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Xenobiotics or their metabolite(s) can be electrophilic or free radicals that elicit a variety of chemical reactions either directly or indirectly effecting the biochemistry of a cell, resulting in oxidative stress and/or cell death. Because drug-induced liver injury (DILI) is the leading cause of death from acute liver failure and the most common cause of drug withdrawals from the market, understanding and identifying hepatotoxic mechanisms have become a major focus area with drug and environmental regulatory agencies. In the following chapters, the in vitro cytotoxic mechanisms of known hepatotoxins were investigated using the ‘Accelerated Cytotoxicity Mechanism Screening’ (ACMS) technique. The following hypothesis was formulated: the ACMS technique can be utilized as a basic toxicological screening model to assess xenobiotic-induced hepatocyte toxicity. Using this technique, the following investigation provides support for the validity of an inflammatory model using glucose and glucose oxidase to generate hydrogen peroxide (H2O2) and its effects on known hepatotoxins, including nitroaromatic drugs (nimesulide, nilutamide, flutamide), aromatic amine drugs (clozapine, thioridazine), ethanol and its metabolite acetaldehyde. Using this inflammatory model, cytotoxicity increased with all examined compounds. This thesis also examined the hepatotoxicity of chlorpromazine (CPZ) and its reactive metabolites. Toxic CPZ metabolites were generated by cytochrome P450 (CYP) or peroxidase/H2O2-catalysed oxidation of CPZ resulting in loss of hepatocyte viability, collapse in mitochondrial membrane potential (MMP) and increased glutathione (GSH) oxidation. Finally, this thesis investigated the molecular targets of acrolein and chloroacetaldehyde (CAA), reactive metabolites of anticancer agents cyclophosphamide/ifosfamide, and the subsequent protective capacities of various protecting agents in both cell- and cell-free models. This study could provide a better understanding into the therapeutic value of protective agents currently being prescribed and agents not currently being used, but which have demonstrated protecting effects against acrolein, CAA or other toxic aldehydes. There are needs for basic toxicology screening models to assess and categorize the hepatotoxic risks associated with xenobiotics, including drugs and environmental toxins. The ACMS technique may be such a model and have a valuable application that may be 1) adaptable for high-throughput screening, 2) useful for supplementing existing in vitro screening techniques as a means to accelerate the pre-clinical screening process and 3) result in a better understanding of environmental pollutants.

PhD

Advisors/Committee Members: O'Brien, Peter, Pharmaceutical Sciences.

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The teratogenic potential of methanol (MeOH) in New Zealand white (NZW) rabbits was examined, with emphasis on the biochemical basis for species differences in susceptibility. A comparison of MeOH and formic acid (FA) elimination profiles was first carried out in NZW rabbits, CD-1 mice and cynomolgus monkeys to evaluate the relationship between MeOH metabolism and its teratogenic potential, and to provide a more accurate assessment of human developmental risk than is currently available. Rabbits and the C3H mouse strain were resistant to MeOH-associated external birth defects, whereas CD-1 and C57BL/6 mice were susceptible. Rabbits were similarly resistant to the entire sprectrum of MeOH-initiated skeletal anomalies found in rodents, but exhibited a novel skeletal defect, notably a decrease in post-lumbar vertebrae, which is not found in rodents. MeOH and FA pharmacokinetics were comparable between susceptible and resistant mouse strains, yet different between resistant rabbits and mice, indicating that adult pharmacokinetics cannot explain strain and species differences in susceptibility to MeOH teratogenicity. Catalase and alcohol dehydrogenase (ADH1) activity were compared in adult and embryonic samples from MeOH-resistant NZW rabbits and susceptible CD-1 mice. Antioxidative and peroxidative catalase activities were substantially lower in rabbits than mice, whereas ADH1-catalyzed EtOH metabolism was faster in rabbits than mice. This suggests that: (1) rabbit MeOH metabolism is catalzyed by ADH1 as opposed to catalase, and (2) MeOH-initiated reactive oxygen species (ROS) formation may contribute to the mechanism of MeOH teratogenicity in susceptible mouse strains. Catalase exhibits antioxidative acivity in all species, but peroxidatively metabolizes MeOH to formaldehyde only in rodents. Humans, and possibly rabbits, however, oxidize MeOH using ADH1. The higher peroxidative catalase activity in mice than rabbits, suggests reduced catalase-mediated MeOH metabolism in the latter species, which would increase catalase capacity for ROS scavenging, and protection, in rabbits.

PhD

Advisors/Committee Members: Wells, Peter G., Pharmaceutical Sciences.

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High chronic fructose consumption is linked to a nonalcoholic steatohepatitis (NASH) type of hepatotoxicity. Oxalate is the major endpoint of fructose metabolism, which accumulates in the kidney causing renal stone disease. Both diseases are life-threatening if not treated. Our objective was to study the molecular cytotoxicity mechanisms of fructose and some of its metabolites in the liver. Fructose metabolites were incubated with primary rat hepatocytes, but cytotoxicity only occurred if the hepatocytes were exposed to non-toxic amounts of hydrogen peroxide such as those released by activated immune cells. Glyoxal was most likely the endogenous toxin responsible for fructose induced toxicity formed via autoxidation of the fructose metabolite glycolaldehyde catalyzed by superoxide radicals, or oxidation by Fenton’s hydroxyl radicals. As for hyperoxaluria, glyoxylate was more cytotoxic than oxalate presumably because of the formation of condensation product oxalomalate causing mitochondrial toxicity and oxidative stress. Oxalate toxicity likely involved pro-oxidant iron complex formation.

MAST

Advisors/Committee Members: O'Brien, Peter J., Pharmaceutical Sciences.

