Later Life Consequences of Subteratogenic Exposure to a Complex PAH Mixture in the Atlantic Killifish (Fundulus heteroclitus)

Subteratogenic and other low-level chronic exposures to toxicant mixtures are an understudied threat to environmental and human health. It is especially important to understand the effects of these exposures for contaminants, such as polycyclic aromatic hydrocarbons (PAHs) a large group of more than 100 individual compounds, which are important environmental (including aquatic) contaminants. Aquatic sediments constitute a major sink for hydrophobic pollutants, and studies show PAHs can persist in sediments over time. Furthermore, estuarine systems (namely breeding grounds) are of particular concern, as they are highly impacted by a wide variety of pollutants, and estuarine fishes are often exposed to some of the highest levels of contaminants of any vertebrate taxon. Acute embryonic exposure to PAHs results in cardiac teratogenesis in fish, and early life exposure to certain individual PAHs and PAH mixtures cause heart alterations with decreased swimming capacity in adult fish. Consequently, the heart and cardiorespiratory system are thought to be targets of PAH mixture exposure. While many studies have investigated acute, teratogenic PAH exposures, few studies have longitudinally examined the impacts of subtle, subteratogenic PAH mixture exposures, which are arguably more broadly applicable to environmental contamination scenarios. The goal of this dissertation was to highlight the later-life consequences of early-life exposure to subteratogenic concentrations of a complex, environmentally relevant PAH mixture.

A unique population of Fundulus heteroclitus (the Atlantic killifish or mummichog, hereafter referred to as killifish), has adapted to creosote-based polycyclic aromatic hydrocarbons (PAHs) found at the Atlantic Wood Industries (AW) Superfund site in the southern branch of the Elizabeth River, VA, USA. This killifish population survives in a site heavily contaminated with a mixture of PAHs from former creosote operations. They have developed resistance to the acute toxicity and teratogenic effects caused by the mixture of PAHs in sediment from the site. The primary goal of this dissertation was to compare and contrast later-life outcomes of early-life, subteratogenic PAH mixture exposure in both the Atlantic Wood killifish (AW) and a naïve reference population of killifish from King’s Creek (KC; a relatively uncontaminated tributary of the Severn River, VA). Killifish from both populations were exposed to subteratogenic concentrations of a complex PAH-sediment extract, Elizabeth River Sediment Extract (ERSE), made by collecting sediment from the AW site. Fish were reared over a 5-month period in the laboratory, during which they were examined for a variety of molecular, physiological and behavioral responses.

The central aims of my dissertation were to determine alterations to embryonic gene expression, larval swimming activity, adult behavior, heart structure, enzyme activity, and swimming/cardiorespiratory performance following subteratogenic exposure to ERSE. I hypothesized that subteratogenic exposure to ERSE would impair cardiac ontogenic processes in a way that would be detectable via gene expression in embryos, and that the misregulation of cardiac genes would help to explain activity changes, behavioral deficits, and later-life swimming deficiencies. I also hypothesized that fish heart structure would be altered. In addition, I hypothesized that the AW killifish population would be resistant to developmental exposures and perform normally in later life challenges. To investigate these hypotheses, a series of experiments were carried out in PAH-adapted killifish from Elizabeth River and in reference killifish. As an ancillary project to the primary aims of the dissertation, I examined the toxicity of weaker aryl hydrocarbon receptor (AHR) agonists in combination with fluoranthene (FL), an inhibitor of cytochrome P4501A1 (CYP1A1). This side project was conducted in both Danio rerio (zebrafish) and the KC and AW killifish.

Embryonic gene expression was measured in both killifish populations over an ERSE dose response with multiple time points (12, 24, 48, and 144 hours post exposure). Genes known to play critical roles in cardiac structure/development, cardiac function, and angiogenesis were elevated, indicating cardiac damage and activation of cardiovascular repair mechanisms. These data helped to inform later-life swimming performance and cardiac histology studies. Behavior was assessed during light and dark cycles in larvae of both populations following developmental exposure to ERSE. While KC killifish showed activity differences following exposure, AW killifish showed no significant changes even at concentrations that would cause overt cardiac toxicity in KC killifish. Juvenile behavior experiments demonstrated hyperactivity following ERSE exposure in KC killifish, but no significant behavioral changes in AW killifish. Adult swimming performance via prolonged critical swimming capacity (Ucrit) demonstrated performance costs in the AW killifish. Furthermore, swimming performance decline was observed in KC killifish following exposure to increasing dilutions of ERSE. Lastly, cardiac histology suggested that early-life exposure to ERSE could result in cardiac structural alteration and extravasation of blood into the pericardial cavity.

