Browsing by Subject "Polycyclic aromatic hydrocarbons"

This dissertation presents a framework to improve bioremediation of soils polluted with polycyclic aromatic hydrocarbons (PAHs). PAHs are of great concern because they are recalcitrant and toxic. PAHs enter the environment from a variety of sources such as incomplete combustion and coal tar distillation. The PAHs focused on in this dissertation have polluted soils as a result of creosote-based wood treatment operations that took place at Holcomb Creosote and Atlantic Wood Industries, Inc. (AWI) both of which are now classified as Superfund sites. There are numerous sites analogous to these two Superfund sites throughout the world which have been polluted through similar wood-treatment operations, as creosote was once industry’s foremost wood preservative.

There is room for existing PAH treatment options, which are mainly physical and chemical in nature, to be expanded to include more sustainable options. Commonly used technologies include excavation, in situ stabilization, and soil washing. Historically, bioremediation strategies relying on bacteria to transform pollutants have been challenged by the tight sorption of heavy- and middle-weight PAHs to soils, as this restricts aqueous phase transport required for bacterial degradation. Multiple studies have demonstrated fungi to be capable of degrading these inaccessible pollutants and other mixtures of hydrophobic pollutants (mycoremediation). Yet, when fungi have been introduced to polluted soils (mycoaugmentation), they have not been able to outcompete the native microbiota long enough to degrade the contaminants of concern over the long term. It is possible that a thorough characterization of the indigenous fungi at a given site may provide some insights into the development of targeted in situ mycoremediation strategies.

Although incorporating site microbes has been generally acknowledged as important for some time, the techniques enabling thorough assessment of microbial ecosystems are relatively new. Consequently, little is known about PAH-associated microbiomes in general, and even less is known about PAH-associated fungal communities. The work presented in this dissertation aims to address this knowledge gap by leveraging recent advances in high-throughput sequencing technology to design and validate targeted mycoremediation strategies. To this end, the overarching goal of this dissertation was to develop and test a framework for incorporating native fungi into a bioremediation strategy to expand such sustainable remediation options to sites where they have not been relevant in the past.

In the first aim of this dissertation research, advances in high-throughput sequencing were used to identify potential biostimulation targets in soils moderately polluted with PAHs. The next generation sequencing (NGS) platform, Illumina, was utilized to sequence the large sub-unit (LSU) gene commonly used as a marker gene in fungal community studies. Relationships were examined between concentrations of over 31 different polycyclic aromatic hydrocarbons and the pollutant-associated communities to test whether there were any fungi capable of tolerating high levels of these toxic contaminants. In this aim, fungal genera were identified that contained species closely related to known PAHs and petroleum hydrocarbon degraders. In all, this work identified 32 targets for biostimulation, based on Spearman rank correlations between prevalence and mid- and high-molecular weight PAHs. Ascomycetes were found to have higher levels of diversity than any other phylum in this subset of biostimulation targets. These data suggest that ascomycete fungi are more likely to be present in heavily polluted soils than basidiomycete fungi (which had previously been subjects of much interest). Overall, this work illustrates that polluted soils harbor fungal biostimulation targets, specifically within Ascomycota.

The second aim of this thesis research was to use the precision bioremediation assessment in highly polluted soils and then to evaluate a range of amendments with the goal of identifying strategies to stimulate the fungal communities that dominate these PAH-associated fungal communities. Here we applied the approach we fine-tuned in the first aim to the AWI soils, as these soils have some of the highest documented PAH-concentrations. Again, Ascomycota were found to be more prevalent in these soils, so an isolate obtained from AWI was used to compare alternative stimulation techniques between three substrates they are known to grow on: chitin, cellulose, and wood. We used anthracene degradation as a proxy for PAH degradation, which we monitored in sacrificial simplified bioreactors responding to the three amendments. T. harzianum is also known to have enzymes which degrade PAHs, but it is unknown which ecological role uses those enzymes, and thus which ecological role we should promote. T. harzianum was grown in the presence of chitin, cellulose, and wood as substrates in liquid culture with anthracene. Chitin was found to stimulate the highest anthracene removal, with a 0.1% (w/v) amendment resulting in ~93% degradation. While ~13% less than chitin, 1% (w/v) cellulose was also found to stimulate ~46% more anthracene degradation than wood, which had no improvement over the abiotic losses (~33% on average). This is notable because the “go to” method for stimulating fungi in the past has been wood supplements. This work provided insight into alternative stimulation strategies to target specific ecological roles that may better degrade PAHs in situ.