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Neural precursor cells self-renew and differentiate throughout development and in response to neural injury in the adult brain. The DNA damage response in NPCs has yet to be characterized. Patients with defective nucleotide excision repair (NER) demonstrate neurodegeneration dismyelination, and microcephaly, suggesting a potential link to defective NPC function with accumulating DNA damage. We observed reduced self-renewal in Csbm/m and Xpam/m NPCs in response to UV damage. Serial passaging resulted in exhaustion of Csbm/m NPCs in the absence of exogenous DNA damage. In vitro neuronal differentiation resulted in abnormal neuritigenesis after UV DNA damage in Csbm/m NPCs, suggesting defects in the terminal differentiation process. Taken together, the results indicate that DNA damage can modulate the apoptotic, self-renewal and differentiation fates of NPCs.

MAST

Advisors/Committee Members: Laposa, Rebecca, Pharmacology.

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Identification of maternal opioid abuse in pregnancy is often difficult to ascertain in the absence of reliable self report. For this reason, physicians and child protection workers often turn to maternal and neonatal hair analysis for the detection of in utero opioid exposures. Since neonatal opiate hair analysis continues to prove difficult due to the scarcity of the hair sample and low drug concentrations, I developed a sensitive method utilizing headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) for the detection of three principal opiates (morphine, codeine, and 6-monoacetylmorphine) in human hair. Moreover, I characterized an at-risk neonatal population for in utero opiate exposures as well as for other drugs of abuse and alcohol. Equipped with a sensitive and specific method for the detection of opiate exposures and understanding the addiction profiles of pregnant women may lead to better clinical and social management and may benefit an at-risk population.

MAST

Advisors/Committee Members: Koren, Gideon, Pharmacology.

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The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene (MC); the prototypical response is induction of drug-metabolizing enzymes. Factors that regulate AHR levels in vivo are poorly understood. It is also not clear how AHR levels affect MC responsiveness. I hypothesize that glucocorticoids enhance hepatic AHR based on previous findings of decreased hepatic AHR protein in hypophysectomised rats and increased AHR levels following glucocorticoid treatment in rodent hepatoma cells. To study this, adrenalectomized (ADX) or SHAM-ADX rats were treated with dexamethasone or vehicle. AHR protein was decreased by 50–60% at 4 days after ADX, but was not altered by dexamethasone. Dexamethasone induced hepatic AHR nuclear translocator (ARNT) mRNA by up to 9-fold, with no corresponding change in ARNT protein. AHR target gene expression was measured in MC-treated ADX rats to assess MC responsiveness given the decrease in AHR protein following ADX. MC-induced hepatic CYP1B1 mRNA was reduced by 50% in ADX rats relative to SHAM. AHR mRNA was increased 4-fold, 6 h after MC in SHAM rats, but no induction was observed in ADX rats. MC-induced 7-ethoxyresorufin O-deethylation activity in ADX rats was 35% of the activity in the MC-treated SHAM group at 6 h. To assess the capacity for hepatic P450-mediated metabolism, NADPH-cytochrome P450 oxidoreductase (POR) was measured. POR activity was decreased by 50-65% following ADX. DEX induced hepatic POR mRNA by up to 7-fold, 6 h after treatment in SHAM, ADX and intact rats. Putative glucocorticoid responsive elements in the rat Por gene were identified, but recruitment of the glucocorticoid receptor to these elements was not detected using chromatin immunoprecipitation. In rat H-4-II-E hepatoma cells, dexamethasone induced POR, but not ARNT, mRNA. I have shown that ADX decreases hepatic AHR protein and subsequently, MC responsiveness is suppressed for some AHR-mediated responses. Decreased POR activity following ADX might contribute to a decreased capacity for P450-dependent metabolism. The novel findings with respect to glucocorticoid regulation of ARNT and POR demonstrate the complexity of AHR-glucocorticoid cross-talk and the need for further study.

PhD

Advisors/Committee Members: Riddick, David S., Pharmacology.

▼ Idiosyncratic drug-induced liver injury (IDILI) continues to be an important issue. There is increasing clinical evidence that most IDILI is immune mediated. Extensive mechanistic studies are needed to better understand these reactions; however, it is impossible to do controlled experiments in humans, and previous animal models did not properly model IDILI. If IDILI is immune mediated, and the major factor preventing liver injury in patients is immune tolerance, then a plausible method to develop an animal model of IDILI would be to impair immune tolerance. Therefore, the objectives of my research were to develop a valid animal model of IDILI, use this model to test mechanistic hypotheses, and to develop better ways to predict risk. In my first two papers I show that immunization of mice with drug-modified hepatic proteins prior to drug exposure paradoxically decreased liver injury unless immune tolerance was impaired during the immunization. In my third paper, I show that PD1-/- mice treated with anti- CTLA-4 and amodiaquine can lead to significant liver injury and liver dysfunction, which is mediated by CD8 T cells. In my fourth paper, I show that this impaired immune tolerance model is able to unlock the potential of isoniazid and nevirapine to cause increased liver injury. Finally, in my fifth paper, I show that this impaired immune tolerance model is also able to distinguish the potential for liver injury of chemically similar drugs, where one drug causes IDILI in humans and the other does not (troglitazone/pioglitazone and tolcapone/entacapone). Overall, I have shown that risk of developing IDILI may be influenced by previous exposures, and I have further developed a model of IDILI, showing its potential for other drugs and as a tool for drug screening. Further characterization and validation of these models is required; however, it is likely that they will make it possible to perform mechanistic studies that have been impossible in the past. Advisors/Committee Members: Uetrecht, Jack, Pharmaceutical Sciences.

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Cadmium (Cd) is a nephrotoxic metal that has a number of consequences for renal function. Cells exposed to Cd may have methods to counteract toxic effects with processes like autophagy. Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome. This study investigated the mechanism of Cd-induced autophagy, and the relationship between autophagy and cell survival. At 10 µM exposure for 24 hours, Cd-induced autophagy was found to be reactive oxygen species-independent, and suppressed with thapsigargin treatment, suggesting the necessity of intracellular Ca2+. Inhibition of the Jun-N-Terminal Kinase resulted in the greatest suppression of Cd-induced autophagy. Autophagy was found to be protective against cell death elicited by the toxic metal, and the absence of autophagy resulted in increased apoptosis in mouse embryonic fibroblast cells. This study adds insight to the mechanism and protective effects of Cd-induced autophagy, and provides the groundwork for future experiments.

