Building toxicity pathway models and estimating transcriptomic points-of-departure (tPODs) in three phylogenetically distant ray-finned fishes exposed to 17alpha-ethinylestradiol and fluoxetine
Date
2021-12-21
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
0000-0002-3213-6805
Type
Thesis
Degree Level
Doctoral
Abstract
The continuous development and production of new chemicals to meet societal needs results in their increasing release and prevalence in the environment. These contaminants of emerging concern (CECs) are continuously discharged from wastewater treatment plants and other sources that are often not equipped to remove these classes of compounds. Thus, the pseudo-persistence of these CECs poses potential toxicological risks to aquatic organisms and, therefore, constitute a significant issue in many aquatic environments. However, current testing frameworks supporting chemical and environmental risk assessment (ERA) are falling short of the global need to rapidly test the increasing numbers of CECs because they rely on the extensive use of live animals, which is time-consuming, costly, and presents significant ethical concerns. Thus, there is an urgent need for the development and implementation of new approach methodologies (NAMs) aimed to replace, reduce, and refine (3Rs) live animal testing while increasing throughput and decreasing costs to improve hazard assessment strategies and support regulatory decision-making. This dissertation aimed to develop and evaluate a NAM system based on short-term embryonic exposure assays to (1) characterize the toxicity pathways of two priority CECs, 17α-ethinylestradiol (EE2) and fluoxetine (FLX) as model chemicals, through cross-organizational level assessments linking molecular mechanistic response patterns with physiological/apical outcomes; and (2) derive transcriptomics points-of-departure (tPODs) that can support quantitative hazard assessment across phylogenetically distant fish. Specifically, fathead minnow (FHM), rainbow trout (RBT), and white sturgeon (WS) were exposed to graded concentrations of each CEC from an early embryonic stage. At 4 days post-hatch (dph), changes across the whole transcriptome and proteome were characterized, and higher organizational-level responses were evaluated at 28 dph (FHM) and 60 dph (RBT and WS). Molecular alterations were then compared to downstream responses to build toxicity pathway models for each chemical. In addition, transcriptomic datasets from 4 dph were used to model dose-response for genes/transcripts and calculate benchmark concentrations (geneBMCs) of toxicant-responsive genes/transcripts. These geneBMCs were used to derive transcriptomics points-of-departure (tPODs) using a number of statistical strategies. These tPODs were then compared to chronic and physiological/apical PODs obtained in this study and from the literature. tPOD estimates were also compared across species.
Results demonstrated that when exposed to EE2, transcriptomic and proteomic profiles of FHM at 4 dph were predictive of histological and apical outcomes at 28 dph. Similarly, transcriptomic profiles of RBT at 4 dph were predictive of histological and apical responses at 60 dph, when exposed to EE2, although proteomic profiles at 4 dph did not correlate well with transcriptomics and apical outcomes. On the other hand, FLX-target genes and proteins were not significantly altered in FHM at 4 dph, but integrated functional analyses of transcriptomics and proteomics revealed molecular processes that were predictive of apical outcomes observed in this study and those reported in the literature. Overall, results showed that transcriptomic responses from whole-body early-life stage (ELS) fish were related to downstream and apical outcomes observed at more advanced life stages. Proteomic responses were limited in predicting relevant responses but added a valuable layer of biological information that enhanced the understanding of the connectivity between molecular events and apical outcomes. Hence, proteomics provided an additional line-of-evidence in support of enhancing our understanding of the mode of action of the tested CECs across biological levels.
Estimated tPODs in all species closely approximated and were protective of chronic apical PODs observed in this study and in the literature, both for EE2 and FLX. tPODs derived from the median of the 20 most sensitive active genes (omicBMC20) were the most protective among tPOD estimates within a species, with FLX tPOD values of 0.02, 0.02, and 0.56 μg/L, and with EE2 tPOD values of 0.06, 0.12, and 2.39 ng/L for WS, RBT, and FHM, respectively. These values were significantly more sensitive than pathway-level BMCs (pathBMC) and were independent of species annotations, which is one of the main limitations when working with non-model species for which no functionally annotated genomes or transcriptomes are available. In general, RBT appeared to be the most sensitive species in terms of tPOD estimates, closely followed by WS, with FHM being the least sensitive to exposure to both chemicals. Regardless, tPOD estimates from ELS fish were within a tight range of concentrations despite minor differences in exposure conditions and data analysis workflow. Overall, this dissertation demonstrated that the fish embryo assays established in this thesis project represent a promising approach to (1) characterize toxicity pathways for CECs that can inform cross-species and chemical hazard assessment, and (2) derive quantitative BMCs (tPODs) that are protective of apical PODs, and therefore show significant promise as a NAM to support chemical hazard assessment and regulatory decision making. Future studies should be directed towards the optimization and validation of this approach using more chemicals and other species that are relevant to ERA.
Description
Keywords
benchmark dose (BMD), animal alternatives, omics, white sturgeon, rainbow trout, fathead minnow
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Toxicology Centre
Program
Toxicology