2017 Progress Report: Organotypic Model of Testis as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials

EPA Grant Number: R835738C004
Subproject: this is subproject number 004 , established and managed by the Center Director under grant R835738
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Predictive Toxicology Center for Organotypic Cultures and Assessment of AOPs for Engineered Nanomaterials
Center Director: Faustman, Elaine
Title: Organotypic Model of Testis as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
Investigators: Faustman, Elaine
Institution: University of Washington
EPA Project Officer: Klieforth, Barbara I
Project Period: December 1, 2014 through November 30, 2018
Project Period Covered by this Report: December 1, 2016 through November 30,2017
Project Amount: Refer to main center abstract for funding details.
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text |  Recipients Lists
Research Category: Safer Chemicals , Health , Human Health

Objective:

The overall goal of this project is to utilize an organotypic in vitro model of testicular development to evaluate the male reproductive toxicity of Engineered Nanomaterials (ENM) using an adverse outcome pathway (AOP) framework. We will use 3-dimensional in vitro testicular co-cultures that have been shown to capture key processes of male reproductive development in order to evaluate the potential for ENMs to alter these processes. We will measure the ability of ENMs to alter cellular differentiation and tissue maturation with a focus on the roles of developmental timing and genetics in influencing susceptibility. We will also explore the role of oxidative stress and inflammation pathways in mediating ENM induced perturbation of development. Finally, we will use toxicokinetic and dynamic models to integrate in vitro our findings into an AOP framework.

Specific Aims and Hypothesis

Hypothesis 1: Differentiation status of organotypic cultures will define the response to ENMs.

Risk Assessment Question: Organotypic cultures that reflect different lifestages will provide an important insight into defining “critical windows of susceptibility” for risk characterization and dose-response.

Hypothesis 2: Assessing oxidative stress responses within organotypic cultures will be a key common response pathway across organ systems to ENMs.

Risk Assessment Question: Basic cellular responses such as oxidative stress response will be less organ and more chemical specific than organotypic cellular responses and will represent a common “molecular initiating event” that defines organotypic response.

Hypothesis 3: Inflammatory pathways within organotypic cultures will have significant and specific impacts beyond general oxidative stress and these can be used to improve our definition of AOP pathways.

Risk Assessment Question: Inflammatory response across time will provide a “molecular initiating event” (biomarker) that goes beyond general oxidative stress to better predict organ specific and strain specific responses.

Hypothesis 4: Genetic (strain) differences between organotypic cultures will be a critical factor in interpreting organ specific responses.

Risk Assessment Question: Genetic background will influence testis organotypic responses to ENMs and this influence will provide important clues for understanding discreet genetic susceptibility factors defining ENM responses.

Hypothesis 5: Using a systems-based Adverse Outcome Pathway analysis with toxicokinetic and dynamic models as a part of the risk assessment framework will allow for cross assay and organ interpretations.

Progress Summary:

Testicular Co-Culture Model: We have optimized testes markers that are specific markers for each cell type in testes co-culture system to allow us to separate specific cell population in three different cell types. We have developed a systems biology approach for characterization of normal development of our testis organotypic cultures with a life stage context, measuring testosterone production and protein expression at days in vitro (DIV) 2, 3, 6, 7, 15 and 16 (Wilder et al. 2018 in preparation). We are preparing a manuscript on cadmium’s developmental toxicity utilizing our mouse testis organotypic culture systems, evaluating cytotoxicity, cell viability and morphology at 24 hours after cadmium treatment at different developmental stage (DIV 2, 6 and 15).

Mouse midbrain micromass: We have utilized in vitro 3D organotypic mouse midbrain micromass culture system to examine adverse effects of silver nanoparticles (AgNPs). We characterized the development of the micromass cultures over time using embryonic midbrains from two mouse strains (C57BL/6 and A/J) (Park et al. 2017). We observed stage-specific protein expressions of proliferation and differentiation and found that C57BL/6 and A/J in vitro mouse micromass systems had similar developmental trends. This study opened a potential for this model to be used for developmental neurotoxicity testing and gene x environment studies by using different mouse strains. We performed our assessment of the toxicological effects of various AgNPs on C57BL/6 and A/J mouse midbrain micromass cultures (Weldon and Park et al. 2018 in revision). Significant dose-response relationships were observed for various AgNPs, and particle sizes, coatings, and developmental stages contributed to susceptibility to AgNP exposures. Strain differences were also observed.

Human Neuronal Progenitor Cells: We have evaluated AgNP effects on proliferating (day 1) and differentiating (day 1 and 7) human neuronal progenitor cells (hNPCs). Proliferating and differentiating hNPCs at day 1 demonstrated significant dose-response curves after exposures to various AgNPs. Similar to what we have found in the in vitro embryonic midbrain micromass system, the hNPCs study also suggested that particle sizes, coatings, and developmental stages were important contributors to adverse effects of AgNPs.