M.Sc.

Advisors/Committee Members: Templeton, Douglas M, Laboratory Medicine and Pathobiology.

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The aryl hydrocarbon receptor (AHR) is a ligand-regulated transcription factor that is activated upon binding to various ligands. The activated AHR modulates the expression of many genes including cytochrome P450s (CYPs) such as Cyp1a1, Cyp1b1, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP-ribose) polymerase (Tiparp). We recently reported that TIPARP is a transcriptional repressor of AHR and Tiparp knockout mice show increased sensitivity to dioxin-induced toxicities. Because of these findings, we examined the sensitivity of Tiparp knockout mice to 3-methylcholanthrene (3-MC), another potent AHR ligand. Tiparp knockout mice treated with 100mg/kg of 3-MC exhibited increased hepatotoxicity, increased lipolysis, and developed chylous ascites compared with treated wildtype mice. No treated Tiparp knockout mice survived beyond day 16; all wildtype mice survived the 30 day treatment. Collectively, this thesis shows that Tiparp knockout mice exhibit increased sensitivity to 3-MC-induced toxicity and lethality supporting our previous findings that TIPARP is an important negative regulator of AHR activity.

M.Sc.

Advisors/Committee Members: Matthews, Jason, Pharmacology.

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N-methyl D-aspartate (NMDA) receptors represent integral signal transducers for excitatory glutamate neurotransmission. While NMDA receptors are critical for synaptic plasticity, the molecular events underlying this process are not fully elucidated. The potential role of NP22, a novel neuronal protein, as a downstream mediator of NMDA receptor function is explored. NP22 protein expression in genetic and pharmacological models of NMDA receptor hypofunction is examined and no significant changes are reported. Characterization of the NP22 protein complex via tandem-affinity and FLAG-purification coupled with mass spectrometry was used and no novel protein interactions are reported. GFP-tagged NP22 colocalization with F-actin decreases in cell processes of transiently transfected HEK293 cells in response to elevated intracellular calcium, while similar colocalization reductions are not seen in stably transfected HEK293 under a comparable treatment regiment. Changes in intracellular calcium affecting NP22 biology can be useful in the ongoing characterization of this novel protein.

MAST

Advisors/Committee Members: Ramsey, Amy J., George, Susan, Pharmacology.

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High chronic fructose consumption has been linked to many diseases. Sugar metabolites, especially glyoxal and methylglyoxal can form advanced glycation products, which contribute to the pathology of diabetic complications. Our objective was to study the metabolism of these metabolites and the associated protein carbonyation and cytotoxicity in isolated hepatocytes. In addition, the effect of oxidative stress on the metabolism of these toxins was also investigated. Methylglyoxal and glyoxal can induce protein carbonylation, which contributes to hepatocyte toxicity. Methylglyoxal, but not glyoxal, was detoxified mainly by the glyoxalase system. Both toxins can be metabolized by mitochondrial aldehyde dehydrogenase. The detoxification of glyoxal was impaired under oxidative stress conditions (i.e. increased hydrogen peroxide level). Glyoxal was found to be a common autoxidation product from glyceraldehyde, hydroxypyruvate and glycolaldehyde. Glyoxal and the reactive oxygen species formation during the autoxidation process contributed to the hepatocyte toxicity of glyceraldehyde, hydroxypyruvate and glycolaldehyde.

MAST

Advisors/Committee Members: O'Brien, Peter J., Pharmaceutical Sciences.

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An animal model of nevirapine (NVP)-induced skin rash was used to test the hypothesis that sulfonation of 12-OH NVP, a metabolite of NVP proven essential for rash development, is the link between 12-OH NVP and the skin rash. Female Brown Norway (BN) rats were co-treated with NVP or 12-OH NVP and sulfation inhibitors dehydroepiandrosterone (DHEA) and salicylamide. Co-treatment with salicylamide markedly decreased formation of the sulfate conjugate but did not prevent development of the rash suggesting that the sulfate is not involved. However, it is not known whether the sulfate formation in the skin was affected. Co-treatments with DHEA decreased the sulfate formation and prevented the rash but also had other effects on NVP metabolism. This implies that the sulfate metabolite is responsible for the rash. Additional studies will be required to resolve these conflicting results.

MAST

Advisors/Committee Members: Uetrecht, Jack, Pharmaceutical Sciences.

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Neonicotinoids are neurotoxic systemic insecticides applied extensively worldwide. The impacts of neonicotinoid exposure on invertebrates are widely studied, however effects on essential vertebrate pollinators like hummingbirds have received little attention. My study into the effects of imidacloprid, a common neonicotinoid, on hummingbirds integrates lab-based and field-based assays with emphasis on cellular, systems, whole-organism, and behavioural levels. Within 2 h of exposure, hummingbirds exhibited a significant depression in energy expenditure (up to 25% +/- 11%). I did not observe effects on feeding behaviour, immune response, or cholinesterase activity in the lab, or stress hormone deposition in feathers collected in the field. I also determined that hummingbirds excrete imidacloprid quickly (elimination half-life of 2.1 h +/- 0.1 h) relative to other bird species. Hummingbirds exist on narrow energy margins, especially during migration and breeding seasons. Therefore, changes in their metabolism following exposures to imidacloprid observed herein could bear important energetic consequences.

M.Sc.

Advisors/Committee Members: Welch Jr., Kenneth C., Cell and Systems Biology.

