Grantee Research Project Results
Final Report: Cell-Free Neurochemical Screening Assays to Predict Adverse Effects in Mammals, Fish, and Birds
EPA Grant Number: R835170Title: Cell-Free Neurochemical Screening Assays to Predict Adverse Effects in Mammals, Fish, and Birds
Investigators: Basu, Niladri , Head, Jessica A , Murphy, Cheryl A. , Neitzel, Richard L.
Institution: University of Michigan , Michigan State University , McGill University
Current Institution: University of Michigan , McGill University , Michigan State University
EPA Project Officer: Aja, Hayley
Project Period: September 1, 2012 through August 31, 2015 (Extended to August 31, 2017)
Project Amount: $1,199,222
RFA: Developing High-Throughput Assays for Predictive Modeling of Reproductive and Developmental Toxicity Modulated Through the Endocrine System or Pertinent Pathways in Humans and Species Relevant to Ecological Risk Assessment (2011) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
The overall objective of this project was to advance an in vitro, cell-free neurochemical screening assay platform, and model data outputs to predict adverse, individual-level reproductive effects associated with toxicant exposure in mammalian, fish and avian wildlife. The overall hypothesis was that several environmental toxicants will emerge to interact with and disrupt the function of neurotransmitter receptors, enzymes and transporters that have critical roles in vertebrate reproduction.
Summary/Accomplishments (Outputs/Outcomes):
Although thousands of anthropogenic chemicals need to be evaluated for regulatory purposes, obtaining whole-animal or population-level data is impractical. Such efforts require animal tests, and are largely descriptive, time consuming, and costly. These limitations have been recognized by the U.S. National Research Council (NRC) in their recent document entitled “Toxicity Testing in the 21st Century: A Vision and a Strategy.” The major outcome of this project was the advancement of an in vitro, cell-free neurochemical screening assay platform as well as refined models (e.g., mathematical; alternative testing) to link molecular and cellular measures with apical ones of regulatory concern. Below is a summary of key accomplishments.
In Study #1, we developed and applied a set of cell-free neurochemical assays to screen many chemicals across many species in a relatively high-throughput manner. The developed platform assessed seven receptors and enzymes that mediate neurotransmission of g-aminobutyric acid, dopamine, glutamate, and acetylcholine. Each assay was optimized to work across 20 vertebrate species (5 fish, 5 birds, 7 mammalian wildlife, and 3 biomedical species including humans). We tested the screening assay platform against 80 chemicals (23 pharmaceuticals and personal care products, 20 metal[loid]s, 22 polycyclic aromatic hydrocarbons and halogenated organic compounds, 15 pesticides). The results showed that cell-free assays can screen a large number of samples in a short period of time in a cost-effective manner in a range of animals not easily studied using traditional approaches.
In Study #2, based on Study #1 results, 10 chemicals (Hg, Pb, Sn, B(a)P, Anthracene, DDE, Prochloraz, EE2, BPA, Triclosan) were selected and dose response curves were obtained for six receptors and enzymes that mediate neurotransmission of g-aminobutyric acid, dopamine, glutamate, and acetylcholine in 8 species (chicken, Japanese quail, cow, rainbow trout, human, dolphin, polar bear, mink). This particular study showed that dose-response curves could be developed for this in vitro, and thus enable quantitative modeling of the in vitro work which can be of use in more sophisticated assessments.
In Study #3, the US EPA E1K library of 800 chemicals suspected of endocrine disrupting potential was studied against three neurochemical receptor binding assays (NMDA, D2, mAChR) in one bird (zebra finch), one fish (rainbow trout), and a mammal (mink). Chemicals tested ranged from pharmaceuticals and dyes to fungicides. The work showed that the assay can be scaled-up to test many chemicals in a relatively short period of time.
In Study #4, we applied the in vitro assay to study complex environmental mixtures (wastewaters) and to compare against whole animal results. Using both in vivo and in vitro approaches, this study shows that wastewater effluents contain agents that can interact with neurochemicals relevant to reproduction and other neurological functions though points to challenges in performing this type of work. Additional work is needed to better resolve in vitro to in vivo extrapolations (IVIVE) as well as cross-species differences.
