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Assessing the toxicity of 17α-ethinylestradiol in rainbow trout using a four-day transcriptomics benchmark dose (BMD) embryo assay

ABSTRACT: There is an urgent demand for more efficient and ethical approaches in chemical risk assessment. Using 17α-ethinylestradiol (EE2) as a model compound, this study established a live-animal alternative embryo benchmark-dose (BMD) assay for rainbow trout (RBT; Oncorhynchus mykiss) to derive transcriptomic points-of-departure (tPODs). Embryos were exposed to graded concentrations of EE2 (0, 1.13, 1.57, 6.22, 16.3, 55.1, and 169 ng/L) from hatch to 4 and up to 60 days post-hatch (dph) to assess molecular and apical responses, respectively. Whole proteome analyses of alevins did not show clear estrogenic effects, while transcriptomics revealed responses that were in agreement with apical effects including excessive accumulation of intravascular and hepatic proteinaceous fluid and significant increases in mortality at 55.1 and 169 ng/L EE2 at later time points. Transcriptomic BMD analysis estimated the median of the 20th lowest geneBMD to be 0.18 ng/L; the most sensitive tPOD. Other estimates (0.78, 3.64, and 1.63 ng/L for the tenth percentile geneBMD, first peak geneBMD distribution, and median geneBMD of the most sensitive overrepresented pathway, respectively) were within the same order of magnitude as empirically derived apical P ODs for EE2 in the literature. This 4-day alternative RBT embryonic assay was effective in deriving protective tPODs for EE2.

ORGANISM(S): Oncorhynchus mykiss

PROVIDER: GSE171788 | GEO | 2021/08/17

REPOSITORIES: GEO

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Project description:Current methods to support chemical hazard assessment face significant challenges given the rapidly growing number of compounds of emerging concern (CEC) requiring assessment. Standard testing strategies rely upon live animal testing, which are time-consuming, expensive, and ethically questionable. These concerns serve as an impetus to develop new approach methodologies (NAMs) that do not rely on live animal tests. This study explored the application of the molecular benchmark dose (BMD) approach in a 7-day embryo-larval fathead minnow (FHM) assay to derive determine transcriptomic points-of-departure (tPODs) which can be useds to predict apical BMDs of fluoxetine (FLX), a highly prescribed and potent selective serotonin reuptake inhibitor frequently detected in surface waters. Fertilized FHM embryos were exposed to graded concentrations of FLX (confirmed at <LOD, 0.19, 0.74, 3.38, 10.2, 47.5 µg/L) for 32 days. Subsets of fish were subjected to omics and locomotor analyses at 7 days post-fertilization (dpf) and to histological and biometric measurements at 32 dpf. Enrichment analyses of transcriptomics and proteomics data revealed significant perturbations in gene sets associated with serotonergic and axonal functions. BMD analysis resulted in tPOD values of 0.56 µg/L (median of the 20 most sensitive gene-level BMDs), 5.0 µg/L (tenth percentile of all gene-level BMDs), 7.51 µg/L (mode of the first peak of all gene-level BMDs) , and 5.66 µg/L (pathway-level BMD). These tPODs are protective of apical locomotor and reduced weight effects (LOEC of 10.2 µg/L) observed in this study and are well within the range of chronic apical BMDs of FLX reported in the literature. Furthermore, the distribution of gene-level BMDs followed a bimodal pattern, revealing disruption of sensitive neurotoxic pathways at low concentrations and metabolic pathway perturbations at higher concentrations. This is one of the first studies to derive tPODs for FLX using a short-term embryo assay at a life stage not considered to be a live animal under current legislations.

Project description:Per- and polyfluoroalkyl substances (PFAS) represent a large group of contaminants of concern based on their widespread use, distribution and persistence in the environment, and potential toxicity. Many of the traditional models for estimating toxicity, bioaccumulation, and other relevant toxicological properties are not well suited for PFAS. Consequently, there is a need to generate hazard information for a large number of PFAS in an efficient and cost-effective manner. In the present study, Daphnia magna were exposed to multiple concentrations of 22 different PFAS for 24 h, in a 96-well plate format. Following exposure, whole body RNA was extracted and pooled extracts, each representing five exposed individuals, were subjected to RNA sequencing. Following analytical measurements to verify PFAS exposure concentrations in-well, and quality control on processed cDNA libraries for sequencing, concentration-response modeling was applied to the data sets for 18 of the tested compounds, and the concentration at which a concerted molecular response occurred (transcriptomic point of departure; tPOD) was calculated. The tPODs, based on average measured concentration of PFAS in the exposure wells, generally ranged from 0.03-0.58 µM (9.9-350 µg/L; interquartile range). In most cases, these concentrations were two orders of magnitude lower than similarly calculated tPODs for human cell lines exposed to PFAS. They were also lower than apical effect concentrations reported for seven PFAS for which some crustacean or invertebrate toxicity data were available, although there were a few exceptions. However, despite being lower than most other available hazard benchmarks, the Daphnia magna tPODs were, on average, four orders of magnitude greater than the maximum aqueous concentrations of PFAS measured in Great Lakes tributaries. Overall, this high throughput transcriptomics assay with Daphnia magna holds promise as a component of a tiered hazard evaluation strategy employing new approach methodologies.

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Assessing the toxicity of 17α-ethinylestradiol in rainbow trout using a four-day transcriptomics benchmark dose (BMD) embryo assay

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