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Oxidative stress and reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), which is detoxified by catalase, are implicated in fetal death and birth defects, but embryonic levels of catalase are only about 5% of adult activity, and its protective role is unknown. Our approach involved the use of mice genetically modified to either: (1) express low levels of endogenous catalase (acatalasemic, aCat); or, (2) express human catalase resulting in elevated levels of embryonic catalase activity (hCat). Using these mouse models we investigated the protective importance of constitutive embryonic catalase against endogenous ROS and the ROS-initiating teratogen phenytoin in embryo culture and in vivo. We hypothesized that aCat mice would be more sensitive to endogenous embryonic and phenytoin-enhanced ROS production, while hCat embryos would be less sensitive. aCat and hCat embryos respectively exhibited reduced and enhanced catalase activity compared to wild-type (WT) controls, with conversely enhanced and reduced spontaneous and phenytoin-enhanced embryopathies and DNA oxidation. Among aCat embryos exposed to phenytoin, embryopathies increased with decreasing catalase activity, and were completely blocked by addition of exogenous catalase. The alterations in phenytoin embryopathies were not due to pharmacokinetic differences, as drug concentrations in maternal and fetal tissues were similar among all strains. However, phenytoin concentrations in fetal brain exceeded those in fetal liver or maternal tissues, which may explain the predominance of cognitive deficits over structural birth defects in children exposed in utero to phenytoin. Similarly in untreated aged mice (about 18 months), female aCat mice showed a substantial loss in motor coordination compared to WT controls in the rotarod test. Following in utero exposure to phenytoin, the effect of altered embryonic catalase activity on postnatal neurodevelopment was assessed by several pre- and post-weaning tests. Catalase deficiency (aCat), independent of drug treatment, reduced performance in surface righting, negative geotaxis tests and rotarod tests. Conversely, high catalase expression (hCat) enhanced performance in the surface righting, negative geotaxis, air righting and rotarod tests. Our results provide the first evidence that the quantitatively minor amounts of antioxidative enzymes like catalase in the embryo and fetus provide important protection against the molecular damage and adverse fetal effects caused by developmental and drug-enhanced oxidative stress. Accordingly, interindividual variation in embryonic/fetal activities of catalase, and possibly other antioxidative enzymes, likely constitute an important determinant of risk for adverse developmental outcomes.

PhD

Advisors/Committee Members: Wells, Peter G., Pharmaceutical Sciences.

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Drug – induced nephrotoxicity is a serious adverse reaction that can have deleterious effects on a patient’s health and well-being. Acyclovir is an example of such an agent that causes the aforesaid effects. The drug induces severe nephrotoxicity in patients. The etiology of acyclovir – induced nephrotoxicity has not been fully elucidated. The overall objective of this thesis is to gain new insight into the pathogenesis of acyclovir – induced nephrotoxicity. Cytotoxicity studies showed that acyclovir induced human renal proximal tubular (HK-2) cell death, in vitro, and that the degree of this toxicity was significantly reduced by co-exposure to 4-methylpyrazole. The results suggest that acyclovir induces direct insult to human renal proximal tubular cells and the toxicity may be caused by the parent drug’s noxious acyclovir aldehyde metabolite. Transepithelial transport studies illustrated that acyclovir does not inhibit the transport of creatinine across porcine renal proximal tubular (LLC-PK1) or HK-2 cell monolayers. The results suggest that acyclovir does not inhibit the tubular secretion of creatinine in vitro, and possibly, in vivo, as well. Therefore, the abrupt, pronounced and transient elevations in the levels of plasma creatinine observed in patients may be solely and genuinely due to reduced GFR as a result of acyclovir – induced nephrotoxicity, and not to a tubular interaction between creatinine and acyclovir. Employing human embryonic kidney cells (HEK293) containing the full-length human ABCG2 gene encoding the wildtype ABCG2 amino acid sequence; cell accumulation studies showed that in the presence of the human breast cancer resistance protein (BCRP) inhibitor, fumitremorgin C (FTC), there was significant intracellular accumulation of acyclovir. The results suggest that acyclovir is a substrate for the efflux transporter and bears several potential implications with respect to the renal transport mechanisms and pathogenesis of the direct tubular damage induced by the drug. Synthesizing all the data, the results contribute to a better understanding of the pathogenesis of acyclovir – induced nephrotoxicity. Moreover, the research highlights the need for future studies that will aid in further elucidation of the underlying cell and molecular mechanism(s) of this toxicity and potential therapies for prevention of the direct renal tubular injury induced by the drug.

PhD

Advisors/Committee Members: Koren, Gideon, Pharmaceutical Sciences.

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Hepatotoxicity is a serious adverse health effect caused by drugs and other chemical toxins generally detected in the later stages of drug development or in whole animal studies. Thus, development of screening approaches available for earlier identification of hepatotoxic molecules is necessary. A novel in vitro- in silico test system for the evaluation of the molecular mechanisms of xenobiotic toxicity in primary hepatocyte systems is presented here. It is well established that hepatocytes in vitro are most representative of hepatotoxicity in vivo, and are most useful for the determination of xenobiotic hepatotoxicity mechanisms at the molecular and cellular level. There is an on-going interest in Quantitative Structure-Activity Relationships (QSAR) in toxicology, as it can identify correlations between chemical structure and biological activity. QSAR can be used to evaluate the effects of metabolism and toxicity as many physicochemical descriptors reflect simple molecular properties that can provide insight into the physicochemical nature of the activity under consideration. QSARs were determined for hepatotoxicity of halobenzenes, p-benzoquinones, α,β-unsaturated carbonyl compounds and nitroaromatics towards isolated hepatocytes. A molecular link was established for their proposed toxicity pathways. For example oxidative activation was linked to EHOMO (energy of the highest occupied molecular orbital) values and hydrophobicity (log P) of the chemicals, while reductive activation was linked with ELUMO (energy of the lowest molecular orbital) values and log P. Such relationships may thus be useful for predicting toxicity of other chemicals of the same mechanism of toxicity. Due to the complexity involved in the phenomena of hepatotoxicity, unravelling of structure-hepatotoxicity relationships is a complicated task. A conceptual framework for QSAR modeling is proposed that involves recognition of molecular initiating events as potential endpoints to improve the prediction potential of QSAR models. Acute toxicity of reactive chemicals could be based on an initial reaction with biomolecules, thus the theory of covalent binding reactivity was used to test this concept. Reactivity assays with thiol and amine surrogate nucleophiles were used to determine susceptibility to toxicity. The derived QSAR expressions suggested that covalent binding reactivity is a good correlate to hepatotoxicity, however only if electrophilicity was the main mechanism of toxicity.