Responses to AHR agonists resulted in a ranking of relative potency for agonists, and determined which agonists, when combined with FL, caused cardiac teratogenesis. These experiments showed interesting species differences for zebrafish and killifish. To probe mechanisms responsible for cardiotoxicity, a CYP1A-morpholino and a AHR2-morpholino were used to mimic FL effects or attempt to rescue cardiac deformities respectively. Findings suggested that the cardiac toxicity elicited by weak agonist + FL exposure was likely driven by AHR-independent mechanisms. These studies stand in contrast to previous research from our lab showing that moderate AHR agonist + FL caused cardiac toxicity that can be partially rescued by AHR-morpholino knockdown.

My findings will form better characterization of mechanisms of PAH toxicity, and advance our understanding of how subteratogenic mixtures of PAHs exert their toxic action in naïve killifish. Furthermore, these studies will provide a framework for investigating how subteratogenic exposures to PAH mixtures can impact aquatic organismal health and performance. Most importantly, these experiments have the potential to help inform risk assessment in fish, mammals, and potentially humans. Ultimately, this research will help protect populations exposed to subtle PAH-contamination.

Neuroimaging assessment of early and late neurobiological sequelae of traumatic brain injury: implications for CTE.

Traumatic brain injury (TBI) has been increasingly accepted as a major external risk factor for neurodegenerative morbidity and mortality. Recent evidence indicates that the resultant chronic neurobiological sequelae following head trauma may, at least in part, contribute to a pathologically distinct disease known as Chronic Traumatic Encephalopathy (CTE). The clinical manifestation of CTE is variable, but the symptoms of this progressive disease include impaired memory and cognition, affective disorders (i.e., impulsivity, aggression, depression, suicidality, etc.), and diminished motor control. Notably, mounting evidence suggests that the pathology contributing to CTE may be caused by repetitive exposure to subconcussive hits to the head, even in those with no history of a clinically evident head injury. Given the millions of athletes and military personnel with potential exposure to repetitive subconcussive insults and TBI, CTE represents an important public health issue. However, the incidence rates and pathological mechanisms are still largely unknown, primarily due to the fact that there is no in vivo diagnostic tool. The primary objective of this manuscript is to address this limitation and discuss potential neuroimaging modalities that may be capable of diagnosing CTE in vivo through the detection of tau and other known pathological features. Additionally, we will discuss the challenges of TBI research, outline the known pathology of CTE (with an emphasis on Tau), review current neuroimaging modalities to assess the potential routes for in vivo diagnosis, and discuss the future directions of CTE research.

Delimiting species without nuclear monophyly in Madagascar's mouse lemurs.

BACKGROUND: Speciation begins when populations become genetically separated through a substantial reduction in gene flow, and it is at this point that a genetically cohesive set of populations attain the sole property of species: the independent evolution of a population-level lineage. The comprehensive delimitation of species within biodiversity hotspots, regardless of their level of divergence, is important for understanding the factors that drive the diversification of biota and for identifying them as targets for conservation. However, delimiting recently diverged species is challenging due to insufficient time for the differential evolution of characters--including morphological differences, reproductive isolation, and gene tree monophyly--that are typically used as evidence for separately evolving lineages. METHODOLOGY: In this study, we assembled multiple lines of evidence from the analysis of mtDNA and nDNA sequence data for the delimitation of a high diversity of cryptically diverged population-level mouse lemur lineages across the island of Madagascar. Our study uses a multi-faceted approach that applies phylogenetic, population genetic, and genealogical analysis for recognizing lineage diversity and presents the most thoroughly sampled species delimitation of mouse lemur ever performed. CONCLUSIONS: The resolution of a large number of geographically defined clades in the mtDNA gene tree provides strong initial evidence for recognizing a high diversity of population-level lineages in mouse lemurs. We find additional support for lineage recognition in the striking concordance between mtDNA clades and patterns of nuclear population structure. Lineages identified using these two sources of evidence also exhibit patterns of population divergence according to genealogical exclusivity estimates. Mouse lemur lineage diversity is reflected in both a geographically fine-scaled pattern of population divergence within established and geographically widespread taxa, as well as newly resolved patterns of micro-endemism revealed through expanded field sampling into previously poorly and well-sampled regions.

