Project Overview

Summary:

Dr. Danielle McDonald at the University of Miami’s Rosenstiel School of Marine and Atmospheric Science, was awarded an RFP-VI grant at $518,043 to conduct the RFP-VI project titled, “The Impact of Deepwater Horizon Oil Exposure on the Vertebrate Stress Response”. The project consisted of 1 principal investigator (McDonald); 2 post-doctorates (Matthew Alloy, Maria Cartolano); 3 PhD students (John Sebastiani, Rumya Sundaram, Maria Cartolano); 2 master’s students (Emily Milton, Jenna Shroyer); 3 undergraduate students (Anastasiya Plotnikova, Anna Barnard, Natalia Ruiz-Huidobro); and 1 high school student (Leandra Toledo).

The proposed work addresses GoMRI Research Theme 3: Environmental effects of the petroleum/dispersant system on the sea floor, water column, coastal waters, beach sediments, wetlands, marshes, and organisms; and the science of ecosystem recovery, with the overall objectives of elucidating the mechanism(s) by which exposure to DWH oil may be interfering with the marine vertebrate stress response and determining the trajectory of recovery. The central hypothesis of this proposal is that there are multiple pathways by which PAHs found in DWH oil may disrupt the marine vertebrate stress response, due to the complexity of both the stress response and the oil mixture, and that recovery will be pathway-dependent.

The central hypothesis is based on past and current research that suggests PAHs found in oil interfere with the typical vertebrate stress response, an adaptive response that serves to promote survival and restore physiological systems to homeostasis. In marine vertebrates, exposure to pollutants typically results in release of the steroid glucocorticoid hormone, cortisol, into the circulation. However, some studies have shown that circulating cortisol does not increase in fish chronically exposed to PAHs. Instead, fish in contaminated environments have reduced cortisol levels at capture compared to fish collected from clean environments, suggesting a disruption in the complicated endocrine axis that regulates cortisol biosynthesis. Exemplifying this issue is the recent finding that bottlenose dolphins captured in Barataria Bay, Louisiana, an area that received substantial oiling from DWH over an extended period of time, have significantly reduced circulating cortisol levels in response to capture stress compared to dolphins captured from Sarasota Bay, Florida, where no oil was observed following the DWH spill. While studies on fish and marine mammals support a disruption in cortisol biosynthesis, the effect of PAHs on the stress response is complicated with other studies showing significant increases in plasma cortisol in response to acute and chronic PAH exposure.

On this background, the ultimate goal of the proposed work is to develop a greater understanding of DWH oil-induced toxicity and ascertain the extent to which the combination of PAHs in DWH oil interferes with the vertebrate stress response. We have developed an Adverse Outcome Pathway (AOP), a mechanism-based model for ecological risk assessment, to inform the scientific questions that will be addressed in the present proposal. The AOP for this proposal links molecular initiating events (MIEs; processes that control cortisol biosynthesis and receptor levels) that may be used as sensitive indicators of DWH oil exposure and predictors of exposure consequences to apical endpoints at the level of the individual (effects on metabolism and behavior) and population (effects on abundance and diversity). Uncertainty in the proposed AOP linkages form the basis for the scientific questions that will be addressed.

To fully assess the impacts of DWH oil on the vertebrate stress response and the trajectory for recovery, the proposed activities integrate molecular, cellular, gland/organ and whole animal physiological and behavioral studies using a benthic marine teleost, the Gulf toadfish, a resident of the Gulf of Mexico. These studies will evaluate multiple pathways (e.g., pituitary fatigue, reduced receptor sensitivity, disruption or overstimulation of cortisol biosynthesis) and the consequence of these changes at the whole animal level (e.g., changes in carbohydrate metabolism, immune capacity and responses to natural stresses such as predation and social interaction). These pathways and endpoints will be evaluated in both short- and long-term exposures to DWH oil and individual PAH constituents. Each exposure will be followed by a complementary recovery period (i.e., no oil or PAH exposure) to assess the recovery trajectory.

The PI has experience with oil toxicity studies on toadfish from efforts during 2013-2015 associated with the Natural Resource Damage Assessment (NRDA). The proposed activities are based on findings from these NRDA related efforts and are an outgrowth of endpoints and AOPs that would not normally be examined in NRDA testing. Dr. McDonald and her team are well-positioned to carry out the proposed research as she has established facilities and extensive experience in marine fish molecular biology, neuroendocrinology, transport and binding kinetics, pharmacology, and whole animal physiology and behavior. In particular, Dr. McDonald is an expert in stress physiology and has authored >20 publications on the stress response and the role of cortisol in the control of fish physiology and behavior. The majority of the methodologies used in the proposed work have been applied successfully by McDonald and her team, ensuring that the proposed work can be completed successfully.

The proposed research is innovative as it will be the first to determine the effect of DWH oil on the many different and integrated components of the stress response. We expect that experimental outcomes will greatly contribute to our overall understanding of the mechanism(s) of DWH oil toxicity on the stress response of marine fish and will allow us to ascertain the trajectory of recovery. Given the conservation in the stress response between marine mammals and fish, studying the impact and mechanism of toxicity of DWH oil on the stress response of marine fish, which can be easily obtained and manipulated experimentally, will also contribute to our understanding of the mode of action in marine mammals, which are of great public concern.

