Browsing by Subject "Toxicology"
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Item Open Access Characterizing and predicting the interaction of proteins with nanoparticles(2023) Poulsen, KarstenNanoparticles are being used or implemented in a wide array of applications including health care, cosmetics, automotive, food, beverage, coatings, consumer electronics, and coatings. Despite this widespread use, we are unable to predict how nanoparticles will interact with biological systems. This is important for both nanotoxicity resulting from human exposure to nanomaterials and the development of new nano-based biotechnologies. The first step in the interaction of nanoparticles with biological systems is often with blood, for biomedical applications, or lung fluid, when nanoparticles are inhaled. In both cases, the nanoparticles are exposed to proteins that form a "corona" by adsorbing on the nanoparticle surface. The subsequent cellular response is determined by this protein corona rather than the bare nanoparticle.Our goal is to develop a predictive capability for protein-nanoparticle interactions. This requires lab automation, large datasets, machine learning, and mechanistic studies. We first developed and validated a semi-automated approach to generate, purify, and characterize protein coronas using a liquid handling robot and low-cost proteomics. Using this semi-automated approach, we characterized the formation of protein coronas with increasing incubation time and serum concentration. Incubation time was found to be an important parameter for corona composition and concentration at high incubation concentrations but yielded only a small effect at low serum incubation concentrations. To better understand how the protein corona affects biological responses, we investigated the response of macrophage cells to titanium dioxide nanoparticles as a function of the protein corona. As in our previous work with serum proteins, we measured the concentration and composition of murine lung fluid proteins adsorbed on the surface of titanium dioxide nanoparticles. We found that a low concentration of lung fluid corona, relative to fetal bovine serum and bovine serum albumin coronas, led to an increased expression of cytokines (interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and macrophage inflammatory protein 2 (MIP-2)), indicating an inflammation response. This underscores the importance of understanding how the composition and concentration of the protein corona governs organism responses to nanoparticle exposures. Our validated high-throughput lab automation work allowed us to synthesize a library of magnetic nanoparticles and characterize their resulting protein coronas. The resulting nanoparticle dataset has 12 unique NP surfaces, seven surface charges, two core sizes, and two core materials. We used this dataset to generate and characterize, via proteomics, a variety of protein coronas varying incubation concentration and purification methods. We used the resulting proteomic dataset in conjunction with a database of protein physicochemical properties to build a machine learning model that identifies the most important nanoparticle and protein properties for protein corona formation. The model was tested using external datasets and found that it can predict human serum and lung fluid coronas on varying nanoparticle surfaces. Overall, this combination of lab automation, machine learning, and mechanistic analysis suggests that a generalizable understanding of the protein corona formation and its effects is forthcoming.
Item Open Access Characterizing Environmental Per- and Polyfluoroalkyl Substance (PFAS) Exposure and Effects in North Carolina Communities(2022) Hall, Samantha MariePer- and polyfluoroalkyl substances (PFAS) are synthetic chemicals used in a wide array of products and applications (e.g., nonstick cookware, waterproof and water-repellent textiles, firefighting foam). Following their decades of use, PFAS have garnered concern as “forever chemicals” due to their extreme persistence in the environment and in humans. PFAS have further elicited concern because they have been linked to adverse health effects in humans, and their huge number (over 12,000 different chemicals) and complex chemistry make them very challenging to analyze and study for exposure and toxicology. Two particular PFAS chemicals, perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS), are drinking water contaminants that can be found in the blood of the vast majority of people. PFOA and PFOS are also linked to toxic effects like kidney and testicular cancer, increased blood cholesterol, and reproductive outcomes. These two chemicals are being phased out of use and federal drinking water standards are likely upcoming. However, the replacements for these two chemicals are much less well-characterized, and many of these newer, replacement PFAS chemicals can be found in the environment of North Carolina due (at least in part) to industrial pollution.
The overarching goal of this dissertation was to characterize the potential exposure and health effects of PFAS in North Carolina communities. The surface water and drinking water in some areas of North Carolina have been found to be contaminated with PFAS; however, there are additional routes of PFAS exposure beyond drinking water, such as ingestion of house dust or placental transfer during pregnancy. This dissertation explores various routes of PFAS exposure and better characterizes the specific PFAS analytes that can be found in North Carolina and the concentrations in which they are present. Additionally, this dissertation evaluates this exposure and potential associations with some adverse health outcomes in a few North Carolina communities.
In Chapter 2, the relationships between PFAS exposure during pregnancy and birth outcomes are explored. This chapter includes data on PFAS concentrations in placenta samples from 120 participants in Durham, North Carolina and evaluates the subsequent associations between placental PFAS exposure and birth outcomes (e.g., infant birth weight, gestational age). A total of 11 PFAS were measured in placental tissues collected in 2010-2011, and the compounds PFOS, PFOA, PFNA, and PFDA were detected in all placenta samples. A few placental PFAS were associated with birth outcomes. The most striking result was that placental PFOS was associated with changes in birth weight, but the direction of change depended on the sex of the infant. For male infants, placental PFOS was associated with lower birthweight, and in female infants, placental PFOS was associated with higher birthweight.
In Chapter 3, the exposure to PFAS through drinking water is evaluated in a community with known PFAS water contamination. This chapter includes data on PFAS concentrations in blood serum and drinking water samples from 49 participants in Pittsboro, North Carolina. The community receives its drinking water from the Haw River, a part of the Cape Fear River watershed. Blood and water samples were collected at two different timepoints to explore temporal variability in contamination. This community was found to have blood levels of PFAS about two to four times higher than the U.S. average. This chapter also includes results on the associations between PFAS blood level and clinical chemistry measurements, such as serum lipids, as indicators of health. Negative associations were found between serum PFOS and PFHxA with decreased electrolytes and decreased liver enzymes. Positive associations were found between serum PFOA and PFHxS with increased total cholesterol and increased non-HDL cholesterol.
In Chapter 4, the effects and toxicokinetics of PFAS in a pregnant rabbit model are evaluated. This chapter includes data from an animal study of 21 pregnant rabbits provided with drinking water that is representative of the PFAS exposure observed in Pittsboro, North Carolina. Rabbits were exposed to this environmentally-relevant mixture of ten different PFAS during and before pregnancy. After exposure, the wastes and tissues were evaluated to measure the PFAS concentration that accumulated. This provided information on where PFAS are distributed in the body after exposure. The liver of the pregnant rabbit was also evaluated to determine if there was an increase in lipids in the liver, or any changes in liver lipid metabolism. For this study, few differences were noted between treated animals and control animals, indicating that the environmentally-relevant dose had little effect on pregnant rabbits. However, due to the lack of PFAS accumulation in blood, tissue, or in wastes, it is likely that the dose of PFAS given through drinking water was too low.
In Chapter 5, the levels of PFAS in indoor house dust were evaluated. This chapter includes data on PFAS concentrations in indoor dust from 184 homes in Durham, North Carolina, as well as 49 fire stations across the U.S. and Canada. House dust and fire station dust PFAS concentrations were then evaluated for associations with characteristics of the building (e.g., square footage, amount of carpeting, age of building construction). Levels of precursor PFAS, such as fluorotelomer alcohols, were typically higher in dust than the perfluoroalkyl acids. This study, along with previous literature, shows that the legacy PFAS in dust has been decreasing, but the precursor PFAS has been increasing in U.S. house dust. Few associations were found between building characteristics and dust PFAS. However, one notable result was that higher 8:2 FTOH was found in dust from buildings with more carpeting, indicating that carpets may be an important source of exposure to fluorotelomer alcohols (possibly from stain-proofing treatment).
Collectively, this dissertation provides important information on the potential exposure and health effects of PFAS in North Carolina communities.
