Browsing by Author "Di Giulio, Richard T"

Modern ecotoxicology draws considerable criticism for its lack of ecological relevance despite its history of interdisciplinary research overlap with the fields of conservation biology and ecology. The overarching aim of this dissertation was to unite the goals of these fields in the context of freshwater pollution by coal-fired power plant (CFPP) effluents, which contribute to the largest point source of environmental pollution in the United States and comprise a substantial threat to receiving ecosystems. CFPPs discharge the by-products of coal combustion, collectively referred to as coal combustion residuals (CCRs), into freshwater rivers and lakes through permitted discharges overseen by the National Pollutant Discharge Elimination System. CCRs are characterized by high concentrations of numerous inorganic elements, many of which are toxic to aquatic organisms. For decades, research on this waste stream has focused on selenium (Se) as an especially toxic trace element for oviparous vertebrates, historically causing severe deformities and species extirpations from which affected ecosystems took decades to recover.

The majority of work described here began with a 2015 field survey of three CFPP-associated lakes in North Carolina from which surface waters, sediments, sediment pore waters, biofilm, zooplankton, and resident fish species were collected and analyzed for their trace element profiles by inductively coupled plasma mass spectrometry (ICP-MS). Initial analyses focusing on Se alone revealed significantly higher tissue liver, muscle, and gonad tissue concentrations in fish from lakes receiving CCR inputs than those from reference systems. At two sites, Mayo Lake and Sutton Lake, water samples and fish tissue concentrations additionally exceeded the US Environmental Protection Agency’s recently revised aquatic life criteria. These analyses were subsequently expanded to consider a more comprehensive suite of ten CCR-associated elements. The results of paired univariate and multivariate analyses of abiotic and biotic compartments for each of the three pairs of lakes showed that CCR-receiving lake sediment pore waters are consistently enriched in manganese, arsenic, selenium, strontium, cadmium, and nickel. From this abiotic compartment, preferential uptake by biological compartments differed among species and among lakes such that only Se was consistently enriched in fish across the three systems. Across all lake pairs, the three fish species differed in their aggregate CCR tissue burdens, with bluegill having the highest burden and largemouth bass having the lowest. A lab-based trophic transfer study of field-collected biofilm and zooplankton to the model fish species, fathead minnow (Pimephalas promelas), supported the hypothesis that diet and trophic position are important mediators of fish tissue trace element composition. CCR loading history, lake size, and water residence time influence the magnitude of trace element accumulation in these systems, with important implications for the legacy of this waste stream as CFPPs are retired and their effluent streams to adjacent water bodies are terminated.

An additional lab-based dietary study of organic selenomethionine exposure sought to describe the bioenergetic consequences of low-level Se exposure to adult zebrafish (Danio rerio) and their offspring. Sex-specific metabolic effects were observed in whole organisms and individual tissue types and metabolic partitioning using the Seahorse Extracellular Flux Analyzer indicated that mitochondrial dysfunction could underlie altered metabolic rates, with implications for the ecological fitness of Se-impacted fish communities. This study also explored maternal and paternal exposure routes of Se exposure to F1 generation embryos and found that both routes of exposure resulted in reduced reserve respiratory capacity relative to control fish.

Inorganic trace elements are incorporated into the lattice of metabolically-stable fish otoliths, providing unique records of fishes’ lifetime exposure history. Two distinct otolith applications are explored here. First, time series otolith concentrations of a subset of CCRs were compared to overlapping time series of CFPP loading data to determine whether changes in system inputs were reflected in otolith uptake. Significant time lags, intra-species variation, and differences among species for Se suggested that otolith chemistry could be reflecting Se input legacies and complex biogeochemical cycling through the food webs from which fish are exposed. In contrast, fish otolith 87Sr/86Sr isotopic ratios did distinguish fish from a CCR-receiving lake in agreement with ratios measured in surface and pore water samples. If this initial proof-of-principle result holds for fish collected from larger reservoirs in which water chemistry is less uniform, otoliths could be powerful biological tracers of freshwater CCR impacts.

