2018 Progress Report: Linking Biological Scales Across Generations: An Estuarine and Marine Model for Measuring The Ecological Impact of Endocrine Disrupting Compounds

EPA Grant Number: R835799
Title: Linking Biological Scales Across Generations: An Estuarine and Marine Model for Measuring The Ecological Impact of Endocrine Disrupting Compounds
Investigators: Brander, Susanne M , Mehinto, Alvine C , Connon, Richard E , White, J. Wilson
Institution: Oregon State University , Southern California Coastal Water Research Project Authority , University of California - Davis
EPA Project Officer: Lasat, Mitch
Project Period: June 1, 2015 through May 31, 2018 (Extended to May 31, 2020)
Project Period Covered by this Report: June 1, 2018 through May 31,2019
Project Amount: $399,884
RFA: Systems-Based Research for Evaluating Ecological Impacts of Manufactured Chemicals (2014) RFA Text |  Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Ecosystems , Safer Chemicals

Objective:

Objective: To determine the effects of bifenthrin and levonorgestrel, and relative positive controls ethinylestradiol and trenbolone, on Menidia beryllina across the molecular (gene expression, DNA methylation) organismal (gross immune response, gonad development), and population levels (sex ratio, population model) over three generations (P, F1, F2). Reared offspring of the exposed parent generation (exposed from fertilization through 21 dph, then reared in clean water) will be examined for epigenetic carry-over effects for the same endpoints. These in vivo responses will be linked to in vitro cellular assays. A mathematical model of fish population dynamics will scale up experimental results to predict the effects of contaminant exposure on the persistence of Menidia populations and on the persistence of other species.

 Experimental Approach: We are using a combination of in vitro cell assays (ER, AR, PR activity), molecular assays including qPCR and analysis of the global DNA methylome (reduced representation bisulfite sequencing), gonad histology, a PHA (phytohaemagglutinin) immune assay, a 21-day reproductive assay (on adult P and F1 generations), and an integral projection model (population dynamics). The unexposed F1 generation will undergo the same assays, the unexposed F2 generation, reared to 21 dph, will undergo the molecular assays and a newly added evaluation of embryonic development described below.

 Anticipated Results: We expect to find that emerging EDCs (bifenthrin, levonorgestrel) produce higher-level effects (e.g. reduced reproductive output, altered immune response and gonadal histology) similar to those of positive controls (ethyinylestradiol, trenbolone), but that the molecular initiating events (assayed in vitro and via qPCR) will differ between those compounds. In vitro effects of the studied EDCs on endocrine related endpoints will likely be of higher magnitude than the effects measured in vivo. We also expect that exposure of P generation fertilized eggs through 21 dph will produce carryover epigenetic effects in both the F1 and F2 generations. Modeling results will likely show emergent population-level effects of different EDC exposures on both persistence and dynamics.

Progress Summary:

At year four we have completed all experimental objectives while also successfully moving lead PI Brander’s research program from North Carolina to Oregon.  The sequenced genome has been annotated, and RRBS run and analyzed. Gene expression analysis, evaluating a suite of 21 genes involved in reproduction, stress and immune response, and growth on 21 dph juveniles was also completed over this time period. This work comprised approximately half of a Ph.D. dissertation completed by B. DeCourten in May 2019. We also brought on a new postdoctoral scholar (K .Major) to lead DNA methylation analyses. A manuscript reporting results of gene expression, a subset of the DNA methylation data, and organism level responses (immune assay, histology, fecundity, hatching rate, growth, etc.) has been submitted to Environmental Science and Technology. To the best of our knowledge M. beryllina is the first euryhaline fish for which RRBS responses linked with higher level effects have been reported. A second manuscript examining DNA methylation results across the entire genome is planned for invited submission to a special issue on epigenetics being published by Frontiers in Marine Science in 2020. Results from this work have been presented at SETAC Sacramento 2018 (B. DeCourten, K. Major), PRIMO 2019 (Pollutant Responses in Marine Organisms, K. Major, Brander) and various regional meetings.

 