Item Open Access Characterizing the Binding Potential, Activity, and Bioaccessibility of Peroxisome Proliferator Activated Receptor Gamma (PPARγ) Ligands in Indoor Dust(2015) FANG, MINGLIANGAccumulating evidence is suggesting that exposure to some environmental contaminants may alter adipogenesis, resulting in accumulation of adipocytes, and often significant weight gain. Thus these types of contaminants are often referred to as obesogens. Many of these contaminants act via the activation (i.e. agonism) of the peroxisome proliferator activated receptor γ (PPARγ) nuclear receptor. To date, very few chemicals have been identified as possible PPAR ligands. In the thesis, our goal was to determine the PPARγ ligand binding potency and activation of several groups of major semi-volatile organic compounds (SVOCs) that are ubiquitously detected in indoor environments, including flame retardants such as polybrominated diphenyl ethers (PBDEs) and Firemaster 550 (FM550), and other SVOCs such as phthalates, organotins, halogenated phenols and bisphenols. Additional attention was also given to the potential activity of the major metabolites of several of these compounds. Since the primary sink for many of these SVOCs is dust, and dust ingestion has been confirmed as an important pathway for SVOCs accumulation in humans, the potential PPAR binding and activation in extracts from environmentally relevant dust samples was also investigated.
Previous studies have also shown that SVOCs sorbed to organic matrices (e.g., soil and sediment), were only partially bioaccessible (bioavailable), but it was unclear how bioaccessible these compounds are from indoor dust matrices. In addition, bioactivation of SVOCs (via metabolism) could exacerbate their PPAR potency. Therefore, to adequately assess the potential risk of PPARγ activation from exposure to SVOC mixtures in house dust, it is essential that one also investigates the bioaccessibility and bioactivation of these chemicals following ingestion.
In the first research aim of this thesis, the bioaccessibility and bioactivation of several important SVOCs in house dust was investigated. To accomplish this, Tenax beads (TA) encapsulated within a stainless steel insert were used as an infinite adsorption sink to estimate the dynamic absorption of a suite of flame retardants (FRs) commonly detected in indoor dust samples, and from a few polyurethane foam samples for comparison. Experimental results demonstrate that the bioaccessibility and stability of FRs following ingestion varies both by chemical and by matrix. Organophosphate flame retardants (OPFRs) had the highest estimated bioaccessibility (~80%) compared to brominated compounds (e.g. PBDEs), and values generally decreased with increasing Log Kow, with <30% bioaccessibility measured for the most hydrophobic compound tested, BDE209. In addition, the stability of the more labile SVOCs that contained ester groups (e.g. OPFRs and 2-ethylhexyl-tetrabromo-benzoate (TBB)) were examined in a simulated digestive fluid matrix. No significant changes in the OPFR concentrations were observed in this fluid; however, TBB was found to readily hydrolyze to tetrabromobenzoic acid (TBBA) in the intestinal fluid in the presence of lipases.
In research aims 2 and 3, two commercially available high-throughput bioassays, a fluorescence polarization PPAR ligand binding assay (PolarScreenTM PPARγ-competitor assay kit, Invitrogen, Aim 2) and a PPAR reporter gene assay (GeneBLAzer PPARγ non-DA Assay, Invitrogen, Aim 3) were used to investigate the binding potency and activation of several groups of SVOCs and dust extracts with human PPARγ LBD; respectively. In the PPAR binding assay (Aim 2), most of the tested compounds exhibited dose-dependent binding to PPARγ. Mono(2-ethylhexyl) tetrabromophthalate (TB-MEHP), halogenated bisphenol/phenols, triphenyl phosphate and hydroxylated PBDEs were found to be potent or moderate PPARγ ligands, based on the measured ligand binding dissociation constant (Kd). The most potent compound was 3-OH-BDE47, with an IC50 of 0.24 μM. The extent of halogenation and the position of the hydroxyl group strongly affected binding. Of the dust samples tested, 21 of 24 samples showed significant PPAR binding potency at a concentration of 3 mg dust equivalents (DEQ)/mL. In the PPAR reporter assay (Aim 3), many SVOCs or their metabolites were either confirmed (based on previous reports) or for the first time were found to be potential PPARγ agonists with various potency and efficacy. We also observed that 15 of 25 dust extracts examined showed an activation percentage more than 8% (calculated activation threshold) of the maximal activation induced by rosiglitazone (positive control). In some cases, activation was as high as 50% of the rosiglitazone activation for the dust extracts with the highest efficacy. Furthermore, the correlation between the reporter assay and the ligand binding assay among the house dust extracts was significant and positive (r = 0.7, p < 0.003), suggesting the binding potency was predicting activation. In research aim 2, the effect of bioactivation on the PPARγ binding potency was also investigated. In vitro bioactivation of house dust extracts incubated with rat and human hepatic S9 fractions was used to investigate the role of in vivo biotransformation on PPAR gamma activity. The result showed that metabolism may lead to an increased binding affinity, as a 3-16% increase in PPARγ binding activity was observed following bioactivation of the dust extracts.
In research aim 4, an effect-directed analysis (EDA) was used to identify compounds likely contributing to the observed PPAR activity among the dust extract. Three dust extracts which showed significant PPAR activity with approximately 25, 30, and 50% of the maximal response induced by rosiglitazone at the highest efficacy were fractionated using normal phase high-performance liquid chromatography (NP-HPLC) and each fraction was individually tested for PPAR activity. Active fractions were then analyzed using gas-chromatography mass spectrometry (GC-MS) and possible compounds identified. Three dust extracts showed a similar PPAR activity distribution among the NP-HPLC fractions. In the most active fractions, fatty acids (FAs) were identified as the most active chemicals. The concentrations of four FAs were measured in the house dust extracts, and the concentrations were found to be highly correlated with the observed PPAR activity. These four FAs were also tested for PPAR activity and found to be partial PPAR agonists, particularly oleic and myristic acid. To tentatively identify sources of FAs, FAs in human/animal hair, dead skin cells, and two brands of cooking oil were analyzed. We found the same FAs in those samples and there concentrations were relatively abundant, ranging from 186 to 14,868 µg/g. Therefore, these results suggest that FAs are likely responsible for the observed PPAR activity in indoor dust. Also, this is the first study reporting on the level of FAs in dust samples. The source of these FAs in dust may be either from the cooking or accumulation of human/animal cells in indoor dust.
In conclusion, this research demonstrates that many SVOCs ubiqutiously detected in house dust, and/or their metabolites, can be weak or moderate PPAR ligands. In addition, chemical mixtures in house dust can effectively bind to and activate PPAR. However, our results suggest FAs are probably responsible for these observations, and likely outcompeting the synthetic environmental contaminants present in the dust extract. Furthermore, bioactivation of contaminants present in house dust can potentially increase their affinity for PPAR. And lastly, the bioaccessibility and stability of SVOCs in house dust after ingestion are likely to modulate the PPAR activity in the environmental mixtures and should be considered in future risk assessments.
Item Open Access Contaminant Interactions and Biological Effects of Single-walled Carbon Nanotubes in a Benthic Estuarine System(2013) Parks, AshleySingle-walled carbon nanotubes (SWNT) are highly ordered filamentous nanocarbon structures. As their commercial and industrial use becomes more widespread, it is anticipated that SWNT will enter the environment through waste streams and product degradation. Because of their highly hydrophobic nature, SWNT aggregate and settle out of aqueous environments, especially in saline environments such as estuaries. Therefore, sediments are a likely environmental sink for SWNT once released. It is important to understand how these materials will impact benthic estuarine systems since they are the probable target area for SWNT exposure in addition to containing many lower trophic level organisms whose survvial and contaminant body burdens can have a large impact on the overall ecosystem. Disruptions in lower trophic level organism survival can have negative consequences for higher trophic levels, impacting the overall health of the ecosystem. It is also important to consider contaminant bioaccumulation, trophic transfer and biomagnification. If SWNT are taken up by benthic invertebrates, there is the possibility for trophic transfer, increasing the exposure of SWNT to higher trophic level organisms that otherwise would not have been exposed. If this type of transfer occurs in environmentally important species, the potential for human exposure may increase. My research aims to determine the magnitude of the toxicity and bioaccumulation of SWNT in benthic estuarine systems, as well as determine how they interact with other contaminants in the environment. This research will contribute to the knowledge base necessary for performing environmental risk assessments by providing information on the effects of SWNT to benthic estuarine systems.
Before investigating the environmental effects of SWNT, it is imperative that a measurement method is established to detect and quantify SWNT once they enter the environment. This research utilized pristine, semiconducting SWNT to develop extraction and measurement methods to detect and quantify these specific materials in environmental media using near infrared fluorescence (NIRF) spectroscopy. Semiconducting SWNT fluoresce in the near infrared (NIR) spectrum when excited with visible&ndashNIR light. This unique optical property can be used to selectively measure SWNT in complex media.
