Investigating the effect of oil spills
on the environment and public health.
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Funding Source: Year 3-5 Investigator Grants (RFP-II)

Project Overview

Using Embryonic Stem Cell Fate to Determine Potential Adverse Effects of Petroleum/Dispersant Exposure

Principal Investigator
Medical University of South Carolina
Marine Biomedicine and Environmental Sciences
Member Institutions
College of Charleston, Medical University of South Carolina


Dr. Demetri Spyropoulos at the Medical University of South Carolina (MUSC) was awarded an RFP-II grant at $1,148,706 to conduct the RFP-II project titled, “Using Embryonic Stem Cell Fate to Determine Potential Adverse Effects of Petroleum/Dispersant Exposure”.  The project consisted of 1 institution (MUSC), 1 principal investigator (Spyropoulos), 3 co-PIs (Baatz, Guillette, Kohno), 1 post-doc (Sifuentos Romero), 6 graduate students (Bernhard, Cantu, Cloy-McCoy, McNAbb, Temkin, Williams), and participation by several research technicians and graduate students.


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).


Overall Summary: RFP-II studies have revealed multiple obesogenic, estrogenic and other hormone disrupting activities either as specific COREXIT dispersant components or as MC252 oil components made bioavailable by dispersant. Most noteworthy is the DOSS dispersant component, which is a potent ligand for the master regulator of stem cells to a fat cell fate (at the expense of bone formation), and drives glucose intolerance and chronic inflammation. Biomarkers for these exposures persist long after DOSS exposure. Biomarkers of the inflammatory pathway driven by DOSS are central to the incidence and prognosis in many cancers. DOSS is commonly found in various foods and personal care products (e.g. in the definition of “homogenized milk”), and is used as a stool softener by 35% of pregnant women (and fetuses carry the lion’s share of impressionable stem cells). Women who take DOSS stool softeners during pregnancy produce breast milk with more DOSS than non-users and importantly, produce less nutritional milk as indicated by biomarker analysis. Therefore, the relative exposure and total exposure to at-risk oil spill cleanup crews is of importance.


 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.


Research Highlights  


RFP-II funded research resulted in 4 Masters degrees, 3 PhDs, 57 outreach products, 8 peer-reviewed publications and 12 datasets submitted to the GoMRI Information and Data Cooperative (GRIIDC), which are/will be available to the public.  Significant outcomes of his research, according to GoMRI Research Theme 5 are highlighted below, with a listing of the most recent publications.


 Our PPARγ and RXRα ligand binding studies have shown very striking results, that suggest that a) these ligand-binding assays can be used to identify obesogenic fractions and b) that dispersed oil and COREXIT alone (EC9500A) carry ligand-binding activities.  For example, while Rosiglitazone (positive control) and COREXIT are additive for PPARγ ligand activity, Tributyltin (positive control) and COREXIT are multiplicative for RXRα ligand activity.  We are currently studying this mechanism. We are also fractionating and solubilizing oil by different methods and studying specific surfactant/dispersant and solvent components of COREXIT to identify ligand binding agents.


We are testing “fat gene reporter” systems, which are molecular switches to speed-up/streamline and improve quantitation (fluorescence/luminescence) of our stem cell to fat cell (obesogen) detection methods (for oil fractions). Reporters with the PPARγ response element (PPRE) and C/EBP  response elements regarding speed of response and quantitative reliability relative to functional assays.


•  Gene Expression Analyses: To characterize cells at each stage of the differentiation process and molecularly quantify obesogenic response, RNA was isolated from alligator and human stem cells cultured under MSC-supporting conditions, preadipocyte culture and cells treated with oil/dispersant mixtures and subsequently differentiated into adipocytes (early and late time points).  cDNA has been generated to be used for quantitative real-time PCR (qRT-PCR) and primer optimization is currently underway.  For this study, well characterized human mesenchymal stem cells (huMSCs) were obtained from Texan A&M. At this time, qRT-PCR results indicate that huMSCs express well characterized MSC-related markers (CD90, CD105 and CD44; with the CD105 marker showing the highest dynamic range), preadipocytes express genes associated with late-stage MSCs/early adipocytes (FSTL1, CD90 and Leptin; with the FSTL1 marker showing the best results) and adipocytes express mature adipocyte markers (AdipoQ, FABP4 and Leptin; with the Leptin marker showing the best results).