In tandem with the decreasing reliance on coal in the US, CFPPs are retiring, excavating their coal ash ponds, and otherwise terminating their CCR effluent streams. As these processes unfold, actively-receiving freshwater ecosystems are undergoing a transition to legacy status. An especially important avenue for future research is how CCR stoichiometry will change over time to reflect the relative transformation, sequestration, and transport of individual elements within and through these recovering systems. The ecotoxicological implications of the legacy CCR waste stream will likely evolve as exposure mixtures change.

Selenium (Se) is a micronutrient necessary for the function of a variety of important enzymes; Se also exhibits a narrow range in concentrations between essentiality and toxicity. Oviparous vertebrates such as birds and fish are especially sensitive to Se toxicity, which causes reproductive impairment and defects in embryo development. Selenium occurs naturally in the Earth's crust, but it can be mobilized by a variety of anthropogenic activities, including agricultural practices, coal burning, and mining.

Mountaintop removal/valley fill (MTR/VF) coal mining is a form of surface mining found throughout central Appalachia in the United States that involves blasting off the tops of mountains to access underlying coal seams. Spoil rock from the mountain is placed into adjacent valleys, forming valley fills, which bury stream headwaters and negatively impact surface water quality. This research focused on the biological impacts of Se leached from MTR/VF coal mining operations located around the Mud River, West Virginia.

In order to assess the status of Se in a lotic (flowing) system such as the Mud River, surface water, insects, and fish samples including creek chub (Semotilus atromaculatus) and green sunfish (Lepomis cyanellus) were collected from a mining impacted site as well as from a reference site not impacted by mining. Analysis of samples from the mined site showed increased conductivity and Se in the surface waters compared to the reference site in addition to increased concentrations of Se in insects and fish. Histological analysis of mined site fish gills showed a lack of normal parasites, suggesting parasite populations may be disrupted due to poor water quality. X-ray absorption near edge spectroscopy techniques were used to determine the speciation of Se in insect and creek chub samples. Insects contained approximately 40-50% inorganic Se (selenate and selenite) and 50-60% organic Se (Se-methionine and Se-cystine) while fish tissues contained lower proportions of inorganic Se than insects, instead having higher proportions of organic Se in the forms of methyl-Se-cysteine, Se-cystine, and Se-methionine.

Otoliths, calcified inner ear structures, were also collected from Mud River creek chubs and green sunfish and analyzed for Se content using laser ablation inductively couple mass spectrometry (LA-ICP-MS). Significant differences were found between the two species of fish, based on the concentrations of otolith Se. Green sunfish otoliths from all sites contained background or low concentrations of otolith Se (< 1 µg/g) that were not significantly different between mined and unmined sites. In contrast creek chub otoliths from the historically mined site contained much higher (≥ 5 µg/g, up to approximately 68 µg/g) concentrations of Se than for the same species in the unmined site or for the green sunfish. Otolith Se concentrations were related to muscle Se concentrations for creek chubs (R2 = 0.54, p = 0.0002 for the last 20% of the otolith Se versus muscle Se) while no relationship was observed for green sunfish.

Additional experiments using biofilms grown in the Mud River showed increased Se in mined site biofilms compared to the reference site. When we fed fathead minnows (Pimephales promelas) on these biofilms in the laboratory they accumulated higher concentrations of Se in liver and ovary tissues compared to fathead minnows fed on reference site biofilms. No differences in Se accumulation were found in muscle from either treatment group. Biofilms were also centrifuged and separated into filamentous green algae and the remaining diatom fraction. The majority of Se was found in the diatom fraction with only about 1/3rd of total biofilm Se concentration present in the filamentous green algae fraction