PhD

Advisors/Committee Members: O'Brien, Peter J., Pharmaceutical Sciences.

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Drug induced idiosyncratic agranulocytosis has been attributed to oxidation by hypochlorite formed by bone marrow myeloperoxidase (MPO). Idiosyncratic liver toxicity could also involve drug oxidative activation by cytochrome P450 (in hepatocytes) or MPO (in Kupffer cells or infiltrating neutrophil/macrophages). Such drug reactive metabolites could cause cytotoxicity or release “danger signals” that attract immune cells which release H2O2 resulting from nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) activation. In vivo animal studies have shown that low level tissue inflammation markedly increased drug-induced tissue toxicity which was prevented by immune cell inhibitors and increased by cell activators. It is suggested that idiosyncratic drugs are much more toxic, taken during symptomless inflammation periods. Furthermore, it is hypothesized that hepatocytes are much more susceptible to some idiosyncratic drugs if they are exposed to hydrogen peroxide (H2O2)/myeloperoxidase or cytokines released by inflammatory cells. A hepatocyte inflammation model, in which hepatocytes were exposed to a non-toxic H2O2 generating system and peroxidase, was found to be much more vulnerable to four idiosyncratic drugs e.g., troglitazone, isoniazid, hydralazine and amodiaquine. The molecular cytotoxic mechanisms for this marked increase in cytotoxicity were investigated as follows: 1) A P450/H2O2-catalyzed pathway not involving oxidative stress e.g., hydralazine and isoniazid; 2) A P450/H2O2-catalyzed oxidative stress-mediated cytotoxic pathway e.g., hydrazine (an isoniazid metabolite) and hydralazine; and 3) A peroxidase/H2O2-catalyzed oxidative stress-mediated cytotoxic pathway e.g,, hydralazine, amodiaquine and troglitazone. Before cytotoxicity ensued, GSH oxidation, protein carbonyl formation and often lipid peroxidation occurred followed by a decrease in mitochondrial membrane potential indicating that oxidative stress was the molecular mechanism of cytotoxicity. In summary, a H2O2-enhanced hepatocyte system in the presence and absence of peroxidase may prove useful for a more robust screening of drugs for assessing the enhanced drug toxicity risk associated with taking drugs during periods of inflammation.

PhD

Advisors/Committee Members: O'Brien, Peter J., Pharmaceutical Sciences.

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Prenatal exposure to xenobiotics can lead to the production of reactive oxygen species (ROS) that alter signal transduction or oxidatively damage cellular macromolecules such as DNA and protein. During development, ROS play an important role in physiological signalling, suggesting that tight regulation of oxidative stress is imperative for normal development. My research employed two models: one to examine the role of ROS-mediated signalling in teratogenesis and the other to examine the role of oxidative DNA damage and repair. Using the industrial plasticizer and environmental contaminant, bisphenol A (BPA), I found that a single high dose exposure during early fetal development can increase ROS production, causing protein and DNA oxidation in fetal brain, accompanied by postnatal increases in hyperactivity and loss of fear conditioning and olfactory discrimination. BPA-exposed mice have increased neocortical cell proliferation that may involve activation of the PI3K/Akt/mTOR pathway based on the observed increase in oxidation of phosphatase and tensin homolog (PTEN). To elucidate the importance of DNA damage during embryonic development, I generated a conditional knockout mouse line for the breast cancer susceptibility gene (Brca) 1. While individuals with decreased BRCA1 develop normally, they have a greater risk of developing breast and ovarian cancers, and Brca1 null mice are not viable, suggesting that the gene is essential for normal development. Using the pro-oxidative drugs ethanol (EtOH) and methamphetamine, we have shown that heterozygous BRCA1-deficient mice develop normally but are more susceptible to ROS-initiated embryopathies in embryo culture. Furthermore, BRCA1-deficient females show greater susceptibility to ROS-initiated neurodevelopmental deficits than their BRCA1-normal littermates. ROS adversely affect embryonic and fetal brain development via both altered biochemical signalling and oxidative damage, the latter of which is modulated by conceptal DNA repair. Further identification of the complex interactions involved in the dynamic balance among ROS production and detoxification, and downstream repair of macromolecular damage, may provide insight into personal risk for developmental deficits.

2018-06-13 00:00:00

Advisors/Committee Members: Wells, Peter G, Pharmaceutical Sciences.

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Drugs with the primary aromatic amine structure are notorious structural alerts in drug development because of their association with a high incidence of idiosyncratic drug reactions (IDRs). The toxicity of aromatic amines has been attributed to the formation of reactive metabolites common to this functional group that can form antigenic substances and redox cycle to induce oxidative stress. To date, there has not been a systematic study to investigate the mechanisms of aromatic amine-induced IDRs. Therefore, in the first part of my thesis, the danger hypothesis was tested through a global screen of hepatic gene expression in rats treated acutely with the aromatic amine drugs sulfamethoxazole (SMX), dapsone (DDS), and aminoglutethimide (AMG). Although one gene, Eiih, was up-regulated by all drugs tested and could be a general biomarker to drugs that induce IDRs, few changes were found that are likely to represent danger signals. To confirm this, a more comprehensive study was performed on the liver with AMG after longer treatment intervals. Despite substantial changes in apoptotic and mitochondrial pathways, there were few immune changes and no histological evidence of damage. These findings suggest that the liver is not a direct target of aromatic amine drugs or, if it is, the default response may be immune tolerance. The presence of myeloperoxidase in neutrophils and antigen presenting cells provides a more plausible mechanism of aromatic amine toxicity through direct activation of the immune system, and this was tested in the second part of my thesis research. A significant increase in activated neutrophils was observed in AMG-treated rats, which was attributed to an increase in granulocyte progenitors in the bone marrow. This effect is opposite to the agranulocytosis that is observed with AMG clinically, but it may have important implications to the early effects preceding the pathology. In the spleen and lymph nodes, T-cell proliferation was found with simultaneous indications of immune modulation through differential intracellular cytokine expression. Moreover, down-regulation of immune activation may be why the AMG-treated rats do not develop hypersensitivity. Further investigations into tolerogenic mechanisms may have broader implications into why IDRs only develop in a subset of patients.