For the third and final aim of this dissertation research, the two most promising amendments were added with and without Trichoderma harzianum spores to test several mycoremediation treatment strategies in soil bioreactors and compare them with a (no carbon added) nutrient stimulation treatment. Pollutants were added as aged Atlantic Wood Industries soil delivering aged pollutants. Triplicate reactors from each treatment were sequenced at time zero, after two weeks, and after one month. At each sampling time, RNA was extracted, converted to cDNA, and submitted to Illumina MiSeq library preparation targeting the LSU region for fungal community analysis in addition to the V4 region of the 16S rDNA for bacterial community analysis. Statistical analyses using DESeq2 identified responders among the groups of reactors subjected to the different biostimulation treatments. Taxa from both the fungal and the bacterial communities responded differentially to the amendments. Fungi were found to comprise the majority of the significant responders. This work also found that mycoaugmented strains were not successful in establishing themselves as prominent members of the active community. This represents one of the earliest studies to directly measure mycoaugmentation failure. These data propose a hypothesis about functional redundancy inhibiting establishment of augmented fungi as already established fungi outcompete them for freshly added nutrients. Over 90% degradation was observed over the course of one month regardless of treatment-interestingly, the highest degradation was found in the nutrient amendment (no carbon added) treatment. These results show similar degradation across the soil bioreactors, yet different microbial growth, which supports the hypothesis that there is community-level functional redundancy and multiple metabolic food webs that result in the observed pollutant degradation.

Overall, this dissertation work demonstrates how significant advances in sequencing technology can be implemented in design and monitoring stages of bioremediation. This work also suggests that significant advances could be possible through the application of targeted metatranscriptomic analysis. Through incorporating such insights as described in this dissertation, this research brings the field of bioremediation one step closer to successfully engineering microbiomes to degrade contaminants of concern.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that accumulate in soils and sediment due to their physicochemical properties. In these environmental matrices, PAHs are predominantly transformed and degraded by the native fungal and bacterial communities. However, microbial degradation of PAHs is a slow process that requires engineered approaches to improve degradation rates to meet remediation criteria.

Engineered bioremediation approaches consist of altering the microbial community by either increasing cell concentrations of specific, targeted organisms or by introducing catabolic genes that confer for a phenotype that can degrade the target contaminant. This approach is called bioaugmentation and is generally applied using the former strategy. Biostimulation is another method, which includes the addition of nutrients that may be limited to microorganisms and can help grow the indigenous microbial community and accelerate contaminant degradation. However, biostimulation is not a targeted approach and may stimulate the entire microbial community, not just organisms capable of degrading the target contaminant.

Bioaugmentation of sediments is challenging due to constraints surrounding the longevity, stability, and delivery of microorganisms. To address the limitations of this remediation approach, the work within this dissertation outlines methods for developing a consortium of PAH-degrading bacteria coordinated within a stable community, as well as a technology for delivering this consortium to creosote contaminated sediments.

The first objective was to identify and isolate PAH-degrading bacteria from creosote contaminated sediment. Sediment was collected from sites along the Elizabeth River, VA and a 16S rRNA amplicon library of sequences was analyzed to generally evaluate the influence of chemical contamination on the bacterial community structure. To detect PAH-degrading organisms within sediment communities, DNA-SIP using uniformly labeled stable isotopes of phenanthrene and fluoranthene were prepared in incubations with Republic Creosoting site sediment. Clones derived from this experiment revealed one prominent degrader of phenanthrene and two prominent fluoranthene degrading bacteria. In an attempt to isolate these and other PAH-degrading organisms for laboratory evaluation, culture-based methods were employed and resulted in the successful isolation of 6 unique bacteria, including one strain which was detected in the DNA-SIP experiments. Overall, it was determined that PAH-degrading bacteria exist in Republic Creosoting site sediments, although not in significant relative abundance compared to other bacteria. This finding suggests that these contaminated sediments could be a good candidate for a bioaugmentation approach.

