Browsing by Subject "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.

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.

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.

Engineered nanoparticles (ENPs) are a part of everyday life. They are incorporated into a wide array of products including sunscreens, clothing, electronics, paints, and automobiles. One particular type of ENP, quantum dots (QDs), are fluorescent semi–conducting nanocrystals, and are touted as the next generation of medical tracers and energy–efficient light bulbs. The continued development and expansion of commercial applications for QDs ensure that they will enter the aquatic environment following manufacture, use, and disposal. Unfortunately, very little information exists on the bioavailability and sub–lethal toxicological effects of QDs in aquatic organisms. The studies described in this dissertation focused on determining the toxicological effects of Lecithin–encapsulated CdSe/ZnS quantum dots in larval and adult Fundulus heteroclitus (the mummichog).

Quantum dot dispersion is greatly influenced by environmental parameters such as pH, natural organic matter concentration, and ionic strength. Lecithin–encapsulated core–shell QDs aggregated and precipitated from suspension in 20 ppt seawater. QD aggregates adhered to the exterior chorion of Fundulus embryos in aqueous embryo exposures, but did not traverse the chorion and deposit into the body of the fry. Incidences of developmental abnormalities increased and hatching rates declined in embryos exposed to the highest concentration tested (100 μg/ml).

Dietary assessments showed that QDs were bioavailable to adult Fundulus. While QDs or their degradation products traversed the intestinal epithelial and were deposited to the liver, less than 0.01% of the cadmium from the QDs was retained in the liver and intestinal tissues. QD uptake did not cause significant changes in hepatic total glutathione or lipid peroxidation levels, nor did it statistically alter the expression of genes involved in metal metabolism and oxidative stress—metallothionein, glutathione–s–transferase, glutathione peroxidase, and superoxide dismutases. There was, however, a clear gender–specific trend in the level of Cu/Zn–superoxide dismutase transcription. In addition, QDs did impact fecundity presumably by feminizing male fish. Vitellogenin transcription was elevated and relative gonad size reduced in male Fundulus consuming 10 μg QD per day. Lastly, QDs or their degradation products were maternally transferred to the eggs following six to eight weeks of parental exposure, thus posing a risk to Fundulus progeny. Based on the results of these studies, it is apparent that chronic exposure to QDs could result in adverse affects in teleosts and other organisms inhabiting estuarine environments.