Grantee Research Project Results
2015 Progress Report: High-Throughput Cellular Assays for Modeling Toxicity in the Fish Reproductive System
EPA Grant Number: R835167Title: High-Throughput Cellular Assays for Modeling Toxicity in the Fish Reproductive System
Investigators: Schultz, Irvin R. , Young, Graham , Nagler, James , Swanson, Penny
Current Investigators: Schultz, Irvin R.
Institution: University of Washington , National Oceanic and Atmospheric Administration , University of Idaho
Current Institution: University of Washington
EPA Project Officer: Aja, Hayley
Project Period: August 1, 2012 through September 30, 2015 (Extended to September 30, 2017)
Project Period Covered by this Report: September 1, 2014 through August 31,2015
Project Amount: $1,199,908
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 is to demonstrate that cellular in vitro assays can provide valid estimates of model parameters used in computational models of fish reproduction. Specific objectives include optimizing rainbow trout pituitary cell culture for assessing toxicant effects on follicle stimulating hormone (FSH) and luteinizing hormone (LH) synthesis. Other objectives are to optimize in vitro ovarian follicle incubations for assessing basal and FSH induced steroidogenesis, optimize rainbow trout primary hepatocyte culture for assessing toxicant effects on vitellogenin (Vg) expression and to incorporate in vitro derived estimates of effect into a computational model of the fish reproductive system.
The development of adverse outcome pathway (AOP) models requires knowledge of dose-dependent perturbations across varying biological scales (cells→organs→systems→ organism→populations). In vitro, cell-based assays are expected to provide the bulk of experimental data for future AOP models while computational or quantitative physiological models are used to extrapolate across biological scales. Our hypothesis is that in vitro assays can provide valid estimates of specific model parameters used in computational models of fish reproduction. To test this, we developed a multi-scale mathematical model of the female rainbow trout hypothalamus-pituitary-ovary-liver (HPOL) axis to extrapolate results from select in vitro studies of tissues comprising the reproductive axis (Gillies et al. 2016). In parallel to model development, we have been testing a series of model endocrine toxicants on isolated pituitary cells, hepatocytes and ovarian follicles. As a demonstration of the complete AOP process, we used the HPOL model with data from trenbolone experiments to simulate effects on oocyte growth and spawning success (Schultz et al. 2013, and Gillies et al. 2016). The model simulations indicate trenbolone effects on E2 synthesis by itself are not sufficient to cause reproductive failure in trout and additional effects occurring simultaneously elsewhere in the system are needed. To date, this work has found that relying on results from a single in vitro assay (e.g. hepatocytes) would not adequately predict the observed effects of trenbolone exposure in vivo.In other words, relying on results from a single in vitro assay (e.g. hepatocytes) would not adequately predict the observed effects of trenbolone exposure in vivo.
Progress Summary:
We have achieved the development and application of a computational model of the trout hypothalamus – pituitary – ovary – liver axis (HPOL).The development of adverse outcome pathway (AOP) models requires knowledge of dose-dependent perturbations across varying biological scales (cells→organs→systems→ organism→populations). In vitro, cell-based assays are expected to provide the bulk of experimental data for future AOP models while computational or quantitative physiological models are used to extrapolate across biological scales. Our hypothesis is that in vitro assays can provide valid estimates of specific model parameters used in computational models of fish reproduction. To test this, we developed a multi-scale mathematical model of the female rainbow trout hypothalamus-pituitary-ovary-liver (HPOL) axis to extrapolate results from select in vitro studies of tissues comprising the reproductive axis (Gillies et al. 2016). The model describes the essential endocrine components of the reproductive axis, including the stage specific growth of maturing oocytes and permits the presence of sub-populations of oocytes at different stages of development. This feature allows the HPOL model to accurately predict oocyte growth and the impact of toxicants that perturb physiological processes associated with oocyte growth and maturation.
In parallel to model development, we have been testing a series of model endocrine toxicants on isolated pituitary cells, hepatocytes and ovarian follicles. Key findings from these studies include estrogen (estradiol-17β; E2) induces the LHβ mRNA levels in pituitary cells from previtellogenic trout at concentrations as low as 0.01 ng/mL (0.037 nM). In contrast, 11-KT had no effect on LHβ mRNA levels and had weak, inconsistent effects on FSHβ mRNA levels. However, effects of 11-KT (the most important male sex hormone in trout) on FSH and LH secretion cannot be ruled out yet. These data suggest that the induction of LHβ in previtellogenic trout is specific to estrogens. Ovarian follicle studies indicated several chemicals affected estrogen synthesis. For example, a significant decrease in E2 production was seen with the pharmaceutical fluoxetine (10 µM), and incubation with the fluoxetine metabolite norfluoxetine showed similar although non-significant trends at concentrations ranging from 0.4 µM to 2.0 µM. The rate of E2 production was drastically reduced after exposure of follicles to environmentally-relevant concentrations of ethinyl estradiol at concentrations as low as 0.1 nM. The synthetic androgen trenbolone was also observed to alter vitellogenin synthesis in hepatocytes, which is in addition to other documented effects on E2 synthesis and FSH release, previously reported by the project (Schultz et al. 2013). As a demonstration of the complete AOP process, we used the HPOL model with data from trenbolone experiments to simulate effects on oocyte growth and spawning success (Gillies et al. 2016). The model simulations indicated trenbolone effects on E2 synthesis by itself are not sufficient to cause reproductive failure in trout and additional effects occurring simultaneously elsewhere in the system are needed. In other words, relying on results from a single in vitro assay (e.g. hepatocytes) would not adequately predict the observed effects of trenbolone exposure in vivo.
Future Activities:
In FY16, additional experiments will be performed with pituitary cells and ovarian follicles to complete chemical testing and to confirm the effects of SSRIs on gonadotropin expression and secretion. Experiments are also planned to determine chemical partitioning and macromolecular binding within the in vitro test systems. Other planned activities will be to draft manuscripts for peer-reviewed publication that summarize the in vitro testing and use of in vitro data with computational models for quantitative adverse outcome pathway modeling.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
| Other project views: | All 24 publications | 6 publications in selected types | All 4 journal articles |
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Gillies K, Krone SM, Nagler JJ, Schultz IR. A computational model of the rainbow trout hypothalamus-pituitary-ovary-liver axis. PLoS Computational Biology 2016;20;12(4):e1004874 (27 pp.). |
R835167 (2015) R835167 (2016) R835167 (Final) |
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Jia Y, Cavileer TD, Nagler JJ. Acute hyperthermic responses of heat shock protein and estrogen receptor mRNAs in rainbow trout hepatocytes. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 2016;201:156-161. |
R835167 (2015) R835167 (2016) R835167 (Final) |
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Schultz IR, Nagler JJ, Swanson P, Wunschel D, Skillman AD, Burnett V, Smith D, Barry R. Toxicokinetic, toxicodynamic, and toxicoproteomic aspects of short-term exposure to trenbolone in female fish. Toxicological Sciences 2013;136(2):413-429. |
R835167 (2013) R835167 (2014) R835167 (2015) R835167 (2016) R835167 (Final) |
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Supplemental Keywords:
recrudescence, maturation, gonadotropin, endocrine disruption, water;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.