Finally, zebrafish (Danio rerio) embryos were exposed to aqueous Se in the form of selenate, selenite, and L-selenomethionine in an attempt to determine if oxidative stress plays a role in selenium embryo toxicity. Selenate and selenite exposure did not induce embryo deformities (lordosis and craniofacial malformation). L-selenomethionine, however, induced significantly higher deformity rates at 100 µg/L compared to controls. Antioxidant rescue of L-selenomethionime induced deformities was attempted in embryos using N-acetylcysteine (NAC). Pretreatment with NAC significantly reduced deformities in the zebrafish embryos secondarily treated with L-selenomethionine, suggesting that oxidative stress may play a role in Se toxicity. Selenite exposure also induced a 6.6-fold increase in glutathione-S-transferase pi class 2 gene expression, which is involved in xenobiotic transformation. No changes in gene expression were observed for selenate or L-selenomethionine-exposed embryos.

The findings in this dissertation contribute to the understanding of how Se bioaccumulates in a lotic system and is transferred through a simulated foodweb in addition to further exploring oxidative stress as a potential mechanism for Se-induced embryo toxicity. Future studies should continue to pursue the role of oxidative stress and other mechanisms in Se toxicity and the biotransformation of Se in aquatic ecosystems.

As the production and use of nanoparticles (NPs) has increased, so has concern over their effects on the environment and aquatic organisms. While the majority of research on the toxicity of NPs has been performed using controlled laboratory conditions, little is known about their effects in more complex environments. The goal of this dissertation is to understand how incorporating increasing levels of environmental complexity affects the study of NP toxicity to early life stage fish. In particular, goals included evaluating the impact of increasing environmental complexity on silver NP (AgNP) toxicity to young Atlantic killifish and zebrafish and exploring how photocatalytic degradation of benzo(a)pyrene (BaP) by TiO2 NPs affected toxicity to young zebrafish.

In order to incorporate high environmental complexity into AgNP toxicity studies, a range of experimental systems were used. First, mesocosms built to simulate freshwater North Carolina wetlands were dosed with gum Arabic-coated AgNPs (GA-AgNPs), polyvinylpyrollidone-coated AgNPs (PVP AgNPs) and AgNO3 as the most ecologically relevant scenario. Mortality induced by samples taken from these mesocosms to embryonic and larval Atlantic killifish was compared to mortality induced by of samples prepared in the laboratory. For embryonic killifish, samples taken from the mesocosms caused significantly less mortality than samples prepared in the laboratory for all types of silver. However, for larval killifish, toxicity of GA-AgNPs from mesocosms was not lower than toxicity of those from the laboratory and toxicity of PVP-AgNPs was actually higher in mesocosm samples. Mortality caused by AgNO3 was significantly less in samples from the mesocosms than from the laboratory, similar to results found in embryos. For larvae, both types of NPs from the mesocosms were more toxic than AgNO3.

In order to understand the difference in results seen between mesocosm samples and laboratory samples, a microcosm approach was used to assess the individual contributions of plants, soil, and natural water to the mediation of AgNP toxicity. In addition, samples were thoroughly characterized in order to understand how these factors were influencing. Silver speciation was assessed using x-ray absorption near edge structures spectroscopy, extent of AgNP aggregation and dissolution using flow field flow fractionation coupled with inductively coupled plasma mass spectrometry (FFFF-ICP-MS), and total silver and dissolved silver quantified using ICP-MS. Organic matter quantity and quality were also measured using total organic carbon (TOC) analysis and fluorescence excitation-emission spectroscopy (EEMS). Ultimately, although plant-derived organic matter was shown to significantly influence silver speciation and AgNP aggregation, these experiments were not able to replicate the interesting results seen with killifish larvae from the mesocosms on a smaller scale. In experiments using zebrafish embryos, the presence of plants was correlated with reduced toxicity. However, this correlation could not be attributed to any of the changes seen in AgNP or silver characteristics due to reduced water column concentrations of silver that also occurred when plants were present.