The fate, bioavailability, bioaccumulation and toxicity of SWNT have not been extensively studied to date. Pristine SWNT are highly hydrophobic and have been shown to strongly associate with natural particulate matter in aquatic environments. In light of this, I have focused my research to examine the influence of sediment and food exposure routes on bioavailability, bioaccumulation, and toxicity of structurally diverse SWNT in several ecologically-important marine invertebrate species. No significant mortality was observed in any organism at concentrations up to 1000 mg/kg. Evidence of biouptake after ingestion was observed for pristine semiconducting SWNT using NIRF spectroscopy and for oxidized 14C&ndashSWNT using liquid scintillation counting. After a 24 hour depuration period, the pristine semiconducting SWNT were eliminated from organisms to below the method detection limit (5 &mug/mL), and the 14C&ndashSWNT body burden was decreased by an order of magnitude to a bioaccumulation factor (BAF) of <0.01. Neither pristine SWNT nor oxidized 14C&ndashSWNT caused environmentally relevant toxicity or bioaccumulation in benthic invertebrates. Overall, the SWNT were not bioavailable and appear to associate with the sediment.
In addition to investigating the toxicity and bioaccumulation of SWNT as an independent toxicant, it is important to consider how they will interact with other contaminants in the environment (i.e., increase or decrease toxicity and bioaccumulation of co&ndashcontaminants, alter the environmental transport of co&ndashcontaminants, induce degradation of co&ndashcontaminants, etc.). I wanted to investigate the effects of SWNT on a complex mixture of contaminants already present in a natural system. New Bedford Harbor (NBH) sediment, which is contaminated with polychlorinated biphenyls (PCBs), was amended with pristine SWNT to determine if the presence of SWNT would mitigate the toxicity and bioaccumulation of the PCBs in deposit-feeding invertebrates. A dilution series of the NBH sediment was created using uncontaminated Long Island Sound (LIS) sediment to test 25% NBH sediment, 50% NBH sediment, 75% NBH sediment, and 100% NBH sediment. The results of this work showed increased organism survival and decreased bioaccumulation of PCBs in treatments amended with SWNT, with the greatest reduction observed in the 25% NBH sediment treatment group amended with 10 mg SWNT/g dry sediment. Polyethylene (PE) passive samplers indicated a reduction of interstitial water (ITW) PCB concentration of greater than 90% in the 25% NBH sediment + 10 mg SWNT/g dry sediment amendment. The ITW concentration was reduced because PCBs were not desorbing from the SWNT. Lower bioavailability leads to reduced potential for toxic effects, supporting the observation of increased survival and decreased bioaccumulation. Once in the sediment, not only are SWNT not bioavailable, they act as a highly sorptive phase, such as black carbon (BC), into which hydrophobic organic contaminants (HOCs), such as PCBS and polycyclic aromatic hydrocarbons (PAHs), can partition, thereby reducing the toxicity and bioavailability of co-occurring HOCs.
To more fully understand the impact of SWNT in this environment, their biodegradability also needs to be investigated. Biodegradation of SWNT could lead to release and/or transformation of sorbed HOCs as well as a change in the inherent transport, toxicity, and bioaccumulation of SWNT in the estuarine environment. Because the persistence of SWNT will be a primary determinant of the fate of these materials in the environment, I conducted experiments to determine if the fungus Trametes versicolor, the natural bacterial communities present in NBH sediment, and municipal wastewater treatment plant sludge could degrade or mineralize oxidized 14C&ndashSWNT. Over a six month time period, no significant degradation or mineralization was observed. In all treatments, approximately 99% of the 14C-SWNT remained associated with the solid phase, with only approximately 0.8% of added 14C present as dissolved species and only 0.1% present as 14CO2. These small pools of non-SWNT 14C were likely due to trace impurities, as no differences in production were observed between treatments and abiotic (killed) controls.
Item Open Access Corrigendum to "The Developmental Neurotoxicity of Tobacco Smoke Can Be Mimicked by a Combination of Nicotine and Benzo[a]pyrene: Effects on Cholinergic and Serotonergic Systems".(Toxicological sciences : an official journal of the Society of Toxicology, 2019-03) Slotkin, Theodore A; Skavicus, Samantha; Ko, Ashley; Levin, Edward D; Seidler, Frederic JThe correct units in Tables S3, S5, and S6 are "fmol/mg protein," whereas no units should be specified in Table S4, which presents data as a ratio.Item Open Access Developmental Neurotoxicity of Silver and Silver Nanoparticles Modeled In Vitro and In Vivo(2010) Powers, Christina MarieBackground: Silver nanoparticles (AgNPs) act as antimicrobials by releasing monovalent silver (Ag+) and are increasingly used in consumer products, thus elevating exposures in human and environmental populations. Materials and Methods: We evaluated Ag+ in a standard model of neuronal cell replication and differentiation, and then determined whether there were similar effects of the ion in vivo using zebrafish. Next, we compared Ag+ and AgNP exposures in the same two models and incorporated the effects of particle coating, size and composition. Conclusions: This work is the first to show that both Ag+ and AgNPs are developmental neurotoxicants in vitro and in vivo. Moreover, although both the soluble ion and the particles impair measures of neurodevelopment, the outcomes and underlying mechanisms of each toxicant are often wholly distinct. Superimposed on the dichotomies between Ag+ and AgNP exposures are clear effects of particle coating, size and composition that will necessitate evaluation of individual AgNP types when considering potential environmental and human health effects. The results presented here provide hazard identification that can help isolate the models and endpoints necessary for developing a risk assessment framework for the growing use of AgNPs.
Item Open Access Effects of chemical exposures on mitochondrial mutagenesis across species(2022) Leuthner, Tess CMitochondria are essential organelles required for all eukaryotic life on earth. Each organelle contains multiple copies of the mitochondrial genome (mtDNA) that encodes genes essential for energy production. Mutations in the mitochondrial genome are associated with mitochondrial diseases and diseases of aging, particularly neurodegenerative diseases, such as Parkinson’s Disease, and cancer. mtDNA mutation rates are often higher than nuclear DNA mutation rates. However, the origin of mtDNA mutations is poorly understood. Mitochondria lack many of the basic DNA repair mechanisms that are in the nucleus, potentially rendering mtDNA vulnerable to DNA damage-induced mtDNA mutations. Very few studies have investigated the impact of chemical exposures, in particular pollutants, on mtDNA mutagenesis, as reviewed in Chapter 4 of this dissertation (published as Leuthner and Meyer, 2021). Therefore, the two research aims of this thesis were designed to investigate the role of chemical exposures on mtDNA damage and mutagenesis in two species, chosen based on particular research strengths that each offered. The overarching hypothesis of this dissertation was that exposure to known nuclear genotoxicants and mutagens would result in the accumulation of mtDNA damage, which would ultimately lead to mtDNA mutations. The first aim of this thesis was to investigate the impact of the ubiquitous pollutant, cadmium (Cd), on mtDNA mutagenesis in the aquatic keystone species, Daphnia pulex. Cd is a known nuclear mutagen and carcinogen, yet the effects of Cd exposure on mtDNA mutations remain unknown. D. pulex offers a number of major advantages for this investigation, as discussed in Chapter 2. A unique aspect for this thesis was the use of a wild, Cd-tolerant population of D. pulex. Remarkably, this population of D. pulex sampled from Simon Lake in Sudbury, Ontario, Canada has adapted to high levels of Cd due to over a century of exposure to pollution from mining and smelting processes. Thousands of generations of experimental evolution were performed under laboratory conditions (an approach termed Mutation Accumulation, or MA, lines) in a Simon Lake isolate and an isolate collected from pristine Buck Lake (Dorset, Ontario, Canada) in both the presence and absence of Cd. This allowed investigation of the effects of Cd on mtDNA mutagenesis in D. pulex populations with very different evolutionary histories. Whole genome sequencing was conducted and mtDNA reads were extracted for analysis of mtDNA mutation frequencies, rates, and signatures. Hundreds of single nucleotide mutates were detected after >2,000 and >12,000 total generations of mutation accumulation, or about 40 to 250 fold more mutations than previous Daphnia mtDNA MA line studies. This afforded the resolution to determine the mechanism of endogenous mtDNA mutagenesis in Daphnia for the first time; these results indicate that endogenous mtDNA mutagenesis is likely mostly driven by polymerase γ error at sites of oxidized and deaminated cytosines (G A/C T). At the earlier timepoint, Cd exposure further increased the rate of this mutation in the Cd-sensitive Buke Lake Daphnia compared to the Cd-tolerant Simon Lake Daphnia by about 3.6-fold. The results of this research