In Study #5, we aimed to develop a quantitative model of the fish reproductive axis that considers upstream neurochemical events, and apply this to study responses in fish that were exposed to pulp and paper mill effluents in vitro. While the model has yet to be validated in a whole animal study, our proof of principle approach highlights the use of computational modeling as a means to integrate results from in vitro studies that assess complex mixtures to predict potentially adverse effects on fish reproduction.
In Study #6, we focused on birds as few modelling attempts in the past have considered this taxa. We anchored this study with data from a whole animal experiment with quail that were exposed to 17β-trenbolone. Trenbolone-acetate is a synthetic anabolic steroid used as a livestock growth promoter and is metabolized to 17β-trenbolone. We characterized molecular-to-physiological-to-organismal measures in these birds, and used it to develop the first avian vitellogenesis model.
In Study #7, we aimed to determine if vitellogenin can be used as an indicator of avian fecundity, and we experimented with a double-dye technique to quantify daily egg yolk production in Japanese quail as a novel, colorimetric biomarker. The findings showed that differential staining holds promise with results suggesting an impairment of yolk production following contaminant exposure.
Conclusions:
In Summary, the collection of studies here yielded several noteworthy outcomes. First, the cell-free neurochemical screen developed was able to compare responses across multiple and diverse animals (n = 20, including many not easily studied) and be scaled-up to study hundreds of chemicals (e.g., we screened the E1K library). The neurochemical screen was used to characterize complex environmental mixtures, though when related to in vivo results there were some important differences and challenges uncovered and thus it will be important to address these in future efforts. Developing quantitative models is of great interest in our field. Here, we extend upon standard endocrine disruption models by involving upstream neurochemical-based changes into the models. We capitalize upon a detailed animal study with Japanese quail to establish the first vitellogenisis model in a bird, and also developed the first visual biomarker (using double-dyed eggs) of endocrine disruption in birds through a novel method. These outcomes help address several grand challenges in our field, and thus help meet the call for new environmental toxicity testing methods that are faster, cheaper, ethical, and more predictive than the status quo.
The work resulted in the establishment of a high-throughput screening assay that can now be used to study multiple ecologically relevant species (birds, fish, mammals) against chemicals of concern as well as complex environmental mixtures. The screening platform can be scaled-up to study thousands of samples, and the work can be adapted to a potentially limitless number of species. The modeling aspects demonstrated portability across taxa, and also a computational tool with predictive potential for reproductive outcomes. An in vivo trenbolone feeding trial with Japanese quail was used to evaluate the in vitro and modelling work. Several publications, presentations, and student dissertations were (and will continue to be) produced.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 19 publications | 4 publications in selected types | All 2 journal articles |
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Arini A, Cavallin JE, Berninger JP, Marfil-Vega R, Mills M, Villeneuve DL, Basu N. In vivo and In vitro neurochemical-based assessments of wastewater effluents from the Maumee River area of concern. Environmental Pollution 2016;211:9-19. |
R835170 (2016) R835170 (Final) |
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Arini A, Mittal K, Dornbos P, Head J, Rutkiewicz j, Basu N. A cell-free testing platform to screen chemicals of potential neurotoxic concern across twenty vertebrate species. Environmental Toxicology and Chemistry 2017;36(11):3081-3090. |
R835170 (Final) |
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Supplemental Keywords:
Screening assay, toxicity testing, comparative biology, risk assessment, ecotoxicology, neurotoxicology, water, animals, Great Lakes region, water pollutants, toxicology, wastewater, vitellogenin, high-throughput screening assays, species sensitivity, biomarkers, receptorsRelevant Websites:
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
- 2016 Progress Report
- 2015 Progress Report
- 2014 Progress Report
- 2013 Progress Report
- Original Abstract
2 journal articles for this project