Due to the differences seen in mesocosm and microcosm results, I hypothesized that ultraviolet (UV) light could be playing a role in mediating AgNP toxicity. AgNPs and AgNO3 were illuminated using a solar simulator prior to dosing zebrafish embryos. UV light was found to decrease GA-AgNP and AgNO3 toxicity and to increase PVP-AgNP toxicity. Ultraviolet-visible light (UV-vis) spectra of AgNPs and AgNO3 after exposure to UV light indicated changes in size distribution and aggregation. These changes could be due to photolysis of coatings leading to increased dissolution of silver and destabilization of the NPs.

I also explored the effect of TiO2 NP facilitated photocatalytic degradation of toxicity of BaP. Due to the unique photocatalytic properties of TiO2 NPs, they are not only being proposed for use as a remediation aid for recalcitrant organic contaminants but also possess the ability to transform organic co-contaminants in the environment and change their toxicity and risk to aquatic organisms. BaP was degraded with TiO2 using a solar simulator before dosing young zebrafish. Degradation resulted in increased cytochrome P450 (CYP) activity, a marker of polycyclic aromatic hydrocarbon (PAH) exposure and toxicity as well as increased mortality. However, these effects ultimately proved to be dependent on the presence of DMSO, a carrier solvent for BaP, and thus their environmental relevance is questionable.

Taking environmental complexity into account is an important part of understanding engineered NP risk. I found that AgNP toxicity is highly dependent on environmental matrix and individual factors such as UV, and that TiO2 NPs have the potential to influence toxicity of organic contaminants. Ultimately, ecologically appropriate and relevant risk assessment of NPs to aquatic organisms will rely on fully characterizing how the fate, behavior, and toxicity of NPs is influenced by environmental factors such as plants, sediments, bacteria, organic matter, ionic strength, and UV light.

Aquatic systems are impacted by a wide variety of natural and anthropogenic stressors. Laboratory studies often focus on impacts of a single stressor, ignoring how these stressors may interact. This dissertation focuses on the interactions between polycyclic aromatic hydrocarbons (PAHs) and two physical stressors (hypoxia and solar radiation). PAHs are ubiquitous environmental pollutants that occur in the environment as complex mixtures, the components of which may interact. Some PAHs are agonists of the aryl hydrocarbon receptor (AhR), which regulates expression of a number of genes (such as CYP1A) involved in metabolism, often of the same compounds that induced the AhR. PAHs that are AhR agonists have been shown to interact synergistically with PAHs that inhibit activity of CYP1A, inducing developmental deformities in fish. AhR shares a dimerization partner, the aryl hydrocarbon receptor nuclear translocator (ARNT), with the protein responsible for regulating hypoxia-induced gene expression, hypoxia-inducible factor 1á (HIF1α), possibly resulting in competition between the two pathways. Competition would result in decreased induction of CYP1A under hypoxic conditions, possibly resulting in synergistic embryonic toxicity between AhR agonist PAHs and hypoxia. In this dissertation, the results are presented of experiments regarding the occurrence of AhR/HIF1α crosstalk in fish and the developmental consequences of co-exposure to hypoxia and PAHs. In vitro testing revealed competition for ARNT in which HIF1α appeared to out-compete AhR. Induction of an AhR-responsive luciferase reporter by several AhR agonists (benzo[a]pyrene (BaP), benzo[k]fluoranthene (BkF), and polychlorinated biphenyl (PCB126)) was reduced under hypoxia and this effect was eliminated by overexpression of ARNT. Induction of a HIF1α-responsive reporter was unaffected by BkF and PCB126. BaP caused an ARNT-independent decrease in both basal and induced HIF1α reporter activity. Attempts to verify this crosstalk pattern in vivo revealed that BaP-induced CYP1A expression was further increased under hypoxic conditions. Induction of hypoxia-inducible genes VEGF and LDHa were unaffected by BaP. The effect of hypoxia on CYP1A expression was not repeated with BkF or pyrene (PY) and the exact conditions that result in hypoxic changes in CYP1A expression remain to be determined. Embryonic toxicity in zebrafish (Danio rerio), including pericardial edema, heart malformations, and death were synergistically induced by hypoxia and BaP, BkF or complex, environmentally relevant PAH mixtures. There was no effect of hypoxia on PCB126 toxicity and hypoxia protected from the pericardial edema induced by PY. CYP1A knockdown mimicked the effects of hypoxia on BkF and PY toxicity and even further exacerbated BkF toxicity under hypoxic conditions, suggesting a role for metabolism in the toxicity. Additionally, since two CYP1A inhibitors (fluoranthene (FL) and á-naphthoflavone (ANF)) had been previously reported to interact synergistically with hypoxia, three other CYP1A inhibitors (dibenzothiophene, carbazole and 2-aminoanthracene) were tested. None of them induced toxicity in combination with hypoxia, indicating that CYP1A inhibition was not the reason for the interaction of FL and ANF with hypoxia.