Research Highlights

Dr. McDonald’s research, which included 80 outreach products and activities, 7 scientific conference presentations and 11 datasets submitted to the GoMRI Information and Data Cooperative (GRIIDC), which are available to the public.  Additionally, at least 4 peer-reviewed publications are currently in various stages of publication.  Significant outcomes of their research (all related to GoMRI Research Theme 3) are highlighted below.

• Continuous waterborne exposure to concentrations of up to approximately 3 µg PAH₅₀/L using a flow-through system designed to maintain concentrations for 28 days did not result in a stimulation of the hypothalamic-pituitary-interrenal (HPI) axis, the endocrine axis responsible for cortisol production and secretion, indicating that toadfish did not perceive oil exposure as a stressor. Similarly, there was no evidence supporting HPI axis hyperactivation or pituitary atrophy in response to a 28 days of oil exposure.
    
• Continuous exposure to environmentally realistic PAH concentrations for 7 days did result in aryl hydrocarbon receptor (AhR) activation and CYP1A induction throughout the HPI axis (i.e., in the pre-optic area (POA) of the hypothalamus, the pituitary and the interrenal cells of the kidney).  CYP1A mRNA expression was elevated in the hypothalamic POA within 4 h of exposure to PAH concentrations of 0.06 µg PAH₅₀/L and in the pituitary and kidney at concentrations of approximately 3 µg PAH₅₀/L.  This elevation persisted for the entire 7-day exposure period and did not return to control concentrations until 1 to 3 days into recovery.  When a second, 28-day exposure was completed, fish that were exposed to approximately 3 µg PAH₅₀/L showed AhR activation and CYP1A induction throughout the hypothalamus and the kidney.  CYP1A mRNA expression was elevated at 28 days exposure with CYP1A mRNA expression returning to control levels by 7 days into recovery (the first time point measured).
  
  
• Despite an early stimulation of the AhR, a significant inhibition in the ability of toadfish to mount a stress response to an acute stressor was only measured after 7-days of recovery from a 7-day exposure to PAH concentrations.  We believe that there were issues with our experimental system, namely that toadfish were experiencing a chronic stressor unrelated to PAHs throughout the exposure, that may have contributed to inconsistencies and variability in the data.
  
  
• Using a more refined experimental approach that better controlled for the chronic stressors that toadfish may have experienced in our experimental system, we determined that there is an inhibition of cortisol secretion at the level of the kidney after exposure to PAH concentrations of 4 µg PAH₅₀/L for 7 days.  This inhibition does not persist - secretion of cortisol by kidney from PAH-exposed fish is slower than controls after 30 and 60 min but is able to catch up within 120 min.  Reflecting this delay in cortisol secretion and not a complete inhibition, cortisol concentrations in vivo in PAH-exposed fish are not significantly different that controls.  PAH exposure tends to cause a downregulation in the mRNA expression of two important proteins, StAR and P450scc, involved in the production of cortisol. Analysis of whether there is also a downregulation of the melanocortin 2 receptor (MC2R), a receptor that mediates a stimulation of cortisol secretion, or decreases in cholesterol availability within the kidney is ongoing (delayed due to the pandemic). Furthermore, PAH exposure causes an inhibition in serotonin-mediated cortisol secretion by kidneys  – serotonin being a neurochemical that is important with respect to behavior and social interactions in fish. We also tested whether the impacts of PAH exposure on the stress response are exacerbated by exposure to chronic stress.  We found this not to be the case; however, fish that are chronically stressed and PAH exposed have significantly lower circulating serotonin concentrations than stressed controls.
  
  
• With respect to whole animal endpoints, AhR activation and CYP1A induction occurred in liver within 4 h of exposure to PAH concentrations of 2.82 µg PAH₅₀/L, elevation persisted for the entire 7-day exposure period and did not return to control concentrations until 1-day into recovery.  PAH concentrations of 0.06 µg PAH₅₀/L and lower did not activate the AhR demonstrating that the hypothalamic-POA may be more sensitive than the liver in terms of CYP1A induction.   Despite the initiation of the molecular response, there were no changes in blood and liver endpoints (e.g., plasma glucose, plasma cholesterol, red blood cell or white blood cell counts, liver glycogen, liver cholesterol, HSI) at these low, environmentally realistic concentrations.  However, there was a significant inhibition in plasma osmolality after 7 days of recovery from a 7-day exposure. When a second, 28-day exposure was completed, fish that were exposed to 3 µg PAH₅₀/L did not show AhR activation at the level of the liver.  Furthermore, there were still no changes in blood and liver endpoints (e.g., red blood cell or white blood cell counts, differential white blood cell counts, liver cholesterol, HSI).  Analysis of plasma glucose, liver glycogen, and plasma osmolality is ongoing (delayed due to the pandemic).

 Proposal Abstract - RFP-VI PI M. Danielle McDonald