•  Obesogen Testing: Dilution curves (first conducted on Pygmy sperm whale and American alligator cells) to establish optimal conditions for stem cell to fat cell induction were applied to human stem cells. (i) Several dilutions of the standard “adipogenic induction cocktail” were tested and two chosen: one that only slightly induced fat cell fate over background (to test for obesogens) and one that more significantly induced fat cell fate (to test for anti-obesogens); (ii) Dose dependent increases in fat cell induction were observed with the pharmaceutical grade obesogen, Rosiglitazone up through 1uM concentrations (50% induction at 100nM, consistent with what has been published for other cell types).  The rate of adipogenesis varied with passage number when preadipocytes were generated for more rapid readout experiments. We are currently determining conditions for both optimal readout and sensitivity to potential obesogens. Cell responsiveness to the alternate pathway environmental obesogen tributyltin are also being assayed.


•  Animal Model Validation: We are performing pilot experiments with positive control obesogens Rosiglitazone (Rx) and Tributyltin (environmental) on our animal models (pregnant mice carrying fat gene reporters and American alligator eggs) to make correlations between in vitro stem cell culture results and in vivo exposure conditions. Rosiglitazone is being used in conjunction with transgenic mice that have been equipped with the fat gene reporter system; thus, changes in responsiveness anywhere in the body can be assessed. Regarding the alligator egg model, pilot experiments indicate that tributyltin is highly embryotoxic at levels deemed ‘moderate’ in rodent animal models. Alternate doses and dosing schemes are currently being considered.


(Temkin, Bowers, Bowden, Baatz & Spyropoulos) We determined if putative obesogens exist within Deepwater Horizon oil or the widely used dispersant COREXIT. Also, we sought to improve the sensitivity of obesogen detection methods to establish a framework for post-toxicological chemical assessment.  Serum-free conditions greatly enhanced sensitivity without reducing specificity in PPARγ transactivation assays. CWAF and COREXIT were found to have significant dose-dependent PPARγ transactivation activities.  From SPE, the 50:50 ethanol:water fraction of CWAF contained this activity and LC-MS indicated major components of COREXIT were present in this fraction.  Molecular modeling predicted several components of COREXIT to be candidate PPARγ ligands.  DOSS was found to be a putative obesogen by transactivation assays, PPAR-driven luciferase induction in vivo and in vitro murine fat cell induction.  Biomarkers of exposure and effect are being pursued in human stem cells, humans and animal models.


(Bernhard, Guillette & Kohno) Obesogens activate the peroxisome proliferator- activated receptor gamma (PPARγ) – retinoid x receptor alpha (RXRα) heterodimer, which together can stimulate adipogenesis. Tributyltin (TBT), a contaminant and agonist for PPARγ-RXRα in mammals, has not been tested in non-mammalian amniotes such as non-avian reptiles.  The American alligator has been investigated for endocrine disruption as a sentinel species, because it exhibits temperature-dependent sex determination (TSD), which is very sensitive to endocrine signaling during development.  To investigate the effects of developmental exposure to obesogen, alligators were exposed in ovo to TBT prior to the thermosensitive period for TSD.  Toxicity and a hatchout delay were observed in TBT-treated eggs, but no effects of the obesogen exposure have been observed.  In vitro transactivation assays for alligator PPARγ- RXRα are underway. These results will reveal whether the American alligator can serve as a good sentinel species for the long-term impacts of oil spill contamination in estuarine habitats.