PhD

Advisors/Committee Members: Uetrecht, Jack, Pharmaceutical Sciences.

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Many drugs are known to cause idiosyncratic drug-induced agranulocytosis, although the mechanism is not well understood. This adverse event is suspected to be the result of the drugâ s reactive metabolite binding to endogenous proteins and creating a foreign body capable of eliciting an immune response. In particular, clozapine has been shown to form reactive metabolites by neutrophil myeloperoxidase (MPO). These studies set out to further characterize MPOâ s role in bioactivating drugs that cause idiosyncratic drug-induced agranulocytosis and to characterize the immune response seen due to clozapine. Overall this work further characterized the early immune response seen due to clozapine treatment using in vitro and in vivo methods. Through this work we have a better understanding of the immune response that may lead to clozapine-induced agranulocytosis. Understanding the clozapine-induced immune response may provide avenues for early detection of clozapine-induced agranulocytosis and ultimately limit the magnitude of this adverse event.

M.Sc.

Advisors/Committee Members: Uetrecht, Jack, Pharmaceutical Sciences.

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We previously observed that postnatal exposure of mice to the procarcinogen 4-aminobiphenyl (ABP) produced liver tumors only in wild-type males, while arylamine N-acetyltransferase deficient males and females of either strain were protected. Others have also observed a sex difference in liver tumors in mice using the procarcinogen diethylnitrosamine (DEN). Reasons for these sex and strain differences are unclear, but differences in acute hepatotoxicity and inflammation may be involved. In this thesis we found that neither ABP nor DEN produced overt hepatotoxicity in postnatally exposed mice, and only DEN caused an increase in levels of the pro-inflammatory cytokine interleukin-6 but was not sex-dependent. The lack of sex difference suggests that sex hormone modulation of inflammation following sexual maturation might favour growth of initiated cells in males. However, the lack of detectable inflammation following ABP exposure may be due to localized responses, or that inflammation may be a DEN-specific effect.

MAST

Advisors/Committee Members: Grant, Denis M., Pharmacology.

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The toxic metal ion cadmium (Cd2+) is pro-oxidant and specifically disrupts the actin cytoskeleton in renal mesangial cells. This study investigated the role of Cd2+-mediated redox modulation of actin through protein S-glutathionylation and the effects of cytoskeletal changes on focal adhesions (FAs) through a Ca2+/calmodulin dependent-protein kinase II (CaMK-II) pathway. Only at low concentrations of Cd2+ (0.5-2 μM) was there an increase in actin glutathionylation, which was a reactive oxygen species-independent, total glutathione-dependent effect. Immunofluorescence of the cytoskeleton suggests that increases in glutathionylation levels occurring under low [Cd2+] are protective in vivo. Higher concentrations (>= 10 μM) of Cd2+ resulted in loss of vinculin and focal adhesion kinase (FAK) from FAs, concomitant with cytoskeletal disruption. Inhibition of CaMK-II preserved cytoskeletal integrity and focal contacts, while decreasing the migration of FAK-phosphoTyr925 to a membrane-associated compartment. This study adds further insight into the Cd2+-mediated effects on the cytoskeleton and FAs.

MAST

Advisors/Committee Members: Templeton, Douglas M., Laboratory Medicine and Pathobiology.

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There is a growing need to develop rapid and cost-effective ecotoxicological tools for risk assessment because traditional methods examine endpoints such as mortality, which do not provide any insight into the mode of action (MOA) of the chemical. Research presented within this thesis illustrates the potential of 1H NMR-based metabolomics as a rapid and routine ecotoxicological tool that can elucidate a chemical’s MOA and also aid in the identification of metabolites of exposure. Metabolomics involves measuring the fluctuations in the endogenous metabolites of an organism within a cell, tissue, bio-fluid or whole organism in response to an external stressor. We focused on the model polycyclic aromatic hydrocarbon (PAH) phenanthrene, and the perfluoroalkyl acids (PFAAs) perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), due to their recalcitrant nature and widespread prevalence in soil environments. 1H NMR-based metabolomics analysis of the exposure of Eisenia fetida earthworms to sub-lethal phenanthrene exposure via filter paper contact tests revealed a concentration-dependent two-phased MOA: a linear correlation between the metabolic response and exposure concentration at low concentrations followed by a plateau in the responses at high concentrations. Alanine, glutamate, maltose, cholesterol and phosphatidylcholine emerged as potential indicators of phenanthrene exposure. An increased energy demand and an interruption in the conversion of succinate to fumarate in the Krebs cycle were observed due to phenanthrene exposure. Sub-lethal PFOA and PFOS exposure to E. fetida via contact tests for two days revealed heightened responses with higher PFOA and PFOS concentrations. Leucine, arginine, glutamate, maltose, and ATP were identified as potential indicators of PFOA or PFOS exposure. E. fetida responses were then investigated after exposure for two, seven and fourteen days to an artificial soil that was spiked with sub-lethal PFOS concentrations. An exposure time-dependent operation of two separate MOAs were identified. Both the contact tests and artificial soil exposure studies identified an elevation in fatty acid oxidation, a disruption in energy metabolism and biological membrane structure, and also an interruption of ATP synthesis following PFOA and PFOS exposure. This thesis illustrates the promise of NMR-based metabolomics as a routine tool for ecotoxicological assessment of contaminated sites.