aim suggest that Cd has a small effect on mtDNA mutagenesis, and that the adapted population is resistant to Cd-induced mtDNA mutations. However, after an additional >10,000 generations of mutation accumulation, there was no effect of Cd on this mutation spectrum, and the number of mtDNA mutations that were present at very low frequency increased significantly compared to the earlier timepoint. A small number of mutations did reach fixation or near-fixation however, and these mutations are discussed individually in Chapter 2. We propose that this result is consistent with natural selection acting on germline mtDNA mutation rates and heteroplasmy. Mitochondria harbor various quality control mechanisms that act in response to stress and mitochondrial dysfunction, such as mitophagy, fission, and fusion. Previous studies indicate that mitophagy may be involved in purifying selection against deleterious mtDNA mutations, in addition to targeted degradation of organelles that contain damaged mtDNA. Therefore, the next research aim of this thesis was to investigate the role of mitophagy on the accumulation of mtDNA damage and mutagenesis after exposure to Cd and another genotoxin, Aflatoxin B1 (AfB1), in the organism Caenorhabditis elegans. C. elegans are often used for MA studies to investigate mutational processes in both the nuclear and mitochondrial genomes, and offer a variety of strengths for such studies, as discussed in Chapter 3. A particular strength for the purposes of this thesis was the ability to work with strains carrying mutations in mitophagy genes. A MA experiment was conducted in wild-type C. elegans and two mitophagy-deficient strains, dct-1 and pink-1, in control, 50µM Cd, and 10µM AfB1 conditions. AfB1 was selected because it causes mtDNA damage that is not expected to be efficiently repaired in mtDNA. After an average of 50 generations of MA, about 10 MA lines were selected for each strain/treatment combination for Duplex Sequencing. Duplex Sequencing is an ultra-sensitive, error-corrected sequencing approach that allows for detection of mutations as low as 1 in 10,000 base pairs. Until the preparation of these samples, no study had yet conducted targeted mtDNA Duplex Sequencing in C. elegans. Wild-type and mitophagy-deficient strains all had mutation spectra indicative of oxidative damage driving mtDNA mutagenesis (GT/CA), contrary to what was observed in Daphnia in Aim 1, and contrary to what has been reported in other organisms. However, this confirmed results from a very recent study that also used mtDNA targeted Duplex Sequencing of wild-type C. elegans. Surprisingly, even though more mtDNA mutations were detected and at a lower frequency than ever previously reported, there was no clear effect of either Cd of AfB1 exposure on mtDNA mutations in any strain, despite a marginally significant increase in G:C A:T mutations in pink-1 AfB1 MA lines compared to wild-type AfB1 MA lines. Overall, this suggests that mitochondria are resistant to exogenous damage-induced point mutations in C. elegans. Further investigations into what mechanisms are responsible for maintaining mtDNA homeostasis that are independent of mitophagy are an exciting future next step. Understanding the impact of chemicals on mtDNA mutations is critical for human and environmental health, as addressed in the Chapter 4 “Mitochondrial DNA mutagenesis: A feature of and biomarker for environmental health,” which has been published as a review. The primary research chapters of this dissertation (Chapters 2 and 3) further contribute to understanding how chemicals impact mitochondrial genome quality and integrity. Collectively, it will be critical to continue to use improved sequencing technologies to continue to investigate the origin and mechanisms of mtDNA mutagenesis resulting from both endogenous and exogenous factors.
Item Open Access Effects of Toxic Metal Exposures and Their Mixtures on Adverse Health Outcomes in the Peruvian Amazon(2022) Berky, AxelNon-essential trace metals (lead, mercury, arsenic, and cadmium) are ubiquitous in our environment and have overlapping routes of exposure, yet mixed trace metal exposures are rarely considered in epidemiological studies. Instead, research often follows a single research question that focuses on a single trace metal of concern and does not incorporate potential co-exposures. The published literature of artisanal small-scale mining in the Amazon is a prime example as it has predominantly focused on mercury exposure, due to its use in the mining process. Once exposures of concern are identified, further studies evaluate health outcomes; however, the health effects cannot be accurately determined without accounting for co-exposures. This verification is becoming more important as there is a growing recognition that mixed trace metal exposures are more common than previously believed.To address the prevalence of mixed trace metal exposures and their health effects in the Peruvian Amazon region of Madre de Dios, I use epidemiological data from the COhorte de NAcimiento de MAdre de Dios (CONAMAD) birth cohort study (2018-Present), and two cross-sectional epidemiological studies (Amarakaeri Communal Reserve study (ACR, 2015), and Etiology and Toxic Metals study (EATM, 2018)). CONAMAD collected survey data along with maternal and cord blood samples at birth, which were processed for minerals and trace metals. The cross-sectional studies collected venous blood for trace metal analysis and hair samples for total hair mercury. Blood samples from the ACR were also processed for amino acids. In-depth demographic and health survey data were collected in all three studies. Structural equation models and random mixed effect models were used to evaluate research questions. The cross-sectional studies demonstrate a high correlation of lead and mercury exposure in communities that rely on wild fish and wild game as protein sources, which is prevalent throughout the Amazon. Consuming a meal of wild game resulted in an estimated lead dose of 500 µg, with those who eat wild game (Yes/No) associated with 1.41 µg/dL (95% CI: 1.20 – 1.70) higher blood lead levels compared to those who do not. This furthers the notion that mixed exposures are likely more common than previously believed. Mixed exposures target the same toxicological pathway, which may lead to synergistic or antagonistic effects. My research found that lead disrupts the arginine pathway and is associated with increased blood pressure. Mercury exposure was a modifier of the arginine pathway, with high blood mercury levels changing the effect of global arginine bioavailability from 17.16 (95% CI: 9.09 – 25.84) to -14.17 (95% CI: -31.88 - -0.33) on systolic blood pressure. Interestingly, mercury was not directly associated with the arginine pathway. Results from the birth cohort demonstrate the importance of nutrition and prenatal care for fetal development, which had a large positive effect on birthweight and gestational age. However, even low maternal lead exposure had detrimental effects on fetal health. A 1% increase in maternal blood lead was associated with a shorter gestational age of 0.05 days (β: -0.75, 95% CI: -1.51 - -0.13), even with the CONAMAD birth cohort having lower blood lead levels than other birth cohorts. There is a need for an integrated approach of nutritional and exposure assessments to better understand neonatal health outcomes.
Item Open Access Endocrine and Neurobehavioral Effects from Flame Retardant Exposure in Early and Juvenile Life Stages of Zebrafish(2015) Macaulay, Laura JeanPolybrominated diphenyl ethers (PBDEs) are a class of flame retardant chemicals that were added to furniture foam, electronics, plastics, and some textiles to reduce their flammability. While PBDEs have been phased out from use in current products, huge reservoirs of products containing PBDEs still exist. It is likely exposure to PBDEs will continue as older products are discarded and recycled. PBDEs are ubiquitous contaminants in indoor and outdoor environments due to their widespread use in many products and their ability to migrate out of treated materials.
Major health effect concerns from PBDE exposure identified in laboratory studies include neurotoxicity, reproductive/developmental toxicity, and thyroid disruption. Importantly, mammals metabolize PBDEs into the hydroxylated polybrominated diphenyl ethers (OH-BDEs), which are structurally similar to endogenous thyroid hormones. Thyroid hormones are essential for metabolic processes, growth, and development, particularly brain development. Multiple studies have demonstrated enhanced potency of OH-BDEs relative to the parent PBDE chemicals, particularly for neurodevelopmental processes. Additionally, in fish species, thyroid hormones are essential for transitioning between larval, juvenile, and adult life stages. Therefore, studying the effects of both PBDEs and OH-BDEs during sensitive developmental life stages (i.e. larval and juvenile development) is warranted. The hypothesis of this thesis research is that PBDE metabolites interfere with thyroid hormone signaling (through interacting with thyroid receptor and deiodinase enzymes) which may result in decreased growth, morphological deficits, and altered neurodevelopment. The objectives of this research project were to evaluate the toxicity of PBDE metabolites and mixtures of PBDEs/OH-BDEs on larval and juvenile zebrafish development, examining both potential modes of action as well as functional consequences of exposure in developing animals.