(McNabb, Guillette & Kohno) Estrogen signals also play a critical role in TSD.  A single exposure to exogenous estrogen leads to sex reversal and skewed sex ratios by inducing ovarian development at a male-producing temperature.  The alligator is potentially an excellent sentinel species because it is an apex predator that does not migrate far from its estuarine habitat during its long lifetime.  We found that COREXIT 9500 could potentially be an alligator estrogen using an in vitro estrogen receptor 1 (ESR1) reporter gene assay.  To pursue this, alligator eggs were exposed to COREXIT 9500 at 0.25, 2.5 and 25 mg/kg egg weight during TSP to investigate the potential endocrine disruption and effects on gonadal development in ovo.  Early findings indicated that exposure to COREXIT in ovo at these doses did not affect viability at hatch-out.  We are now investigating this more thoroughly by interrogating specific components of COREXIT and fractions generated by our team members.


(Temkin, Bowers, Bowden, Baatz & Spyropoulos) In addition to identifying DOSS as a potent peroxisome proliferator-activated receptor-γ (PPARγ) agonist and driver of adipogenesis in vitro (published) and in mice in vivo (manuscript in preparation) we determined that Span80 substantially activates retinoid X receptor-α (RXRα) and drives adipogenesis in vitro (manuscript under review for publication). Thus, both arms in the master regulation of adipogenesis are stimulated by components of COREXIT.  Mixing experiments indicate greater than an additive effect for these compounds when combined, further bolstering the notion that COREXIT is likely to be obesogenic.  Through collaboration with Drs. Vijay John and Srini Raghavan, we analyzed their two-component dispersant (LT), which shows reduced obesogenic activity compared to COREXIT.  This demonstrates proof of principle that dispersants without endocrine disrupting activity can be developed.  Our SURP summer student Caitlin Sojka further found DOSS to have activity on other steroid hormone receptors.


(Cantu, Spyropoulos, Bowden) Developed new method to monitor eicosanoid (inflammatory/anti-inflammatory bioactive lipids) in non-traditional species; used to investigate chorioallantoic membrane in oviparous species.  Propose using this model as a sentinel for placental function in mammalian species.  Submitted manuscript detailing method in January 2016 to General and Comparative Endocrinology. Investigated eicosanoid milieu in stem cells exposed to DOSS and Rosiglitazone, data in progress; however, preliminary results indicate that DOSS and Rosi alter the extracellular signals that iPSC cells express in media.  Examined plasma based biomarkers associated with pansteatitis in Mozambique tilapia and published manuscript detailing these biomarkers in PLoS One.   Accepted February 25th, 2016.


(McNabb, Guillette & Kohno) Identified Estrogenic and obesogenic activity in the American alligator and Diamondback terrapin.  Long-term effects of oil and dispersants as endocrine disrupting contaminates (EDCs) has not been evaluated in wildlife.  Using nuclear hormone receptor transactivation assay with alligator and terrapin estrogen receptor 1 (ESR1), ESR2, PPARγ and RXRα, EDCs activities of COREXIT EC9500A and CWAF (chemical-enhanced water accommodated faction of crude oil with COREXIT) were identified in the wildlife.  These activities are under functional evaluation with experiment in ovo and in vitro.  Nicole A. McNabb conducted a project investigating the endocrine activities of COREXIT in the Diamondback terrapin and Daphnia magna. Cloned and sequenced estrogen receptors (ESR1 and ESR2) from Diamondback terrapins (Malaclemys terrapin) and used transactivation assays to determine activity by COREXIT on these terrapin receptors to compare to prior results obtained on alligator receptors. Performed Daphnia toxicity tests (immobilization, reproduction, and short-term screening method for juvenile hormone activity) using COREXIT.  Publication currently in progress.


(Williams, Guillette & Kohno) Cameron E. Williams performed in vitro transactivation assays to examine the effects of CWAF exposure on estrogen receptors (ESR1 and ESR2) in the American alligator, Alligator mississippiensis. Examined gonadal differentiation during in vitro exposure to CWAF in the American alligator.  Gonad-adrenal-mesonephos complexes were dissected and cultured with exposure to E2 or CWAF to analyze the estrogenic effects of CWAF on sex determination and differentiation.  SURP student, Amelia G. Brumbaugh, conducted a project entitled ‘Obesogenic Activity of Oil Dispersant Components in the American Alligator (Alligator mississippiensis) Following the Deepwater Horizon Oil Spill’.