A second stressor known to interact with PAHs to induce toxicity is solar radiation. While most studies of the effects of solar radiation on PAH toxicity have focused on the effects of UV light on PAHs already absorbed by an organism (photoactivation), less attention has been paid to the toxic effects of photomodified PAHs. We exposed carbazole to ambient sunlight and subsequently exposed developing zebrafish to the resulting mixture. Photo-exposed carbazole caused developmental toxicity including edema, heart malformations, craniofacial malformations and death that were not caused by parent carbazole; these effects were severely exacerbated by hypoxia and significant mortality was also observed. Additionally, photo-exposed carbazole induced expression of CYP1A and GSTp, likely resulting from agonism of the AhR and toxicity of this mixture was alleviated by morpholino knockdown of AhR. Some photoproducts were identified, but none of them appeared to be involved in the toxicity or supposed AhR induction observed with photoexposed carbazole. The results of these experiments underscore the importance of consideration of the interactive effects of physical and chemical stressors when assessing risks to wildlife populations inhabiting polluted areas.

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.

Polycyclic aromatic hydrocarbons (PAHs) are important contaminants that are found in increasing amounts in aquatic ecosystems. One of the sites that that is contaminated by extremely high levels of PAHs is the Atlantic Wood Industries Superfund Site on the Elizabeth River, VA. The Atlantic killifish (Fundulus heteroclitus) from this site exhibit increased levels of antioxidants, increased sensitivity to hypoxia, and increased expression of enzymes involved in glycolytic metabolism, suggesting that exposure to PAHs in the environment may induce changes in mitochondrial function and energy metabolism. Normal mitochondrial activity is crucial to an organism's survival. Therefore, gaining a better understanding of how mitochondria are affected by environmental contaminants such as PAHs is a pressing research objective. As a first step in understanding changes in cellular bioenergetics of aquatic organisms in response to PAHs, this research focused on the effect of benzo[a]pyrene (BaP), a representative PAH, on mitochondria the killifish model and on comparison of the mitochondria of the PAH-adapted killifish from the Elizabeth River Superfund Site to reference site fish. In order to assess the extent of mitochondrial DNA damage in the killifish, a PCR-based assay (LA-QPCR) for nuclear and mitochondrial DNA (nDNA, mtDNA) damage was adapted to this model and validated in with UV exposure and BaP exposure studies, as well as with ex situ study examining DNA damage in killifish inhabiting the Elizabeth River Superfund site. With the newly adapted LA-QPCR, mtDNA and nDNA damage in the killifish from the Elizabeth River Superfund site and from a reference site (King's Creek, VA) that were treated with BaP were examined. Similar increases in mitochondrial and nuclear DNA damage were observed in King's Creek fish treated with BaP. Killifish from the Elizabeth River showed high levels of basal nDNA and mtDNA damage compared to fish from the reference site, but the level of damage induced due to BaP treatment was much lower in Elizabeth River killifish. Laboratory-reared offspring from both populations showed increased BaP-induced damage in mtDNA, relative to nDNA. Similar to the adult experiment, the Elizabeth River larvae had higher levels of basal DNA damage than those from the reference site, but were less impacted by BaP exposure. Results suggest that BaP exposure can have important energetic consequences and that multi-generational exposure in the wild may lead to adaptation that dampens DNA damage arising from BaP exposure. Since the toxic effects of many PAHs are the result of bioactivation by cytochrome P4501A (CYP1A), the existence of enzymes that can potentially metabolize PAHs in mitochondria was verified. Using Western blot, protein similar in size to microsomal CYP1A was identified with monoclonal antibody against scup CYP1A in the mitochondrial fraction from adult male killifish livers. The size of the protein in the mitochondria was the similar to that of microsomal CYP1A. Fish dosed with BaP had increased EROD activity in the liver mitochondrial fraction compared to controls. In killifish larvae dosed with BaP and benzo[k]fluoranthene (BkF), CYP1A protein levels as well as enzyme activity were elevated. However, fish from the Elizabeth River Superfund site showed recalcitrant mitochondrial CYP1A protein levels and enzyme activity in a similar manner to microsomal CYP1A. Finally, the hypothesis that energy metabolism of BaP-treated fish may be different from the control group and that killifish from the Elizabeth River Superfund site may also have altered energy metabolism compared to reference site fish was tested. Respiration of killifish embryos treated with BaP from both populations was measured. Compared to the King's Creek control fish, all other treatment groups showed decrease in oxygen consumption, indicating lower respiration rate. However, when activities of key enzymes involved in glycolysis (PK) and anaerobic metabolism (LDH) in adult killifish liver and muscle were measured, no differences in the enzyme activities were observed in BaP-treated group compared to the control group. Moreover, metabolomic analysis on BaP treated King's Creek and Elizabeth River killifish showed no difference in the profile in all four treatment groups. The findings in this thesis contribute to the understanding of how BaP, a common environmental pollutant in the aquatic ecosystem, targets the mitochondria in fish model. Nevertheless, deeper examination of how BaP may impact mitochondrial function in killifish and potentially influence adaptation of killifish at a highly contaminated site is necessary. Further studies will elucidate whether such impacts can potentially affect the energy budget and organism level fitness in populations in the wild.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants formed from the incomplete combustion of fossil fuels and are found in the environment as complex mixtures. PAHs are developmentally toxic to fish, causing yolk sac edema, hemorrhaging, craniofacial malformations and cardiac defects including impaired heart looping, elongated heart, decreased blood flow, and pericardial effusion. Previous research has shown that many of the toxic effects of PAHs are mediated through the aryl hydrocarbon receptor (AHR), which upregulates phase I and II metabolic genes, but the underlying mechanisms of PAH-induced toxicity are not yet known. The primary goal of this dissertation was to better understand the molecular mechanisms by which PAH mixtures cause developmental toxicity in fish. To this end, the zebrafish (Danio rerio) was used as a developmental model. Simple mixtures consisting of a PAH that is an AHR agonist (benzo[a]pyrene or benzo[k]fluoranthene) and a PAH that is a cytochrome P450 1 (CYP1) inhibitor (fluoranthene) were used in these experiments along with the dioxin-like compound 3,3',4,4',5-pentachlorobiphenyl (PCB-126). Morpholino gene knockdown was used to examine the role of specific genes in response to PAHs, gene expression changes in response to PAH exposures were examined via QPCR, quantification of pericardial effusion was used as a metric for cardiac toxicity, and CYP1 activity was measured as an indication of AHR pathway induction. First, PAH mixtures consisting of an AHR agonist (BkF) and a CYP1 inhibitor (FL) induced cardiac toxicity that was preceded by upregulation of CYP1 and redox-responsive gene expression, and these effects were dependent upon the AHR2. Second, knockdown of glutathione s-transferase pi class 2 (GSTp2), part of phase II metabolism, exacerbated PAH-induced toxicity but did not affect PCB-126-induced toxicity. Third, knockdown of another isoform of the AHR, AHR1, exacerbated PAH- and PCB-126-induced toxicity and increased CYP1 activity but did not affect CYP expression in response to these agonists. Simultaneous knockdown of AHR1A and AHR2 did not exacerbate nor ameliorate PAH-induced toxicity but did prevent PCB-126-induced toxicity. Fourth, to examine AHR2-dependent and AHR2-independent gene induction in zebrafish hearts in response to PAHs, microarrays were used. Gene expression changes caused by PAHs were largely AHR2-dependent and consisted of genes involved in cell adhesion, oxidation-reduction, and TGF-&beta signaling processes as well as genes involved in heart structure and function. These findings help to elucidate how PAHs elicit deformities during development and highlight differences between PAHs and other AHR agonists. Additionally, these experiments have identified other genes in addition to AHR2 that are involved in mediating or responding to the toxicity of PAHs.