In the first aim of this thesis research, structural relationships were examined between eleven different halogenated phenolic compounds (OH-BDEs, OH-PCBs, halogenated phenols, and TBBPA) to test developmental toxicity in zebrafish from 0-6 days post fertilization (dpf). In addition, follow up studies were performed with the most toxic compound, 6-hydroxy- 2,2’,4,4’-tetrabromodiphenyl ether (6-OH-BDE-47), to examine effects on TH-mediated morphological development and to better understand its mechanism of action in zebrafish. Thyroid disrupting agents including propylthiouracil, iopanoic acid, and native thyroid hormones were also used as positive controls for morphologic studies. Exposures to 6-OH-BDE-47 (10 nM to 100 nM) during development resulted in severe delays, similar to exposures from the T3 and thyroid disrupting agents. Lower jaw deformities and craniofacial cartilage malformations were also observed following exposure to 6-OH-BDE-47 at doses greater than 50 nM. Of interest, these developmental delays were rescued by overexpression of TRβ mRNA during the exposure period. These data indicate that OH-BDEs can adversely affect early life development of zebrafish and suggest they may be impacting thyroid hormone regulation in vivo through downregulation of the thyroid hormone receptor.
In the second aim of this dissertation research, neurobehavioral performance was monitored in larval and juvenile fish following a developmental exposure to 6-OH-BDE-47. 6-OH-BDE-47 has been identified as a neurotoxicant in previous cell based assays, and was identified as overtly toxic to zebrafish larvae in Aim 1 of this research. Developmental exposures (0-6 dpf) to 6-OH-BDE-47 resulted in decreased larval swimming activity at 6 dpf, with persisting impacts on behavior at 45 dpf. Young adult fish, when tested at 45 dpf, exhibited increased fear/anxiety response in the novel tank diving task and hyperactivity in a test of sensorimotor habituation. These data indicate that exposures to PBDE flame retardants and their metabolites during critical developmental windows can alter long term cognitive responses more than a month after the exposure has ceased.
Finally, for the third aim of this dissertation research, zebrafish undergoing larval-juvenile metamorphosis were exposed to a mixture of PBDEs (30-600 µg/L DE-71) and OH-BDEs (1-300 nM) from 9-23 dpf. Metamorphosis is a unique developmental period in fishes which is partially mediated by thyroid hormones. Juvenile animals, like larval animals, represent a sensitive and unique subpopulation of animals. At the end of the exposure period (23 dpf), a subset of fish were reared in clean water until 45 dpf for neurobehavioral testing. Fish samples were collected at 3 time points throughout the experiments, Days 12, 23, and 45. Tissue accumulation of test chemicals was monitored, and juvenile fish treated with the High Mixture were found to accumulate over 100 µg/g ww ∑PentaBDEs. The highest mixture treatment was found to be acutely toxic to zebrafish juveniles, resulting in >85% mortality within 14 days of exposure. Fish treated with 30 nM 6-OH-BDE-47 or the lower mixture exhibited reduced morphology scores relating to fin, pigmentation, and swim bladder maturation. In addition, reduced skeletal ossification and caudal area was observed at earlier time points with treatment to 6-OH-BDE-47. These alterations were accompanied by increases in chondrogenic gene expression, declines in osteogenic gene expression, and increases in thyroid receptor expression. Approximately 3.5 weeks after the exposure period, juvenile fish were tested on neurobehavioral tasks of novel tank exploration and sensorimotor habituation, however, no significant treatment related effects on task performance were observed. Collectively, these data suggested that the larval/juvenile development stage is a sensitive developmental window which can be adversely impacted by PBDE/OH-BDE exposure.
Item Open Access Engineering a Biofilm for the Biodegradation of Polycyclic Aromatic Hydrocarbons in Estuarine Sediment(2019) Volkoff, SavannahPolycyclic 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.
Item Open Access Environmental toxicant effects on the dopaminergic system in the nematode Caenorhabditis elegans: Strengths and limitations of various approaches to analysis(2019) Smith, Latasha LIn order to develop a better understanding of the role environmental toxicants may play in the onset and progression of neurodegenerative diseases, it has become increasingly important to optimize sensitive methods for quickly screening toxicants to determine their ability to disrupt neuronal function. The model organism Caenorhabditis elegans can help with this effort. This species has an integrated nervous system producing behavioral function, allows for easy access for molecular studies, has a rapid lifespan, and is an inexpensive model. This project focuses on the development and optimization of a low-cost efficient behavioral tool for the identification of environmental toxicants with actions that disrupt dopamine function in the nematode C. elegans. Several dopamine-mediated locomotory behaviors, Area Exploration, Body Bends, and Reversals, as well as Swimming-Induced Paralysis and Learned 2-Nonanone Avoidance, were compared to determine the best behavioral method for screening purposes. These behavioral endpoints were also compared to morphological scoring of neurodegeneration in the dopamine neurons. We found that in adult worms, Area Exploration is more advantageous than the other behavioral methods for identifying DA-deficient locomotion and is comparable to neuromorphological scoring outputs. For larval stage worms, locomotion was an unreliable endpoint, and neuronal scoring appeared to be the best method. We compared the commonly used dat-1p::GFP reporter strains BY200 and BZ555, and we further characterized the dopamine-deficient strains, cat-2 e1112 and cat-2 n4547. We found that BY200 and BZ555 have differential responses in DA-targeted neurodegeneration using the model toxicant 6-hydroxydopamine, with BY200 appearing to have somewhat increased sensitivity to treatment. In contrast to published results, we found that the cat-2 strains slowed on food almost as much as N2s. Rather than being null mutants, both showed decreased levels of cat-2 mRNA and DA content, with cat-2 e1112 having the greatest reduction in DA content in comparison to N2. Ultimately, we concluded that while behavioral analysis is a promising endpoint, it is probably best used as a quick screening tool, with follow-up studies assessing affects to neuron morphology. A more informative readout of toxicant effects to the DA system would be best accomplished using a combination of the assays outlined in this project. We offer a table at the conclusion of this study comparing strengths, limitations, costs, and equipment needs for the methods of analysis of the dopaminergic system in C. elegans undertaken in this project.
Item Open Access Exposure, Metabolism, and in Vitro Effects of Isopropylated and Tert-butylated Triarylphosphate Ester (ITP & TBPP) Flame Retardants and Plasticizers(2019) Phillips, AllisonFollowing the phase-out of polybrominated diphenyl ethers (PBDEs) in the early 2000s, organophosphate esters (OPEs) emerged as PBDE substitutes and have been applied to furniture foam, electronics, building materials, and some plastics to reduce their flammability. Although they have been used for quite some time in hydraulic fluids, isopropylated and tert-butylated triaryl phosphate esters (ITPs & TBPPs) have been more recently introduced as flame retardant (FR) replacements for the pentaBDE mixture in polyurethane foam (PUF). In addition to their use as FRs, ITPs and TBPPs are also used as plasticizers.
ITPs and TBPPs comprise a family of aryl organophosphate esters with varying degrees of isopropylation and tert-butylation. Individual ITP and TBPP isomers have been widely detected in indoor house dust, and recent biomonitoring studies demonstrate that human exposure to these compounds is widespread. Due to concerns about their persistence, bioaccumulation, and potential toxicity (P, B, & T), the U.S. Environmental Protection Agency (EPA) listed ITPs as one of five high priority chemicals fast-tracked for expedited risk assessment under the 2016 Toxic Substances Control Act (TSCA) reform.
As such, studying the exposure, metabolism, and in vitro effects of these compounds is especially timely. The hypothesis of this research dissertation is that ITP and TBPP isomers may exhibit some of the same P, B, & T properties that motivated the phase out of PBDEs. The main objectives of this research project were to generate meaningful data to fill gaps in our knowledge of ITP and TBPP isomers, and to contribute to the ongoing risk assessment of these compounds.