PhD

Advisors/Committee Members: Simpson, Myrna J., Chemistry.

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The in vivo teratogenicity of methanol (MeOH) and its underlying molecular mechanisms were investigated, particularly the pathogenic contribution of reactive oxygen species (ROS) and oxidative stress. First, MeOH pharmacokinetics were compared among different strains of mice (CD-1, C3H and C57BL/6J) and species (New Zealand White rabbits and cynomolgus monkeys) to determine the potential contribution of metabolism to teratological risk. The mechanism was evaluated directly by manipulating important ROS-relevant pathways including: (1) altering a key antioxidative enzyme (genetically altered mice with high or low catalase activity), (2) inhibiting glutathione [GSH] synthesis using buthionine sulfoximine (BSO), and (3) blocking ROS effects with a free radical spin-trapping agent (phenylbutylnitrone, PBN). Rabbits and one mouse strain (C3H) were resistant to MeOH teratogenicity. Sensitive (C57BL/6J, CD-1) and resistant (C3H) mouse strains exhibited similar MeOH and formic acid (FA) pharmacokinetics, suggesting that developmental risk is not dependent upon MeOH or FA metabolism. MeOH did not increase embryonic ROS formation or DNA oxidation, nor was MeOH teratogenicity altered in C57BL/6J mice expressing elevated catalase levels, or by pretreatment with PBN, suggesting that ROS do not contribute to the in vivo mechanism of MeOH teratogenesis. This apparent ROS independence was in contrast to results from other studies implicating a ROS-dependent mechanism, based on different models of toxicity. These species- and strain-dependent results raise questions regarding the determinants of risk and the relevance of different animal models to the human situation. The spectrum of MeOH-initiated birth defects was expanded, and the incidences of multiple anomalies increased, by GSH depletion. GSH is both a ROS scavenger and a cofactor for formaldehyde dehydrogenase (ADH3), which detoxifies the MeOH metabolite formaldehyde. The results from this collection of studies suggest that formaldehyde is a proximate teratogen in vivo, and that GSH protects by serving as a cofactor for ADH3.

PhD

Advisors/Committee Members: Wells, Dr. Peter G., Pharmaceutical Sciences.

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Consumption of alcohol (ethanol, EtOH) during pregnancy can result in a spectrum of anomalies termed the Fetal Alcohol Spectrum Disorders (FASD), characterized by structural and behavioural deficits, while in utero exposure to methanol (MeOH), a structurally similar alcohol, enhances structural malformations in rodents. The mechanisms underlying these toxicities are unclear, however reactive oxygen species (ROS) have been implicated. I hypothesize that prenatal exposure to EtOH or MeOH enhance the formation of ROS, which oxidatively damage fetal cellular macromolecules (DNA, protein, lipids) and/or alter fetal redox-sensitive signal transduction, causing structural birth defects and postnatal neurodevelopmental deficits. If so, then altering the balance of pathways of ROS formation and detoxification, and repair of oxidatively damaged DNA, should determine teratological risk. This hypothesis was tested using the free radical spin trapping agent phenylbutylnitrone (PBN), and pharmacological and genetic modulations of the ROS-forming enzyme NADPH oxidase (NOX), the antioxidative enzyme catalase and the DNA repair enzyme oxoguanine glycosylase 1 (OGG1). Embryonic EtOH exposure oxidatively damaged embryonic DNA and enhanced structural malformations, which were: (1) blocked by the free radical spin trapping agent PBN; (2) decreased in transgenic mice expressing human catalase (hCat); (3) exacerbated in acatalasemic (aCat) mice; and, (4) exacerbated in OGG1 knockout mice in a genotype-dependent fashion. Fetal EtOH exposure enhanced fetal brain DNA oxidation and postnatal behavioural deficits in CD-1 mice, and in OGG1 knockout mice in a gene dose-dependent fashion; both effects were blocked by PBN. Interestingly, untreated OGG1 knockout mice exhibited a learning deficit compared to wild-type littermates, constituting the first report of a phenotype for these mice. Embryonic MeOH exposure enhanced embryopathies, embryonic protein oxidation and upregulated mRNA and protein expression of embryonic ROS-initiating NOXs in CD-1 embryos, providing the first evidence of NOX involvement in MeOH embryotoxicity. Embryopathies were respectively decreased and increased in hCat and aCat embryos. These results suggest that ROS are involved in the teratological mechanism of EtOH and MeOH, and that embryonic catalase, NOX, and DNA damage and repair are determinants of risk.

PhD

Advisors/Committee Members: Wells, Peter G., Pharmacology.