Most of the research on bioremediation has focused on organisms in isolation and existing in a free-floating, or planktonic, cellular state. The second objective of this dissertation was to confirm the PAH-degrading capabilities of isolated bacteria and to coordinate these organisms into a biofilm structure, which provides protection and additional community benefits to participating microorganisms. To this end, we employed a high-throughput, reproducible assay to confirm whether or not isolated bacteria are capable of coordinating within a biofilm. We also used culture-based methods and performed incubations with multiple types of PAHs to determine if the isolated organisms can interact with PAHs of various size and ring number. Finally, we used a metabolic assay for the novel application of assessing the respiration capacity of the isolated PAH-degrading bacteria in the biofilm conformation, to determine if these organisms are metabolically active when they are situated within a biofilm. We found that all of the organisms isolated were capable of forming a biofilm that was metabolically active. Many of these organisms demonstrated the ability to degrade phenanthrene and fluoranthene, but only a few showed the potential for degrading pyrene. These results confirmed that the isolated organisms from Republic site sediment can degrade PAHs and form a biofilm structure, which will be beneficial for their application to sediments in a bioaugmentation strategy.

The final aim of this work was to evaluate the use of an activated-carbon amendment based technology for the delivery of a bacterial consortium to PAH-contaminated sediment. While validated for use as a remediation technology and delivery strategy for organisms capable of degrading polychlorinated biphenyls (PCBs), this approach has not yet been tested for use with sediments contaminated with PAHs.

A large number of air pollutants exist in a gaseous phase, some of which are considered highly carcinogenic compounds. Yet these compounds have not been as well studied as airborne particulate matter (e.g., PM10 or PM2.5). Polycyclic aromatic hydrocarbons (PAHs), especially nitrated PAHs (nitro-PAHs), are a well-known class of toxic environmental pollutants that result from industrials processes and incomplete combustion of organic fuels. This study explores meteorological and covariate effects on urinary metabolites of nitro-PAHs, namely amino-PAHs, during air pollution exposure with and without HEPA and/or ESP filtration. Urinary concentrations of cotinine (a biomarker of tobacco smoke exposure) and five specific amino-PAHs were measured including: 1-aminopyrene (1-AP), 1-naphthylamine (1-AN), 2-naphthylamine (2-AN), 2-aminofluorene (2-AF), and 2-aminophenanthrene (2-APhe). Eighty-nine subjects were assigned to one of two intervention groups in Broad Town offices located in Changsha City, China. Participants experienced periods of air pollution exposure with and without combinations of HEPA and ESP air filtration. Linear mixed effects modeling highlights the potential associations between urinary amino-PAH concentrations and covariates such as temperature and relative humidity. Additionally, both HEPA and ESP did not appear to significantly impact gas-phase levels of urinary amino-PAHs. Such findings may bring the efficacy of ESP filtration systems into question. Especially when weighing the health costs of increased ozone exposure and lack of gas-phase pollutant filtration to ESP system’s improved PM2.5 reduction. The results underscore a need for nitro-PAH related research and the potential need of gas-phase air filtration solutions that remove carcinogenic and mutagenic gas-phase air pollutants from indoor air.

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.

Cancers are a complex set of related diseases with wide ranging etiologies, and humans are exposed to a milieu of environmental exposures that may contribute to disease development. Breast cancer in particular is a complex and multifactorial disease, yet prior research has largely focused on studying exposures to one factor/contaminant at a time, which does not reflect the real-world environment. In addition, investigating incidence of total breast cancer has the potential to mask the impacts of environmental factors on 1) the development of different subsets of breast cancer, varying in hormone receptor status and 2) progression of these cancers to advanced and more aggressive stages. Patients with distant metastatic breast cancer have poor prognosis despite aggressive, multidisciplinary treatment regimens compared to carcinoma in situ or early-stage breast cancer. This reinforces the unmet need to identify risk factors associated with advanced breast cancers to reduce incidence and improve overall breast cancer outcomes. The objective of these studies was thus to determine environmental risk factor associations with breast cancer by stage, and to utilize a real world environmental exposure chemical mixture to interrogate potential impacts on breast tumor progression by hormone receptor status.