In the first aim of this thesis research, the maternal transfer of Firemaster® 550 (FM 550), a commercial mixture containing ITP isomers and brominated FRs, was investigated in dosed Wistar rats. Gestational and lactational transfer were examined separately, with dams orally exposed to 300 or 1000 µg of FM 550 for 10 consecutive days during gestation (gestational day [GD] 9-18) or lactation (postnatal day [PND] 3-12). Levels of parent compounds were measured in dam and pup urine. The two brominated components of FM 500, 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB) and bis (2-ethylhexyl)-2,3,4,5-tetrabromophthalate (BEH-TEBP), underwent both gestational and lactational transfer. Triphenyl phosphate (TPHP) and ITPs were rapidly metabolized by the dams and were not detected in whole tissue homogenates. However, diphenyl phosphate (DPHP) and mono-isopropylphenyl phenyl phosphate (ip-PPP) were detected in urine from the dosed animals. This study was the first to confirm ip-PPP as a urinary metabolite of ITPs and establish a pharmacokinetic profile of FM 550 in a mammalian model.
In the second aim of this thesis research, the contribution of individual ITP and TBPP isomers was quantified in four commercial flame retardant mixtures: FM 550, Firemaster® 600 (FM 600), an ITP mixture, and a TBPP mixture. Findings suggested similarities between FM 550 and the ITP mixture, with 2-isopropylphenyl diphenyl phosphate (2IPPDPP), 2,4-diisopropylphenyl diphenyl phosphate (24DIPPDPP), and bis(2-isopropylphenyl) phenyl phosphate (B2IPPPP) being the most prevalent ITP isomer in both mixtures. FM 600 differed from FM 550 in that it contained TBPP isomers rather than ITP isomers. ITP and TBPP isomers were also detected and quantified in house dust standard reference material, SRM 2585, demonstrating their environmental relevance.
The third aim of this thesis research investigated phase I and II in vitro metabolism of TPHP, 4-tert-butylphenyl diphenyl phosphate (4tBPDPP), 2-isopropylphenyl diphenyl phosphate (2IPPDPP), and 4-isopropylphenyl diphenyl phosphate (4IPPDPP) at 1 and 10 µM doses using human liver subcellular fractions. Parent depletion and the formation of known metabolites, including DPHP, hydroxyl-triphenyl phosphate (OH-TPHP), ip-PPP, and tert-butylphenyl phenyl phosphate (tb-PPP), were monitored via gas chromatography/mass spectrometry (GC/MS) and liquid chromatography tandem mass spectrometry (LC/MS/MS). Tb-PPP and its conjugates were identified as the major in vitro metabolites of 4tBPDPP, accounting for up to 33% of the initial parent dose. While the mass balance between parents and metabolites was conserved for TPHP and 4tBPDPP, approximately 20% of the initial parent mass was unaccounted for after quantifying metabolites of 2IPPDPP and 4IPPDPP that had authentic standards available. Two novel ITP metabolites, mono-isopropenylphenyl diphenyl phosphate and hydroxy-isopropylphenyl diphenyl phosphate, were tentatively identified by high-resolution mass spectrometry (HRMS) and screened for in recently collected human urine. This study provided insight into recent human biomonitoring and epidemiological studies and contributed to our understanding of the biological fate of ITP and TBPP isomers.
Finally, the fourth aim of this thesis research evaluated ITPs, TBPPs, and related commercial mixtures for their effect on the activity of purified human liver carboxylesterase (hCE1). Four of the 15 OPEs tested had IC50 values lower than 100 nM, including TPHP, 2-ethylhexyl diphenyl phosphate (EHDPP), 4-isopropylphenyl diphenyl phosphate (4IPPDPP), and 4-tert-butylphenyl diphenyl phosphate (4tBPDPP), as did four commercial flame retardant mixtures tested. Because hCE1 is critical for the activation of imidapril, an ACE-inhibitor prodrug prescribed to treat hypertension, the most potent inhibitors, TPHP and 4tBPDPP, and an environmentally relevant mixture (house dust) were further evaluated for their effect on imidapril bioactivation in vitro. TPHP and 4tBPDPP were potent inhibitors of hCE1-mediated imidapril activation (Ki = 49.0 and 17.9 nM, respectively), as were extracts of house dust (100 µg/ml), which caused significant reductions in imidapril activation. Combined, these data suggest that exposure to OPEs can affect pharmacotherapy.
Collectively and in context of other recently published findings, this thesis research suggests that ITPs and TBPPs may be regrettable substitutes for PBDEs.
Item Open Access From molecular mechanisms to functional impact: Developing integrated analyses in neurotoxicology - The 16th biennial meeting International Neurotoxicology Association and 8th meeting of the NeuroToxicity Society.(Neurotoxicology, 2019-05) Westerink, Remco HS; van Thriel, Christoph; Levin, Edward D; Shafer, Timothy JItem Open Access Genetic Sensitivity to Mitochondrial Toxicity(2017) Luz, Anthony LincolnMitochondria are the main cellular producers of ATP, and play key roles in cellular signaling and apoptosis. Mitochondria also contain their own genomes (mtDNA), which encode 13 subunits of the electron transport chain (ETC), 22 tRNAs, and 2 rRNAs, making mtDNA integrity critical to both mitochondrial and organismal health. Mitochondria are dynamic organelles that fuse and divide to maintain mitochondrial shape, number, and size. However, mitochondrial fission and fusion also play a major role in the mitochondrial stress response. For example, mildly damaged mitochondria can fuse with healthy mitochondria allowing contents to mix, resulting in the generation of healthy mitochondria, which is known as functional complementation. Alternatively, when mitochondria become damaged beyond repair, they are targeted for autophagosomal degradation, or mitophagy. The overall importance of fission, fusion, mitophagy, and mtDNA is demonstrated by the fact that deficiencies in these processes and mtDNA content cause human disease. Interestingly, the age of onset, and severity of clinical manifestations of mitochondrial disease vary from patient to patient, even in individuals harboring identical mutations. These observations suggest a role for the environment in the development and progression of certain mitochondrial diseases; however, the relationship remains poorly understood.
To investigate the role of environmental toxicants in the development, progression, and exacerbation of mitochondrial disease I have taken two approaches using the in vivo model organism Caenorhabditis elegans. First, ten known and suspected mitochondrial toxicants (2,4-dinitrophenol (DNP), acetaldehyde, acrolein, aflatoxin B1 (AfB1), arsenite, cadmium, cisplatin, doxycycline, paraquat, rotenone) were screened for exacerbation of larval growth delay in wild-type, fission-, fusion-, and mitophagy-deficient nematodes using the COPAS Biosort. Second, a C. elegans model of mtDNA depletion was developed using chronic low-dose ethidium bromide exposure. Five toxicants (AfB1, arsenite, paraquat, rotenone, ultraviolet C radiation (UVC)) were tested for exacerbation of mitochondrial function (assessed via changes in steady-state ATP levels) in nematodes with reduced mtDNA content. Mitochondrial health was then further assessed for some of the identified gene-environment interactions. Mitochondrial respiration was measured using the Seahorse XFe24 Extracellular Flux Analyzer, while steady-state ATP levels were assessed using transgenic luciferase expression nematodes and traditional extraction protocols. Gene expression, mtDNA, and nuclear DNA copy number were assessed using real-time PCR, while enzyme activity was assessed using microplate reader-based assays.
Results from the fission, fusion, and mitophagy toxicant screen revealed that fusion-deficient nematodes were sensitive to a variety of toxicants (DNP, AfB1, arsenite, cisplatin, paraquat, rotenone), while pink-1 mitophagy-deficient nematodes were sensitive to rotenone, and fission- and pdr-1 mitophagy-deficient nematodes were only mildly sensitive to paraquat, and rotenone, respectively. As mitochondrial disease is rare, but chronic arsenite exposure is widespread, we further investigated the mechanisms underlying arsenite sensitivity in fission- and fusion-deficient nematodes. Although not sensitive in the larval growth assay, fission-deficient nematodes were sensitive to arsenite later in life in both reproduction and lethality assays. Seahorse and ATP analysis revealed that arsenite disrupts mitochondrial function in fusion-deficient nematodes at multiple life stages (L4, 8- and 12-days of age), while enhancing mitochondrial function in 8-day old wild-type nematodes, and has minimal effect on mitochondrial function in fission-deficient nematodes. Lastly, arsenite inhibited both pyruvate and isocitrate dehydrogenase activity in fusion-deficient nematodes, suggesting a disruption of pyruvate metabolism and Krebs cycle activity underlie the observed mitochondrial dysfunction. These results suggest that deficiencies in mitochondrial fusion may sensitive individuals to arsenite toxicity.
