This project will establish an in vitro test for the influences of oil/dispersed oil and breakdown products on long-term health; guiding safer, more effective oil spill contingency plans. The in vitro tests established will be amenable to high throughput modification and go beyond acute cytotoxicity testing to predict life-long consequences of oil/dispersant on different higher life forms in the environment.
Research Theme #5 Goal: To utilize species-specific stem cells in high-throughput in vitro models to study potential adverse effects of oil/dispersant fractions on economically burdensome health issues originating in fetal development in humans and sentinel species (terrestrial, marine & estuarine).
Specific Objectives: To apply the new technology of induced pluripotent stem cells (iPSCs) to address critical barriers in the study of health in marine, estuarine and terrestrial animals; specifically, to bypass the inaccessibility, slow rate and cost of rigorous scientific studies in vivo via high throughput in vitro assays to test the “fetal origins of adult disease” scenario. Fetal development is especially sensitive to the impacts of environmental exposures. The project focus will be on the adverse impacts of oil/dispersant fractions and their metabolites (e.g. PAHs) on differentiation, metabolism and reproductive capacity (going beyond cytotoxicity studies). For this, the research team has developed novel pig and phylogenetically linked pygmy sperm whale (PSW) iPSCs and mouse embryonic stem cells (ESCs) that can served as high-throughput models to complement conventional in vivo animal models to test oil/dispersant exposure during development. Whale (marine mammal) and alligator (estuarine vertebrate) exhibit differing metabolism of environmental contaminants accumulated in adipose tissue when compared to terrestrial mammals. As a proof of principle, the research team compared iPSCs and ESCs from these organisms in a standard adipogenic differentiation assay. Preliminary results indicate that adipogenesis of PSW iPSCs is quite distinct from pig, which will guide new avenues in toxicity testing by comparing and understanding the mechanisms of adipogenesis in different species. This leads us to:
Hypothesis: Molecular and cellular responses of species-specific stem and primary cells following exposure to oil/dispersant fractions (and their metabolites) will be useful predictors of organism-specific/common developmental and adult pathologies that extend beyond simple “cell death” assessment.
In this proposal, the team plans to utilize ESCs, iPSCs and primary cells in high-throughput in vitro systems to identify and study the impacts and mechanisms of oil/dispersant compounds on cellular homeostasis and differentiation. Further, in vivo animal model systems will be used to validate these systems through the following Research Objectives:
Research Objective 1: To establish an in vitro model to evaluate the impacts of oil/dispersed oil on stem/iPSC adipogenic programming & differentiation. This will examine the influences of oil/dispersed oil on epigenetic programming and differentiative capacities in stem cells. Part 1A will study epigenetic programming by measuring adipogenic chromatin remodeling, gene expression, cellular stress & energetic changes. This will be performed after cytotoxicity testing of oil/dispersed oil fractions in iPSCs, since very limited data is available for these higher organisms. Part 1B will examine the influences of oil/dispersant on adipogenic differentiation of preadipocytes from pig (terrestrial) and PSW (marine) iPSCs and American alligator (estuarine) fetal tissue. The goal of this aim is to go beyond toxicity testing of oil/dispersed oil fractions to look at impacts on fetal origins of adult diseases.
Research Objective 2: To validate the in vitro systems in vivo for development of safer dispersants. Mouse and alligator embryos and pregnant females will be exposed to oil/dispersed oil sub-fractions and/or identified compounds. Influences on adipogenesis and sexual development (newborns/juveniles) and adult health (mice) will be quantified. Due to cost and time, only the most promising results from Objective 1 will be considered. The goal is to determine if the in vitro model established in Objective 1 can lead to high throughput testing methods for the development of safer/more effective dispersants.