Browsing by Subject "polycyclic aromatic hydrocarbon"
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Item Open Access Biodegradation of a Sulfur-Containing PAH, Dibenzothiophene, by a Mixed Bacterial Community(2009) Cooper, Ellen M.Dibenzothiophene (DBT) is a constituent of creosote and petroleum waste contamination, it is a model compound for more complex thiophenes, and its degradation by mixed microbial communities has received little attention. The chemical characteristics, environmental fate and ecotoxicology of DBT degradation products are not well understood. This research investigated DBT degradation in an enrichment culture derived from creosote-contaminated estuarian sediment using a suite of assays to monitor bacterial populations, bacterial growth, degradation products, DBT loss, and toxicity. Ultraviolet (UV) irradiation was evaluated as a sequential treatment following biodegradation. Additionally, to advance SYBR-Green qPCR methodology for characterizing mixed microbial communities, an alternative approach for evaluating qPCR data using a sigmoidal model to fit the amplification curve was compared to the conventional approach in artificial mixed communities. The overall objective of this research was to gain a comprehensive understanding of the degradation of a model heterocyclic PAH, DBT, by a mixed microbial community, particularly within the context of remediation goals.
DBT biodegradation was evaluated in laboratory scale cultures with and without pH control. The microbial community was monitored with 10 primer sets using SYBR-Green quantitative polymerase chain reaction (qPCR). Twenty-seven degradation products were identified by gas chromatography and mass spectrometry (GC/MS). The diversity of these products indicated that multiple pathways functioned in the community. DBT degradation appeared inhibited under acidic conditions. Toxicity to bioluminescent bacteria Vibrio fischeri more than doubled in the first few days of degradation, was never reduced below initial levels, and was attributed in part to one or more degradation products. UV treatment following biodegradation was explored using a monochromatic (254 nm) low-pressure UV lamp. While DBT was not extensively photooxidized, several biodegradation products were susceptible to UV treatment. At higher doses, UV treatment following DBT biodegradation exacerbated cardiac defects in Fundulus heteroclitus embryos, but slightly reduced toxicity to V. fischeri.
This research provides a uniquely comprehensive view of the DBT degradation process, identifying bacterial populations previously unassociated with PAH biodegradation, as well as potentially hazardous products that may form during biodegradation. Additionally, this research contributes to development of unconventional remediation strategies combining microbial degradation with subsequent UV treatment.
Item Open Access Mitochondria as a Target of Benzo[a]pyrene Toxicity in a PAH-adapted and Naive Population of the Atlantic Killifish (Fundulus Heteroclitus)(2009) Jung, DawoonPolycyclic 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.
Item Open Access Molecular Mechanisms Underlying Adaptation to PAHs in Fundulus heteroclitus(2010) Clark, BryanChronic 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.
Item Open Access Persistent Effects of Polycyclic Aromatic Hydrocarbon Exposure Across Generations: a Bioenergetic and Mitochondrial Perspective(2018) Kozal, Jordan SierraPolycyclic 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.