Future Activities:

We have focused on expanding the applications of our testis co-culture system and in vitro neurotoxicity models to answer this project’s research questions through development of organotypic cultures to evaluate reproductive and developmental toxicity, modification of our previous co-culture system for immature mouse testes, characterization of normal testes development, and evaluation of perturbed conditions using various chemicals including cadmium. We are finalizing our characterization of life-stage specific AOPs for reproductive and developmental toxicity (papers in progress; Wilder et al. 2018). We plan to analyze gene expression for our mouse testis co-culture systems and compare them to our previous rat testis study (Wegner et al. in preparation)

For our mouse midbrain micromass and human neuronal progenitor cells, we plan to calculate benchmark doses for each AgNPs examined in C57BL/6 and A/J mouse micromass system, and compare with other organ systems such as hepatocytes or with other species like human using results from human-derived NPCs. We will finalize our assessment of the benchmark dose of various silver nanoparticles on hNPCs (Park et al 2018 in preparation). We will also investigate toxicological effects and benchmark doses of cadmium on proliferating and differentiating hNPCs and compare results with the ones obtained from mouse testis co-culture system.

References:

Liang S, Yin L, Shengyang Yu K, Hofmann MC, Yu X. High-Content Analysis Provides Mechanistic Insights into the Testicular Toxicity of Bisphenol A and Selected Analogues in Mouse Spermatogonial Cells. Toxicol Sci. 2017 Jan;155(1):43-60. doi: 10.1093/toxsci/kfw178.


Journal Articles on this Report : 6 Displayed | Download in RIS Format

Other subproject views: All 35 publications 6 publications in selected types All 6 journal articles
Other center views: All 127 publications 34 publications in selected types All 33 journal articles
Type Citation Sub Project Document Sources
Journal Article Harris S, Hermsen SA, Yu X, Hong SW, Faustman EM. Comparison of toxicogenomic responses to phthalate ester exposure in an organotypic testis co-culture model and responses observed in vivo. Reproductive Toxicology 2015;58:149-159. R835738 (2016)
R835738 (2017)
R835738C004 (2015)
R835738C004 (2017)
R834514 (Final)
R834514C003 (2015)
R834514C003 (Final)
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  • Journal Article Harris S, Wegner S, Hong SW, Faustman EM. Phthalate metabolism and kinetics in an in vitro model of testis development. Toxicology in Vitro 2016;32:123-131. R835738 (2016)
    R835738 (2017)
    R835738C004 (2015)
    R835738C004 (2016)
    R835738C004 (2017)
    R834514 (Final)
    R834514C003 (Final)
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  • Journal Article Harris S, Shubin SP, Wegner S, Van Ness K, Green F, Hong SW, Faustman EM. The presence of macrophages and inflammatory responses in an in vitro testicular co-culture model of male reproductive development enhance relevance to in vivo conditions. Toxicology In Vitro 2016;36:210-215. R835738 (2016)
    R835738 (2017)
    R835738C004 (2016)
    R835738C004 (2017)
    R834514 (Final)
    R834514C003 (Final)
  • Full-text from PubMed
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  • Journal Article Wegner SH, Yu X, Pacheco Shubin S, Griffith WC, Faustman EM. Stage-specific signaling pathways during murine testis development and spermatogenesis: a pathway-based analysis to quantify developmental dynamics. Reproductive Toxicology 2015;51:31-39. R835738 (2016)
    R835738 (2017)
    R835738C004 (2015)
    R835738C004 (2017)
    R834514 (2015)
    R834514 (Final)
    R834514C003 (2015)
    R834514C003 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
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  • Journal Article Weldon BA, Park JJ, Hong S, Workman T, Dills R, Lee JH, Griffith WC, Kavanagh TJ, Faustman EM. Using primary organotypic mouse midbrain cultures to examine developmental neurotoxicity of silver nanoparticles across two genetic strains. Toxicology and Applied Pharmacology 2018 (April 17), 10 pp. [epub ahead of print]. R835738 (2017)
    R835738C004 (2017)
    R835738C005 (2017)
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  • Journal Article Weldon BA, Griffith WC, Workman T, Scoville DK, Kavanagh TJ, Faustman EM. 2018. In vitro to in vivo benchmark dose comparisons to inform risk assessment of quantum dot nanomaterials. Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology 2018;10(4):e1507. R835738 (2017)
    R835738C004 (2017)
    R835738C005 (2017)
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  • Supplemental Keywords:

    Reproductive and Developmental Toxicity, Chemical Screening, testicular development, in vitro model

    Relevant Websites:

    Predictive Toxicology Center Exit

    Progress and Final Reports:

    Original Abstract
  • 2015 Progress Report
  • 2016 Progress Report
  • Final

  • Main Center Abstract and Reports:

    R835738    Predictive Toxicology Center for Organotypic Cultures and Assessment of AOPs for Engineered Nanomaterials

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R835738C001 Airway Epithelium Organotypic Culture as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
    R835738C002 Organotypic Model of Human Kidney as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
    R835738C003 Organotypic Models of Mammalian Liver as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
    R835738C004 Organotypic Model of Testis as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
    R835738C005 Integrating Liver, Kidney and Testis Nanomaterial Toxicity using the Adverse Outcome Pathway Approach