Results thus far show that early-life exposure to all EDCs except for ethinylestradiol (EE2) significantly reduced fecundity in the parental generation. Conversely, only EE2 skewed parental sex ratios (feminized) relative to controls. Sex ratios in the F1 generation were not altered. In both the parental and F1 generations, differences in adult immune response are apparent between bifenthrin other treatments, with bifenthrin treated fish having relatively elevated T-cell proliferation. Both EE2 and bifenthrin had increased atresia in adult parental ovaries, but this effect did not carry over to the F1. Findings from the F0, F1, and F2 generations demonstrate that exposure to EDCs increased growth in the parental generation, and that androgenic treatment groups (Levo, TB) maintain this growth through the subsequent F1 generation. F2 juveniles (21 dph) treated with EE2 are significantly longer compared to other treatments, indicating a potential epigenetic effect on growth. Larval deformities, while not evident in the directly exposed parental generation, are significantly higher across all categories (craniofacial, skeletal, cardiovascular) in EE2 and TB-exposed F1 embryos and larvae, and craniofacial deformities are significantly higher in F2 LV-exposed early life stages. For hatching and larval survival, TB treated parental embryos had a lower percent hatched, as did BF, TB, and LV treated F1 embryos. In the F2, the EE2, TB, and LV treatments had a significantly lower percent survival. In summary, potential epigenetic effects on growth, deformities, and survival were observed across different combinations of treatments. While altered expression of endocrine-related genes was evident, more responses were measured via RRBS (reduced representation bisulfite sequencing). Differential methylation was seen across all three generations, both in the promoter region and within the gene body.

We are also making progress toward modeling goals based on White et al. (2017), and are now in the process of completing the model. Finally, Brander will be co-chairing and K. Major presenting in a third consecutive session on epigenetic effects in aquatic organisms at the 2019 Society of Environmental Toxicology and Chemistry (SETAC) meeting. A focus paper in Environmental Toxicology and Chemistry published by Brander, Connon, and A. Biales of the EPA, has consistently been the most highly read paper in Brander’s department on ResearchGate over the past year. Resulting support from this project has also made continued work on related research evaluating combined exposure to elevated temperatures and endocrine disruptors, as well as additional collaborations with UC Davis possible, as well as a newly awarded EPA STAR on New Approach Methods using silversides and newly generated cell lines as a model for responses across a salinity gradient.

Future Activities:

We have had a second no-cost extension approved, given the move of Lead PI Brander and Co-PI White from the University of North Carolina, Wilmington to Oregon State University. The new OSU contract was finalized this past spring. In year five, we will complete the submission of all manuscripts. At this point the only remaining work is the planned integral projection model, for which preliminary analysis has already been completed and will be presented at SETAC Toronto.


Journal Articles on this Report : 7 Displayed | Download in RIS Format

Other project views: All 8 publications 8 publications in selected types All 8 journal articles
Type Citation Project Document Sources
Journal Article Brander SM, Gabler MK, Fowler NL, Connon RE, Schlenk D. Pyrethroid pesticides as endocrine disruptors: molecular mechanisms in vertebrates with a focus on fishes. Environmental Science & Technology 2016;50(17):8977-8992. R835799 (2016)
R835799 (2017)
R835799 (2018)
  • Abstract from PubMed
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  • Abstract: ACS-Abstract
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  • Journal Article Brander SM, Biales AD, Connon RE. The role of epigenomics in aquatic toxicology. Environmental Toxicology and Chemistry 2017;36(10):2565-2573. R835799 (2017)
    R835799 (2018)
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  • Abstract: Wiley-Abstract
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  • Journal Article DeCourten BM, Brander SM. Combined effects of increased temperature and endocrine disrupting pollutants on sex determination, survival, and development across generations. Scientific Reports 2017;7(1):9310 (9 pp.). R835799 (2015)
    R835799 (2017)
    R835799 (2018)
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  • Journal Article White JW, Cole BJ, Cherr GN, Connon RE, Brander SM. Scaling up endocrine disruption effects from individuals to populations: outcomes depend on how many males a population needs. Environmental Science & Technology 2017;51(3):1802-1810. R835799 (2015)
    R835799 (2016)
    R835799 (2017)
    R835799 (2018)
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  • Journal Article Frank DF, Miller GW, Harvey DJ, Brander SM, Geist J, Connon RE, Lein PJ. Bifenthrin causes transcriptomic alterations in mTOR and ryanodine receptor-dependent signaling and delayed hyperactivity in developing zebrafish (Danio rerio). Aquatic Toxicology 2018;200:50-61. R835799 (2018)
    R835432 (Final)
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  • Journal Article DeCourten BM, Connon RE, Brander SM. Direct and indirect parental exposure to endocrine disruptors and elevated temperature influences gene expression across generations in a euryhaline model fish. PeerJ 2019;7:e6156. R835799 (2018)
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  • Journal Article Frank DF, Brander SM, Hasenbein S, Harvey DJ, Geist J, Connon RE. 2019. Exposure to environmentally relevant bifenthrin concentrations results in behavioral and transcriptomic alterations during early development of Menidia beryllina. Aquatic Toxicology 206:1-13. R835799 (2018)
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    Supplemental Keywords:

    Menidia beryllina, epigenetic, methylation, RRBS, qPCR, systems biology, ecotoxicology

    Progress and Final Reports:

    Original Abstract
  • 2015 Progress Report
  • 2016 Progress Report
  • 2017 Progress Report