These studies begin by evaluating the associations between breast cancer summary stages and the Environmental Quality Index (EQI), which includes a range of environmental factors across five overarching environmental domains. Our analysis found total and localized breast cancer incidence were increased in counties with poor environmental quality compared to those with good environmental quality. These overall environmental trends were largely driven by land quality, which was associated with incidence of early-stage disease (carcinoma in situ and localized breast cancer), in addition to total breast cancer, especially in urban counties.

Taking advantage of more granular patient data, these studies further evaluated the odds of localized, regional, or distant metastatic breast cancer in categories of environmental quality using women with carcinoma in situ as registry-based controls. Overall environmental quality was not associated with invasive breast cancer; however, poor land environmental quality was associated with increased odds of all invasive breast cancer types, particularly in more rural communities (distant metastatic breast cancer was 5-8% more likely compared to carcinoma in situ). Cumulatively, epidemiologic analyses indicate significant associations between poor overall and land environmental quality and breast cancer incidence as well as odds of invasive breast cancer. Furthermore, associations differed by breast cancer summary stage, rural-urban status, and environmental domain thus identifying a critical need to assess cumulative environmental exposures in the context of cancer stage.

Emerging evidence suggests the role of environmental chemicals, in particular endocrine disrupting chemicals (EDCs), in incidence and progression of breast cancer, which can impact survival outcomes. Polycyclic aromatic hydrocarbons (PAHs) are a toxic and ubiquitous class of environmental chemicals, many of which exhibit endocrine disrupting activity. They are products of fuel combustion from human and natural sources. PAH exposure is widespread, and many PAHs are considered carcinogenic. Urinary metabolite data were collected from 9517 individuals from the U.S. CDC National Health and Nutrition Examination Survey years 2005-2014 for four parental PAHs naphthalene, fluorene, phenanthrene, and pyrene. We utilized these urinary biomarkers to estimate PAH intake, and regression models were fit for multiple demographic and lifestyle variables, to determine variable effects, interactions, odds of high versus low PAH intake. Smoking and secondhand smoke (SHS) exposure accounted for the largest PAH intake rate variability (25.62%), and there were strongest interactions between race and ethnicity and smoking or secondhand smoke exposure, reflected in a much greater contribution of smoking to PAH intake in non-Hispanic Whites as compared to other racial and ethnic groups. Increased risk of high PAH intake was observed in older age groups, obese persons, college graduates, midrange incomes, smokers, and those who were SHS exposed. Among the non-smoking population, effects of other demographic factors lessened, suggesting a highly interactive nature. Results suggest that there are demographic subpopulations with high PAH intake because of different smoking behaviors and other exposures.

The PAH-contaminated Atlantic Wood Industries, Inc. (AWI) Superfund site in Portsmouth, Virginia provides a model for studying a real-world complex PAH mixture, and its extrapolation to cancer risk and PAH exposure in the general population. Cancer risk at the Superfund site due to sediment-derived PAHs was examined and PAH sources in the general population upon PAH mixture exposure were then evaluated. The PAH mixture was assessed for potential carcinogenicity using US EPA’s OncoLogic ranking tool and the US EPA list of priority PAHs. Cancer risk due to PAH exposure was calculated for Superfund site users and compared to the US EPA assessment. Human intake and health endpoints of PAHs within the mixture were extracted from USEtox chemical fate database. Eleven PAH compounds within the mixture were of carcinogenic concern, and seven PAHs conveyed significant excess cancer risk at the Superfund site and in the general population, wherein PAH-contaminated seafood ingestion was a main route of exposure.