Chronic exposure to toxicant mixtures is a serious 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), which are toxic, ubiquitous, and increasingly prevalent. Furthermore, estuarine systems are of particular concern, as they are highly impacted by a wide variety of pollutants; fish there are often exposed to some of the highest levels of contaminants of any vertebrate populations, along with other stressors such as fluctuations in water level, dissolved oxygen, and temperature. A population of Fundulus heteroclitus (the Atlantic killifish or mummichog, hereafter referred to as killifish) inhabits a Superfund 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 better understand the mechanism(s) by which Elizabeth River killifish resist the developmental toxicity of a complex mixture of PAHs and to investigate the tradeoffs associated with this resistance. Because the aryl hydrocarbon receptor (AHR) pathway plays an important role in mediating the effects of PAHs, one major hypothesis of my work was that suppression of the AHR response plays an important role in the resistance of Elizabeth River killifish. For this reason, investigation of the activation of the AHR pathway, as measured by CYP induction, is a unifying thread throughout the work. Another major hypothesis of this work is that adaptation to PAHs has secondary consequences for Elizabeth River killifish, such as altering their response to other xenobiotics. To investigate these hypotheses, a series of experiments were carried out in PAH-adapted killifish from the Elizabeth River and in reference fish. The morpholino gene knockdown technique was modified for use in killifish; we demonstrated that CYP1A knockdown exacerbates PAH-driven cardiac teratogenesis and AHR2 (but not AHR1) knockdown rescues PAH-driven cardiac teratogenesis. Using acute toxicity tests of larval killifish, we showed that Elizabeth River killifish are less sensitive than reference larvae to chlorpyrifos, permethrin, and carbaryl. These results demonstrated that the adaptation was able to protect from multiple xenobiotics, not just PAHs. Using the in ovo ethoxyresorufin-o-deethylase (EROD) assay and a subjective cardiac deformity screen, we showed that the adaptation was spread throughout the killifish subpopulations of the Elizabeth River estuary. However, the adaptive response varied greatly among the subpopulations, which showed that AHR pathway suppression was not required for some level of protection from PAH toxicity. Finally, using the quantitative real-time PCR, the EROD assay, and cardiac deformity screening, we demonstrated that the adaptation was heritable for two generations of fish reared in clean laboratory conditions. The findings in this dissertation will help to reveal how mixtures of PAHs exert their toxic action in un-adapted organisms. Furthermore, these studies will hopefully demonstrate how chronic exposure to PAH mixtures can affect organisms at the population and even evolutionary level. Perhaps most importantly, they will help us to better predict the consequences and tradeoffs for organisms and populations persisting in PAH-contaminated environments.