We are now investigating this more thoroughly by interrogating specific components of COREXIT and fractions generated by our team members.  Advanced LC MS/MS method to measure DOSS in urine (Cantu, Temkin and Bowden)


(Temkin, Bowers, Bowden, Baatz & Spyropoulos) PhD candidate Lexi Temkin is now finalizing a manuscript for submission, which presents a multimodal approach to demonstrating that DOSS is a bona fide obesogen in vivo. Apart from the physical manifestations (phenotypes of increased adiposity, decreased bone mineral density, glucose intolerance, etc.), the most compelling evidence comes in the form of “Post- Exposure Persistent Biomarkers” (PEPBs); In particular, blood-derived molecules indicative of obesity, diabetes and chronic inflammation that persist several months after the culmination of DOSS exposure. PEPBs from mouse exposure studies and other PEPBs are now being compared in biological fluids (blood, urine, breast milk, cord blood) from humans. Lexi is following this by pursuing the epigenetic changes in the genome that are linked to these PEPBs and, excitingly looking into “antidotal” exposures that might reverse these epigenetic changes.


(Cantu, Spyropoulos, Bowden) PhD candidate Theresa Cantu is completing her work on human induced pluripotent stem cell (hiPSC) exposures to obesogens to complement her work on chicken and alligator egg exposures. We use iPSCs as a rapid in vitro surrogate for studying the Developmental Origins of Health and Disease. DOSS exposure in particular induces a number of lipidomic, epigenetic and gene expression changes. And Theresa has focused on correlating gene expression changes to inflammatory lipid changes. Looking at 103 eicosanoids, Theresa has zeroed in on those most reproducibly changed and associated with chronic (unresolved) inflammation; many of which are now highly associated with obesity, diabetes and cancers.


(Temkin, Cantu, Boggs and Bowden) We are now interrogating specific components of COREXIT in biological samples. Advanced LC MS/MS method to measure DOSS in urine, blood and breast milk.


Other Products Or Deliverables:


1) Public education and outreach linkages: Previous GoMRI RFP-II funding established multiple routes for education and outreach. These include 1) weekly CEA- STEM (Comparative Embryonic Approaches to Stem Cell Research) meetings; 2) bi- weekly REACH (Reproduction, Endocrinology and Childhood Health) meetings; and 3) MOES (Marine Organism and Environmental Sciences) Journal Clubs have brought together Medical University clinicians, researchers and students, College of Charleston faculty and students and NOAA/NIST/DNR personnel for stimulating scientific interactions and an enhanced resource base. MUSC graduate student outreach includes:


2) Presidential Scholars Program, partnered with the Low Country Pregnancy Center to aid underserved pregnant women make healthy choices; 2) the Graduate Students Reaching out With Science (GROWS) program for public school children; 3) Kiawah Island Nature Conservancy and Environmental Sciences Committee for the Board of Directors and citizens involved in wildlife ecology; 4) Society for Advancement of Chicanos and Native Americans in Science; 5) The Low Country Paddlers Club; 6) various local and national news stories and websites featuring RFP-II study findings (e.g. Huffington Post - Can Oil Spills Make You Fat?).

3) Diversity initiatives: This proposal will support all students of diverse race, ethnicity, socioeconomic status, sex and gender preference..


4) Leveraged Funding based on GoMRI research results, two NIH R01 and one NCI R21 grant applications (PI Spyropoulos) are under review by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) and NCI. MUSC SCTR Leveraged Funding Project is underway: “The Obesogenic Potential of DOSS: a Novel Low-Risk/High-Benefit Approach to Managing Neonatal Obesity.” 6/1/2016-5/31/17

PDF Proposal Abstract

This research was made possible by a grant from BP/The Gulf of Mexico Research Initiative.