination alone when toxicity is expressed on the basis of dissolved organic carbon (DOC). Nitrate-spiked UV+chlorine treatment produced greater cytotoxicity than nitrate-spiked chlorine alone or ambient UV+chlorine samples, on both a DOC and total organic halogen basis. Samples treated with UV+chloramine were more cytotoxic than those treated with only chloramine using either dose metric. This study demonstrated the combination of cytotoxicity and DBP measurements for process evaluation in drinking water treatment. The results highlight the importance of dose metric when considering the relative toxicity of complex DBP mixtures formed under different disinfection scenarios.