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BaP) are ubiquitous environmental contaminants. PAHs are toxicologically important for both humans and wildlife in large part due to their mutagenic, carcinogenic, and teratogenic properties. While the effects of adult and developmental exposures to PAHs are relatively well characterized, the potential for PAHs to have effects across generations is an emerging concern in the field of environmental health. In epidemiological studies, prenatal exposure to PAHs is associated with adverse birth outcomes as well as later life metabolic, neurological, and reproductive disorders— which have become global human health epidemics. These findings have been validated in animal models, with reduced survivorship, increased morphological deformities, and alterations in behavior, physiology, and disease risk in multiple subsequent generations. However, the mechanisms underlying the multigenerational effects of PAHs are poorly understood. This dissertation focuses on mitochondrial contributions to the maternal and cross-generational toxicity of PAHs.

Mitochondria are essential to the development, health, survival, and reproduction of all aerobic organisms. The importance of maintaining mitochondrial function for health is supported by the prevalence of mitochondrial diseases, which clinically manifest in at least 1 in 4,300 people. Mitochondrial diseases often present with metabolic, neurological, and reproductive consequences, similar to those associated with prenatal PAH exposures. Mitochondrial DNA (mtDNA) is maternally inherited and undergoes bottlenecks (i.e. reductions in mtDNA copy number per cell) during oogenesis and early embryonic development, creating potential for maternal and cross-generational inheritance of mitochondrial diseases. Inheritance of mitochondrial dysfunction across generations has been established for genetic, pharmacological, and dietary etiologies. Notably, mitochondria are important targets of environmental contaminants such as PAHs, which affect bioenergetics at multiple levels of biological organization. However, the potential for environmental toxicant-induced mitochondrial dysfunction to have persistent effects across multiple generations is still largely uncharacterized. This is the knowledge gap we address in this dissertation.

To this end, we evaluate the persistent bioenergetic effects of BaP – a model PAH and known mitochondrial toxicant – in F1 (maternally exposed) and F2 (cross-generationally exposed or germline exposed) generations following a chronic maternal (F0) dietary exposure using the model teleost Danio rerio. Maternally exposed F1 embryos exhibit reduced mitochondrial DNA integrity, reduced mitochondrial function and efficiency, and impaired antioxidant defense systems during development, largely in the absence of effects in exposed F0 females. Metabolic shifts during development create potential for disease pathologies and reduced organismal fitness later in life. In F1 adults, mitochondrial dysfunction presents in cardiac tissue with reductions in mitochondrial reserve capacity. Cardiac function and plasticity are key determinants of fitness in the environment, and impaired function confers disease risk in humans. Maternally BaP exposed F1 fish also exhibit altered locomotor activity throughout life and reduced fear/anxiety behaviors as adults.

PAH-induced changes in mitochondrial function and metabolic plasticity persist in the F2 embryos, two generations removed from the original BaP exposure, suggesting cross-generational reductions in fitness may follow a single exposure event. Metabolic consequences occur in F2 embryos at F0 exposure levels that do not cause significant dysfunction in the F1 generation, with important implications for evaluating the risk associated with the persistent effects of pollution (e.g. lasting impacts of oil spill events) in the environment.

The ability to adjust metabolism is crucial for organisms to effectively respond to a variety of natural and anthropogenic stressors, suggesting that organisms with alterations in fundamental bioenergetic processes may be more sensitive to secondary stressors. Herein we demonstrate that F2 organisms with a cross-generational history of exposure exhibit altered metabolic response to thermal stress, reduced thermal tolerance, and fitness tradeoffs. Cross-generational exposure to BaP potentiates metabolic effects under thermal stress even in the absence of effects at baseline temperature. Taken together, these data suggest that exposure to PAHs such as BaP affects mitochondrial function, organismal physiology, behavior, and secondary stress response capacity across generations, creating potential for downstream population and ecosystem level effects.