Lastly, I have found that reducing mtDNA content 35-55% only mildly sensitized nematodes to certain secondary toxicant exposures, including UVC and arsenite. Alternatively, reduced mtDNA content did not sensitize nematodes to acute or chronic paraquat or AfB1 exposure, and provided resistance to rotenone. However, we also found that EtBr can induce cytochrome P450s (CYPs), which play a major role in rotenone metabolism; thus, it is likely that induction of CYPs and not reduced mtDNA content is responsible for rotenone resistance. These results suggest that individuals with reduced mtDNA content may be sensitive to certain toxicant exposures, but also highlight the robust mechanism that exist to maintain the integrity of mitochondria and mtDNA.
Collectively, these results suggest individuals suffering from mitochondrial disease caused by mutations in mitochondrial fission, fusion, or mitophagy genes, or by depletion of mtDNA, may be especially sensitive to certain environmental toxicant exposures, including arsenic. Arsenic’s pervasive contamination of drinking water results in chronic exposure for over 100 million people worldwide; thus, dramatically increasing the probability of exposure for individuals suffering from mitochondrial disease, and warrants further investigation in the human populous.
Item Open Access Genomic Signatures of Disease and Environmental Exposure in the Peripheral Blood(2011) LaBreche, Heather GarrenMy thesis research has centered on the concept of the peripheral blood cell (PBC) as an indicator of disease and environmental exposure. The PBC is not only easily accessible and constantly replenished, but it provides a snapshot of an individual's health. Doctors have long utilized PBCs as indicators of health based on count, morphology or the expression of particular cell surface markers. Using these methods, PBCs can serve as indicators of infection, inflammation or certain types of hematological malignancies. Now PBCs can be characterized as a function of their gene expression profiles in response to disease and toxicant exposure. Advances in cDNA microarray technology have made it possible to analyze global gene expression in small volumes of whole blood, or even in a sorted population of blood cells. The resulting gene expression data can serve as a molecular phenotype, or signature, of disease or toxicant exposure. These signatures serve a twofold purpose. First, they act as biological markers (biomarkers) that can indicate the presence of disease or aid monitoring the response to treatment. Second, they provide insight into the underlying biological mechanisms that are at work, by revealing genes, networks and pathways that are affected by the disease or toxin. This paradigm has been applied in a number of contexts, including infection, inflammation, leukemia, lymphoma, neurological disorders, cardiovascular disease, environmental exposures and solid tumors.
In the work presented here, we describe signatures of lead (Pb) exposure and breast cancer based on peripheral blood gene expression. Our objective in generating a blood-based signature of lead exposure was to develop a potential predictor of past and present exposure. This is particularly relevant because of continued widespread lead exposure through both environmental and occupational sources. Pb causes significant toxicities in a number of different organ systems including the hematological, endocrine, neurological and renal systems. Pb is considered a potential carcinogen due to evidence that it causes cancer in animal models and contributes to an elevated cancer risk in humans. Pb is thought to contribute to cancer risk indirectly through a variety of mechanisms, such as inhibition of DNA synthesis and repair, oxidative damage, interaction with DNA-binding proteins and tumor suppressor proteins, causing chromosomal aberrations and alterations to gene transcription. In addition, it has been shown to exacerbate the effects of other mutagens. Recent work also indicates that even low-level Pb exposure (defined here as levels below the threshold of detection of many common tests or below the level set by the CDC as an "elevated blood lead level" in children, or 10µg/dL) can impact health, especially in children, who are more susceptible to these negative health consequences.
We hypothesized that we could detect subtle and lasting changes in the PBC transcriptome that correlated to Pb exposure. We used a mouse model of per os Pb exposure to generate signatures corresponding to two different doses of Pb. One dose reflected a high-level exposure and the other a low-level exposure. We also analyzed the gene expression changes following removal of the Pb source. We were able to generate robust, dose-specific signatures of Pb exposure. This supports the growing body of evidence that even low levels of Pb exposure can have biological effects, and that there is likely no safe level of exposure. We also utilized a collection of pathway signatures to identify those pathways that were activated or repressed in response to Pb exposure compared to controls. We observed an increase in interferon-gamma pathway activity in response to low-level Pb exposure and an increase in E2F1 pathway activity in response to high-level Pb exposure. These results support previous findings that low-level Pb exposure can increase interferon-gamma production, whereas high-level Pb has been shown to increase DNA synthesis. The Pb signatures we report here were not predictive of a past lead exposure. These results suggest that the effect of Pb exposure on PBC gene expression is transient, perhaps due to the rapid turnover of blood cells and the absorption of Pb by the bones. We have proposed further studies to identify cells in the bone marrow that may serve as indicators of past Pb exposure based previous reports on the lasting effects of genotoxic stress on this tissue.
We also describe a predictor of human breast cancer based on peripheral blood gene expression. The objective of this study was to identify and characterize PBC gene expression patterns associated with the presence of a breast tumor. This work has the potential to make a significant impact on breast cancer screening and diagnosis. Despite the success of mammography in reducing mortality from breast cancer, many cancers go undetected due to factors such as breast density, age of the woman, or type of cancer. A blood-based breast tumor predictor would potentially offer an easy and noninvasive means of detecting primary breast cancer as well as monitoring patients for recurrences or metastases. In addition, the concept of using a blood-based biomarker for cancer detection would have positive implications for other types of cancer. For instance, patients with ovarian cancer are typically diagnosed at a late stage because of the absence of definitive symptoms and the lack of effective screenings methods.
We were able to successfully identify robust predictors of both mouse mammary tumors and human breast tumors based on PBC gene expression. The human breast tumor predictor exhibits a high level of sensitivity and specificity in distinguishing breast cancer patients and controls in an independent validation cohort. However, the true novelty in this study is that it integrates a factor modeling approach and a transgenic mouse model of breast cancer to identify biologically meaningful gene expression changes in the mouse PBC transcriptome. These genes were then used as the starting point for developing a human breast cancer predictor. This establishes an experimental system in which we can address questions that are inherently difficult to answer in human studies, such as whether this predictor is useful in detecting breast tumors early or in monitoring patients for recurrence or metastasis. In fact, our work suggests that tumor-associated gene expression changes in the PBCs can be detected in asymptomatic mice. Our results support those of previous studies, which identified blood gene expression profiles that are associated with a variety of solid tumors, including breast cancer. However, the sensitivity and specificity of our predictor are higher than that of the previously reported breast cancer signature. This may suggest that our strategy of using a mouse model to first identify informative genes allowed us to focus on those genes most relevant to the presence of a breast tumor and overcome the influence of the high degree of variation in blood gene expression in our human population. In order to be clinically useful, the predictor we report here would need to be tested in additional, large validation sets to establish its utility in an early detection setting and its specificity in distinguishing breast cancer from other cancer types as well as other potentially confounding conditions such as infection and inflammation. We describe some preliminary experiments in the mouse model intended to address these important questions.
Item Open Access Halogenated Organophosphate Flame Retardants: Developmental Toxicity and Endocrine Disruptive Effects(2015) Dishaw, Laura VictoriaFollowing the phase out of polybrominated diphenyl ethers (PBDEs), manufacturers turned to several alternative flame retardants (FRs) to meet flammability standards. Organophosphate FRs (OPFRs), and in particular tris (1,3-dichloropropyl) phosphate (TDCPP), have been increasingly detected in textiles and foam padding used in a variety of consumer products including camping equipment, upholstered furniture, and baby products. Like PBDEs, OPFRs are additive, meaning that they are not chemically bound to the treated material and can more readily leach out into the surrounding environment. Indeed, OPFRs have been detected in numerous environmental and biological matrices, often at concentrations similar to or exceeding that of PBDEs.
Although OPFRs have been in use for several decades, relatively little is known regarding their potential for adverse human and environmental health consequences. However, based on their structural similarity to OP pesticides, they may have analogous mechanisms of toxicity. OP pesticide toxicity is classically associated with cholinesterase inhibition, resulting in cholinergic intoxication syndrome. OPFRs have been shown to be ineffective cholinesterase inhibitors, however chlorpyrifos (CPF) and other OP pesticides have been shown to elicit adverse effects on developing organisms through other mechanisms.