Exploring Mechanisms of Bacterial Adaptation to Seasonal Temperature Change

This research examines three potential mechanisms by which bacteria can adapt to different temperatures: changes in strain-level population structure, gene regulation and particle colonization. For the first two mechanisms, I utilize bacterial strains from the Vibrionaceae family due to their ease of culturability, ubiquity in coastal environments and status as a model system for marine bacteria. I first examine vibrio seasonal dynamics in temperate, coastal water and compare the thermal performance of strains that occupy different thermal environments. Our results suggest that there are tradeoffs in adaptation to specific temperatures and that thermal specialization can occur at a very fine phylogenetic scale. The observed thermal specialization over relatively short evolutionary time-scales indicates that few genes or cellular processes may limit expansion to a different thermal niche. I then compare the genomic and transcriptional changes associated with thermal adaptation in closely-related vibrio strains under heat and cold stress. The two vibrio strains have very similar genomes and overall exhibit similar transcriptional profiles in response to temperature stress but their temperature preferences are determined by differential transcriptional responses in shared genes as well as temperature-dependent regulation of unique genes. Finally, I investigate the temporal dynamics of particle-attached and free-living bacterial community in coastal seawater and find that microhabitats exert a stronger forcing on microbial communities than environmental variability, suggesting that particle-attachment could buffer the impacts of environmental changes and particle-associated communities likely respond to the presence of distinct eukaryotes rather than commonly-measured environmental parameters. Integrating these results will offer new perspectives on the mechanisms by which bacteria respond to seasonal temperature changes as well as potential adaptations to climate change-driven warming of the surface oceans.

Autonomic cardiovascular dysregulation as a potential mechanism underlying depression and coronary artery bypass grafting surgery outcomes.

BACKGROUND: Coronary artery bypass grafting (CABG) is often used to treat patients with significant coronary heart disease (CHD). To date, multiple longitudinal and cross-sectional studies have examined the association between depression and CABG outcomes. Although this relationship is well established, the mechanism underlying this relationship remains unclear. The purpose of this study was twofold. First, we compared three markers of autonomic nervous system (ANS) function in four groups of patients: 1) Patients with coronary heart disease and depression (CHD/Dep), 2) Patients without CHD but with depression (NonCHD/Dep), 3) Patients with CHD but without depression (CHD/NonDep), and 4) Patients without CHD and depression (NonCHD/NonDep). Second, we investigated the impact of depression and autonomic nervous system activity on CABG outcomes. METHODS: Patients were screened to determine whether they met some of the study's inclusion or exclusion criteria. ANS function (i.e., heart rate, heart rate variability, and plasma norepinephrine levels) were measured. Chi-square and one-way analysis of variance were performed to evaluate group differences across demographic, medical variables, and indicators of ANS function. Logistic regression and multiple regression analyses were used to assess impact of depression and autonomic nervous system activity on CABG outcomes. RESULTS: The results of the study provide some support to suggest that depressed patients with CHD have greater ANS dysregulation compared to those with only CHD or depression. Furthermore, independent predictors of in-hospital length of stay and non-routine discharge included having a diagnosis of depression and CHD, elevated heart rate, and low heart rate variability. CONCLUSIONS: The current study presents evidence to support the hypothesis that ANS dysregulation might be one of the underlying mechanisms that links depression to cardiovascular CABG surgery outcomes. Thus, future studies should focus on developing and testing interventions that targets modifying ANS dysregulation, which may lead to improved patient outcomes.

Revisiting the monoamine hypothesis of depression: a new perspective.

As the incidence of depression increases, depression continues to inflict additional suffering to individuals and societies and better therapies are needed. Based on magnetic resonance spectroscopy and laboratory findings, gamma aminobutyric acid (GABA) may be intimately involved in the pathophysiology of depression. The isoelectric point of GABA (pI = 7.3) closely approximates the pH of cerebral spinal fluid (CSF). This may not be a trivial observation as it may explain preliminary spectrophotometric, enzymatic, and HPLC data that monoamine oxidase (MAO) deaminates GABA. Although MAO is known to deaminate substrates such as catecholamines, indoleamines, and long chain aliphatic amines all of which contain a lipophilic moiety, there is very good evidence to predict that a low concentration of a very lipophilic microspecies of GABA is present when GABA pI = pH as in the CSF. Inhibiting deamination of this microspecies of GABA could explain the well-established successful treatment of refractory depression with MAO inhibitors (MAOI) when other antidepressants that target exclusively levels of monoamines fail. If further experimental work can confirm these preliminary findings, physicians may consider revisiting the use of MAOI for the treatment of non-intractable depression because the potential benefits of increasing GABA as well as the monoamines may outweigh the risks associated with MAOI therapy.