Most research tends to focus on tumor etiology and the effect of single chemicals, offering little insight into the effects of realistic complex mixture exposures on tumor progression. To address this issue, the final portion of this work investigated the effect of the previously mentioned AWI Superfund site-derived PAH-enriched EDC mixture in a panel of normal mammary epithelial cells and breast cancer cell models. Cells or organoids in culture were treated with EDC mixture at doses estimated from U.S. adult intake of the top four PAH compounds within the mixture from the National Health and Nutrition Examination Survey database. Physiologic doses of this PAH mixture (6, 30, 300nM) demonstrated increased aryl hydrocarbon receptor (AhR) expression and CYP activity in estrogen receptor (ER) positive, but not normal mammary epithelial cells or ER negative breast cancer cells. In addition, upregulated AhR signaling corresponded with increased cell proliferation and expression of anti-apoptotic and antioxidant proteins XIAP and SOD1. A mathematical model was employed to validate PAH-mediated increases in AhR and XIAP expression in the MCF-7 ER-positive cell line. Furthermore, the PAH mixture caused significant growth increases in ER-negative breast cancer cell derived 3D tumor organoids, providing further evidence for the role of a real world-derived PAH mixture in enhancing a tumor proliferative phenotype.

Results from these studies have implications for cancer control and prevention and targeted PAH exposure reduction initiatives by identifying vulnerable subpopulations, and further identify key consequences of PAH exposure and how it may contribute to breast cancer biology and progression.

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 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.

Organisms are routinely subjected to a variety of environmental and chemical perturbations simultaneously. Often, these multi-stressor exposures result in unpredictable toxicity that occurs through unidentified mechanisms. Here, we focus on polycyclic aromatic hydrocarbons (PAHs) and hypoxia, two environmental and physiological stressors that are known to co-occur in the environment.

Simultaneous exposures to PAHs and hypoxia result in exacerbated toxicological phenotypes in several fish species. For example, while exposures to the individual stressors resulted in few observed effects, zebrafish embryos simultaneously exposed to hypoxia and individual PAHs as well as PAH mixtures developed severe cardiovascular deformities and trunk abnormalities. This dissertation aims to better understand how these two stressors interact, what other phenotypes of toxicity are associated with co-exposures, and how wild-caught fish, including PAH-resistant fish, respond to co-exposures of PAHs and hypoxia.

Several locations in the Elizabeth River (VA, USA) are highly contaminated with PAHs, due to the release of creosote mixtures from wood treatment facilities. Interestingly, some populations of Atlantic killifish (Fundulus heteroclitus) inhabiting the Elizabeth River (ER) are resistant to PAH-induced teratogenesis. However, evolutionary resistance to PAHs due to chronic PAH exposure is associated with reduced fitness and increased susceptibility to other environmental stressors in at least one PAH-resistant killifish population. The first objective of this dissertation was to investigate this association further by examining a previously under-studied population captured from a creosote-contaminated site in the Elizabeth River, Republic (Rep). We assessed PAH toxicity and effects on energy metabolism in Rep killifish in comparison with killifish from the reference site Kings Creek (KC). We also investigated the effects of hypoxia as a secondary stressor on KC and Rep killifish; we assessed phenotypes of co-exposure in killifish, as well as whether PAH-resistance inferred greater vulnerability to hypoxia during embryonic development.

Initially, killifish captured from the Rep and KC sites were exposed to an individual PAH-type chemical, beta-naphthoflavone (BNF), a simple mixture of benzo(a)pyrene (BaP) and fluoranthene (FL) or a real-world complex PAH mixture known as Elizabeth River sediment extract (ERSE). Wild-caught adults and F1 embryos were assessed for CYP1A activity, F1 embryos were assessed for cardiovascular deformities and alterations in bioenergetic metabolism, and F1 juveniles were also assessed for alterations in bioenergetic metabolism. Following exposures to simple and complex PAH mixtures, Rep killifish exhibited several phenotypes associated with PAH resistance including decreased incidences of developmental cardiovascular deformities and recalcitrant cytochrome P450 1A (CYP1A) activity, results which led us to classify them as “PAH-resistant.” Rep embryos and juveniles also exhibited signs of altered metabolic rates. Rep embryos had significantly higher routine metabolism compared to KC embryos and juvenile Rep killifish had significantly lower maximal metabolic rates and aerobic scopes than KC juveniles. These results suggest that populations of killifish that have adapted or evolved to withstand the toxicity associated with PAHs consequently have altered energetic metabolism or demands. Such consequences could result in an enhanced vulnerability to other environmental and anthropogenic stressors in PAH-resistant killifish.