The main objective of this research project was to evaluate the toxicity of four structurally similar OPFRs (TDCPP; tris (2,3-dibromopropyl) phosphate, (TDBPP); tris (1-chloropropyl) phosphate (TCPP) and tris (2-chloroethyl) phosphate (TCEP)) in comparison to chlorpyrifos (CPF), a well-studied OP pesticide. A combination of in vitro and in vivo models was used to elucidate potential mechanisms as well as functional consequences of exposure in developing organisms.
In the first research aim, a series of in vitro experiments with neurotypic PC12 cells was used to evaluate the effects of four structurally similar OPFRs (TDCPP, TDBPP, TCEP, or TCPP) and CPF on neurodevelopment. The effects of TDCPP were also compared to that of BDE-47, a major component of the commercial PentaBDE mixture. In general, TDCPP elicited similar or greater effects when compared to an equimolar concentration of CPF. All OPFRs tested produced similar decrements in cell number and altered phenotypic differentiation, while BDE-47 had no effect on cell number, cell growth, or neurite growth.
For the second research aim, zebrafish (Danio rerio) were used to evaluate the effects of the same suite of chemicals on early development. TDCPP, TDBPP, and CPF elicited overt toxicity (e.g., malformations or death) within the concentration range tested (0.033-100 µM). TDBPP was the most potent with 100% mortality by 6 days post fertilization (dpf) at ≥3.3 µM. CPF and TDCPP showed equivalent toxicity with malformations observed in at 10 µM and significant mortality (≥75%) at ≥33 µM. There was no overt toxicity among TCEP- and TCPP-exposed fish. All test chemicals affected larval swimming behavior on 6 dpf at concentrations below the overt toxicity threshold. Parent chemical was detected in all in embryonic (1 dpf) and larval (5 dpf) tissues. TDCPP and TDBPP showed rapid and extensive metabolism.
Finally, for the third aim, juvenile (45-55 dpf) zebrafish were exposed to CPF (1 µg/g food) or TDCPP (Low TDCPP = 1 µg/g food; High TDCPP = 40 µg/g food) via diet for 28 days followed by a 7 day depuration period where all treatments received clean food. A dietary exposure was chosen to more closely recapitulate exposure in humans. Samples were collected at seven time points throughout the experiment: days 0, 7, 14, 21, 28, 30, 35. Whole tissues were collected for tissue accumulation and histopathology endpoints. Viscera and brain were dissected and flash frozen separately for DNA damage analyses.
Tissue measurements of CPF, TDCPP, and the metabolite bis (1,3-dichloropropyl) phosphate (BDCPP) were often below the method detection limit, however when present there was a trend towards increased accumulation with treatment and time. On Day 7 Low TDCPP caused a dramatic but transient increase in DNA damage in both viscera and brain that returned to control levels by Day 14. Similar results have been seen previously with other genotoxicants and may be due to CPF and High TDCPP inducing an adaptive response prior to the 7 day sampling point. All treatments shifted the neurohypophysis to adenohypophysis ratio (NH/AH; Day 7 only) and significantly increased thyroid follicle activation (Day 14). Finally High TDCPP affected gonad maturation, causing a significant increase in ovary follicle development (Day 14) and a transient but marked decrease in testes maturity (Day 7). Taken together these data suggest that dietary exposure to TDCPP and CPF elicits DNA damage in brain and viscera and alters endocrine function in juvenile zebrafish. Importantly, analyses were restricted to the first three time points (Days 0, 7, and 14) due to the emergence a disease among the experimental colony. Although these samples were collected prior to the disease becoming apparent, it remains a potential confounder of the current results.
Item Open Access Impacts of Cosmetic Ingredients on Larval Barnacles: A Study & Discussion of How Cosmetic Ingredients Affect Marine Life(2019-04-24) Boden, AlexandraIn recent years, scientists have discovered toxicity of active ingredients in sunscreens to marine life such as coral reefs. While the research findings have brought about significant policy changes in places like Hawaii and Palau, little attention has been given to the hundreds of other ingredients that are commonly found in such cosmetics. Through 24-hour exposure studies with barnacle larvae, this study documents toxicity and settlement inhibition of sunscreens and 5 common “inactive” ingredients. Results indicate that “inactive PDMS additives” are toxic to barnacle larvae inhibit settlement. Sunscreens are even more potent, therefore suggesting the combination of ingredients that makeup sunscreens pose a greater risk to marine life than individual components. The Master’s Project products are an analysis and discussion of the research conducted, as well as a short video translating the research for science communication purposes.Item Open Access Impacts of Mountaintop Removal Coal Mining on the Mud River, West Virginia: Selenium Accumulation, Trophic Transfer, and Toxicity in Fish(2014) Arnold, Mariah ChristineSelenium (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.
Item Open Access Incorporating Environmental Realism into the Toxicity of Nanoparticles to Early Life Stage Fish(2015) Bone, Audrey JayneAs 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.
Item Open Access Integrated Bayesian Network Models to Predict the Fate and Transport of Natural Estrogens at a Swine Farrowing CAFO(2012) Lee, BoknamNatural steroidal estrogen hormones in swine wastes generated from Concentrated Animal Feeding Operations (CAFOs) have become a potential pollutant to many aquatic environments due to their adverse impacts on the reproductive biology of aquatic organisms. In North Carolina, the swine CAFO industry is a major agricultural economic enterprise that is responsible for the generation of large volumes of waste. However, there is limited scientific understanding regarding the concentration, fate, and transport of the estrogenic compounds from these swine facilities into terrestrial and aquatic environments. To address this issue, my research involved the development and application of integrated Bayesian networks (BNs) models that can be used to better characterize and assess the generation, fate, and transport of site-specific swine CAFO-derived estrogen compounds. The developed model can be used as decision support tool towards estrogen risk assessment. Modularized and melded BN approaches were used to capture the predictive and casual relationships of the estrogen budget and its movement within and between the three major systems of a swine farrowing CAFO. These systems include the animal barns, the anaerobic waste lagoon, and the spray fields. For the animal barn system, a facility-wide estrogen budget was developed to assess the operation-specific estrogen excretion, using an object-oriented BN (OOBN) approach. The developed OOBN model provides a means to estimate and predict estrogen fluxes from the whole swine facility in the context of both estrogen type and animal operating unit. It also accounts for the uncertainties in the data and in our understanding of the system. Next, mass balance melding BN models were developed to predict the natural estrogen fates and budgets in two lagoon compartments, the slurry and the sludge storage. This involved utilizing mass balance equations to account for the mechanisms of flushing, sorption, transformation, settling, and burial reactions of estrogen compounds in the slurry and sludge storages. As an alternative approach, a regression based BN melding approach was developed to both characterize estrogen fate and budgets as a result of the sequential transformation processes between natural estrogen compounds and to assess the seasonal effects on the estrogen budgets in the two different lagoon compartments. Finally, a dynamic BN model was developed to characterize rainfall-driven estrogen runoff processes from the spray fields. The dynamic BN models were used to assess the potential risk of estrogen runoff to adjacent waterways. In addition, the dynamic model was used to quantify the effects of manure application rates, rainfall frequency, the time of rainfall and irrigation, crop types, on-farm best management practices, seasonal variability, and successive rainfall and manure application events on estrogen runoff.
The model results indicate that the farrowing barn is the biggest contributor of total estrogen as compared to the breeding and gestation operating barns. Once the estrogen reaches the anaerobic lagoon, settling and burial reactions were shown to be the most significant factors influencing estrogen levels in the slurry and sludge, respectively. The estrogen budgets in the lagoon were also found to vary by season, with higher slurry and sludge estrogen levels in the spring as compared to the summer. The risk of estrogen runoff was predicted to be lower in the summer as compared to the spring, primarily due to the spray field crop management plans adopted. The results also indicated that Bermuda grass performed more favorably when compared to soybean, when it came to retaining surface water runoff in the field. Model predictions indicated that there is a low risk of estrogen runoff losses from the spray fields under multiple irrigation and rainfall events, unless the time interval between irrigation was less than 10 days and/or in the event of a prolonged high magnitude rainstorm event. Overall, the estrone was the most persistent form of natural estrogens in the three major systems of the swine farrowing CAFO.
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