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 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.

Approach:

Preparation of 3D Co-Cultures at important windows of development. Testicular cells will be isolated from male rats at postnatal day 5 or postnatal day 18. 

Evaluation of survival and proliferation. Cell death will be evaluated by lactate dehydrogenase (LDH) release assay (Promega) as well as a “3 color” fluorescence assay to simultaneously measure live (calcien AM stained), dead (propidium iodide stained) and total nuclei (hoechst stained). Proliferation will be evaluated through a combination of previously optimized methods, including BrDU Index, and PCNA expression. Ultimately we will develop SOPs for high-throughput measurement of proliferation in 96-well plates using either Luminex beads to

measure PCNA expression or the CyQuant proliferation assay (LifeTechnologies).

Evaluation of tissue differentiation and spermatogenesis. Testicular tissue differentiation and early spermatogenesis can be demonstrated by the expression of protein markers associated with mature Sertoli cells (androgen receptor, FSH receptor), functional Leydig cells (3β-HSD, LH receptor), spermatogonial germ cells (c-kit) and germ cells poised for meiosis (stra8). Progression through spermatogenesis in co-cultures isolated at PND 18 can also be demonstrated by expression of markers of spermatocytes ( sycp3, spo22, RAD51, ) and mature sperm (prm1, prm2). 

Uptake of ENM into 3D-TCS organtypic cultures (dosimetry measurements): Because of the widely varying structures, forms and charges on ENMs it is essential to correct for uptake. We have focused on metals and metal containing or contaminated ENMs for this application and thus we can use some specific techniques. We will work closely with Project 5 to identify critical missing data.

Oxidative stress assessment: Our organotypic cultures will be assessed for both mitochondrial, oxidative and ER dependent responses using RT-PCR probes. These assessments will be done on both of the differentiation states.

We will generate dose-response curves in the testis organotypic cultures for both A/J and C57Bl/6 mice. We will start with the C57Bl/6 mice and will generate these results for the 2 different lifestages, for the various ENM and for both acute and 14 day exposures.

Rationale:

A critical public health question is whether and how organisms respond differentially to environmental insult at different life stages. Nowhere is this question more relevant than with reproductive and developmental toxicity. In our testis cultures we will examine two stages that represent significant lifestages. First, we will examine pre-meiotic testis at a time that represents continued proliferation and early markers and processes that establish the mature testis. Second, we will examine later stages where meiotic processes are in completion. We will determine if ENM differentially affect these two times of testis maturation. This research has direct significance for determining sensitive lifestages for risk protection and will determine if in vitro organotypic cultures where the diverse cell types are present can identify in vivo windows of sensitivity and cellular and molecular targets.

There is significant literature on ENMs and their induction of oxidative stress. We anticipate that there will be significant differences in the early versus late differentiated testis cultures in their response to ENMs, especially when their response is corrected for uptake of the ENM. There are possible differences in uptake following differentiation thus ICP-MS dosimetry will be used to correct for uptake and exposure. In our laboratory oxidative stress has had multiple effects on our cells inducing as well as inhibiting cell differentiation. Metals are also well known for inducing metal response genes MT1/2 as well as oxidative stress during differentiation.

Many ENM have been identified as eliciting an inflammatory response. However, these responses have been highly dependent upon the type of ENM, the tissues examined and the times of assessment. For the testis, the overall role that inflammation may play in reproductive toxicity is just beginning to be recognized and has been proposed for diverse male reproductive toxicants such as phthalates (Di butyl) and for benzo (a) pyrene. These chemicals have been shown to affect macrophage immune-endocrine function and testosterone production but the underlying mechanisms responsible for these effects have yet to be elucidated. It is unclear whether these toxicants directly disrupt testicular macrophages to mediate toxicity or whether macrophage isruption/dysfunction is an intermediate step in the AOP pathway. 

The testis organotypic cultures will allow us to examine the role of inflammation more directly following ENM exposures. ENMs may generate inflammatory responses by initiating innate immune response or via Toll receptor activation as has been reported for QDs and titanium dioxide nanomaterials. Cells that are damaged can release endogenous molecules that can cause inflammation via TLR activation by release of damage-associated molecular patterns (DAMPs). It is also important to remember that our assessments will need to be made in the context of oxidative stress response as we present in H2 as the transcription factor NFkB can be activated by oxidative stress and cause subsequent pro-inflammatory gene transcription.

There is a large literature showing dramatic strain differences in reproductive and developmental toxicity following metal exposures across mouse strains. Preliminary studies from our Nanotoxicology Center has shown that these two strains represent significantly different responses across several metal based ENM responses (immunological-cytokines response in vitro and neutrophilic response-in vivo) and so these were chosen for our initial comparisons across projects.

Expected Results:

We will establish and standardize our operating procedures for Testis Organotypic Cultures for a 14 day time window and at two different lifestages to reflect potential windows of susceptibility for testis. Our output will include an assessment of background responses occurring in vitro over this assessment period and a comparison with in vivo conditions. We will also be able to standard methods for assessing toxicological impact. 

Although metals are of great toxicological interest, only a few have been assessed during Phase 1 and 2 of the published ToxCast Assays. This research will add to the body of knowledge about metal responses in vitro in a target tissue. It will also significantly increase our knowledge of ENM in reproductive tissues in vitro. In collaboration with Project 5 we will have a mathematical model and framework to test relevant AOP responses for a broad array of environmental agents beyond the ENM and their associated metals.

We will produce innovative organ based testing methods and identification of technology transfer ideas across our model systems. We anticipate transfer of RiboTag methodologies by the end of year 2. These efforts will provide new information and methods to inform timely decisions about metals and ENMs.

Publications and Presentations:

Publications have been submitted on this subproject: View all 5 publications for this subprojectView all 74 publications for this center

Journal Articles:

Journal Articles have been submitted on this subproject: View all 4 journal articles for this subprojectView all 13 journal articles for this center

Progress and Final Reports:

2015 Progress Report
2016 Progress Report


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