We further explored this hypothesis by subjecting Rep and KC killifish to hypoxia as a secondary stressor. Again, Rep and KC killifish were exposed to ERSE, this time with and without diurnal hypoxia for the extent of embryonic development (14 days). Killifish embryos were assessed for CYP1A activity, cardiovascular deformities, heart rate, hatch rate, larval body length, and other larval abnormalities.

Hypoxia suppressed the induction of CYP1A usually seen with PAH exposures. However, it did not exacerbate the frequency or severity of cardiovascular deformities in either population. Regardless, hypoxia exposure lowered the heart rate of embryos from both killifish populations. Additionally, there were severe effects of co-exposures on hatch rates, which was especially evident in Rep embryos.

However, PAH-resistant embryos appeared to be at an advantage when the secondary stressor was hypoxia: Rep embryos co-exposed to hypoxia and PAHs had greater rates of survival and hatch rates, and fewer incidences of severe cardiovascular deformities than KC embryos. These results directly contrast with other work which has found that larval PAH-resistant killifish are more susceptible to hypoxia than larvae from reference killifish populations. While PAH and hypoxia co-exposures did not cause interactive toxicity in many of the endpoints assessed, the effects of co-exposures on hatch rates and heart rates could have severe implications for embryonic bioenergetics and larval survival, leaving exposed embryos more vulnerable to other types of stress or unable to meet energetic demands.

The second objective of this dissertation, which utilized zebrafish (Danio rerio) as a model organism, was to identify other pathways that could either influence or be influenced by co-exposures of PAHs and hypoxia. Endpoints of interest targeted the oxidative stress response and mitochondrial function due to their known roles in PAH and hypoxia toxicity and visible effects on bioenergetics seen during completion of the previous objective. We hypothesized that co-exposures would not only cause interactive toxicity in these two pathways, but that these pathways might play a role in the exacerbated toxicity seen with co-exposures.

Zebrafish embryos were exposed to ERSE and hypoxia, both simultaneously and individually. They were assessed for a range of endpoints targeting mitochondrial function and oxidative stress directly after removal from 24-hour exposures and up to 18 hours of recovery time in clean media; assays used in this work included measurements of oxygen consumption and mitochondrial function using the Seahorse Extracellular Flux Analyzer, mitochondrial DNA damage and copy number, ATP content, mitochondrial-specific superoxide production, apoptosis, antioxidant enzyme activity, alterations in gene expression, and developmental deformities.

Hypoxia exposures resulted in drastic reductions in parameters relating to mitochondrial respiration, ATP turnover, and mitochondrial DNA copy number. Hypoxia exposures also resulted in increases in superoxide radical generation and inductions of catalase enzyme activity. PAH exposures reduced embryonic ATP production and content, altered mitochondrial membrane dynamics, and induced DNA damage. They also resulted in inductions of glutathione reductase and glutathione-s-transferase enzyme activity indicating an alteration in glutathione usage or balance in exposed embryos.

However, while PAH and hypoxia exposures caused a diverse array of effects, there appeared to be very little interaction between the two stressors in the co-exposure group. Co-exposed embryos exhibited phenotypes associated with both types of exposures, but there were rarely any visible interactions due to co-exposures. Regardless, both pathways are clearly responsive to individual exposures and co-exposures of PAHs and hypoxia. Additionally, co-exposed embryos exhibited toxicity associated with both stressors, which indicates that the co-exposures pose a greater risk to organismal health compared to exposures of the individual stressors alone.

Combined, these results highlight several key factors regarding co-exposures of not just PAHs and hypoxia, but potentially other multiple-stressor scenarios as well: 1) co-exposures can cause toxicity through unexpected mechanisms and result in unpredictable toxicological phenotypes; 2) developmental stage during exposures and timing of exposures are often critical in determining the toxic effects of stressors in developing organisms; and 3) even similarly related model organisms can exhibit different responses to the same exposures. Therefore, it is extremely important that we continue to investigate different combinations of stressors with a broad array of model organisms and a range of developmental time points in order to truly understand how multiple stressor scenarios will impact ecosystem health.