▼ Cytochrome P450 monooxygenases (P450s) are important enzymes involved in the metabolism of a variety of xenobiotics, including insecticides and plant allelochemicals, and endogenous compounds, including juvenile hormones, ecdysteroids and fatty acids, in insects. Despite rapid advances in revealing various P450 genes in insects, our knowledge on the role of these genes in detoxification of insecticides is very limited. This research was to perform a genome-wide analysis of P450 genes and evaluate the role of selected P450 genes in detoxification of three commonly used pyrethroid insecticides in the yellow fever mosquito (Aedes aegypti). Our genome-wide analysis of revealed 159 P450 genes that can be classified into 18 families and 63 subfamilies. These genes are distributed in four clans, including 11 genes in the CYP2 clan, 80 in the CYP3 clan, 58 in the CYP4 clan and 10 in the mitochondrial CYP clan. The largest families are CYP6, CYP9, CYP4 and CYP325. The intron-exon organization of the genes is very diverse among the gene families, and the highest conservation of gene structures was observed in the CYP6 and CYP9 families predominantly containing single-intron genes. The phylogenetic analysis suggested that the CYP6 and CYP9 families might be derived from a common ancestor. The expression patterns of five transcripts including three individual genes (CYP6AA5, CYP6AL1 and CYP9J32) and two alternative splicing variants (CYP4J16A and CYP4J16B) of CYP4J16 were investigated in various tissues and at different developmental stages of the mosquito. Our results indicated differential expressions of these transcripts in different tissues and at different developmental stages examined. Furthermore, the exposure of the mosquitoes (larvae and adults) to each of three pyrethroid insecticides (permethrin, cypermethrin and deltamethrin) resulted in either down or up-regulation of these transcripts. Functional analyses of the selected P450 transcripts were conducted by using RNA interference (RNAi) followed by insecticide bioassay. RNAi was achieved by feeding mosquito larvae with chitosan/double stranded RNA (dsRNA) nanoparticles or injecting dsRNA to the adults. For the larvae, we obtained relatively low repressions of the P450 transcripts but the repressions were sufficient for carrying out our functional studies. Our study showed increased mortalities by 41.2% to cypermethrin when CYP6AA5 was silenced and 46.0% to permethrin when CYP9J32 was silenced. Similarly, the injection of dsRNAs in adults resulted in significant repressions of the P450 transcripts, and subsequent insecticide exposures led to a 29.3% increase in the adult mortality to cypermethrin when CYP6AA5 was silenced. Our further analysis of the nuclear receptor HR96 in the up-regulation of the P450 genes showed that when HR96 was silenced by RNAi, the up-regulation of CYP4J16B by cypermethrin was reduced by 10.1-fold but silencing HR96 did not affect the up-regulation of other P450 genes examined. These results suggest that HR96 is likely involved in… Advisors/Committee Members: Kun Yan Zhu.

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Mother's milk provides multiple benefits to the offspring. However, xenobiotics transferred into breast milk may pose a risk to the nursing infant. The breast cancer resistance protein (BCRP) actively transports xenobiotics into breast milk. BCRP also transports nutrients, like riboflavin, and together with recently identified riboflavin transporters (RFT), may provide a mechanism for riboflavin secretion into breast milk. Expression of RFT in the mammary gland remained unknown. Our objective was to characterize Bcrp and Rft mRNA expression in the mammary gland of FVB/N mice, and investigate a strategy to decrease excretion of BCRP-transported xenobiotics into the milk using riboflavin intervention. Rft and Bcrp mRNA were upregulated in the mammary gland of lactating mice. An intravenous riboflavin administration significantly reduced the levels of BCRP-transported cimetidine in milk. This study demonstrates the use of riboflavin to exploit the function of mammary BCRP in order to reduce xenobiotic secretion into breast milk.

MAST

Advisors/Committee Members: Ito, Shinya, Pharmacology.

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Polybrominated diphenyl ethers (PBDEs) are synthetic chemicals that are added to a variety of consumer products as flame-retardants. The ubiquitous nature and endocrine disrupting properties of PBDEs are a public concern. A pilot study was performed to investigate whether in utero PBDE exposure, as measured in maternal hair, is associated with genitourinary tract malformations in male infants. In addition, we compared PBDE levels in maternal and infant hair and used segmental analysis to investigate how PBDEs varied along the shaft. Preliminary results suggest a trend toward an elevated PBDE body burden in mothers whose infants were born with genitourinary tract malformations; this was significant for some PBDEs. The sum of PBDEs (ΣPBDEs) in maternal hair did not correlate with infant hair levels; children had significantly greater levels. A significant increase in the ΣPBDEs was observed in distal hair suggesting hair PBDEs may be reflective of both internal and external exposure.

MAST

Advisors/Committee Members: Koren, Gideon, Pharmacology.

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Tese de mestrado, História (História Antiga), Universidade de Lisboa, Faculdade de Letras, 2012

Nesta dissertação, discutiu-se como o Estado romano lidou com os cristãos em seu exército, bem como as diversas posições de eclesiásticos sobre a questão e como o problema foi resolvido após a conversão de Constantino. Contrariando a tese formulada por Edward Gibbon no século XVIII, foi explicado como o cristianismo na verdade fortaleceu o “espírito militar” e contribuiu na formação de uma identidade militar tardia. Para tal esforço foi realizada uma contextualização histórica da guerra romana dos seus primórdios ao fim do século IV, levando em conta diversas fontes que tratam do tema em questão. De entre essas fontes se destaca o Epitoma rei militaris de Flávio Vegécio (séc. IV d.C.), que foi analisado em sintonia com o contexto político da basileia, termo aplicado ao sistema imperial romano tardio. Não obstante uma visão crítica sobre os breviários e epítomes do Baixo Império, Vegécio não se resumiu a compilar informações acerca da estrutura, organização e disciplina da legião clássica. Em um período conturbado, em que os velhos símbolos pagãos eram resignificados, a cristianização da milícia constituiu um dos derradeiros processos de coesão e renovação do ideal dos soldados romanos, do qual o novo sacramentum fornece importante prova. Palavras-chave: Exército romano – Cristianismo – Basileia – Vegécio – Século IV d.C.

In this thesis, we discussed how the Roman state dealt with the Christians in their army, and the various ecclesiastical positions on the issue and how the problem was resolved after the conversion of Constantine. Contrary to the thesis formulated by Edward Gibbon in the eighteenth century, it was explained how Christianity actually strengthened the “military spirit” and contributed to the formation of a late military identity. For such an effort was made into a historical context of the Roman war of its beginnings to the end of the fourth century, taking into account various sources dealing with the issue in question. Among these sources highlight is the Flavius Vegetius’ Epitoma rei militaris (fourth century A.D.), which was analyzed in line with the political context of basileia, a term applied to the Late Roman imperial system. Despite a critical view of the breviary and epitomes of the Lower Empirer, Vegetius was not reduced to complein information about the structure, organization and discipline of classical legion. In a troubled period in which the old pagan symbols were resignified, the Christianization of the militia was one of the last processes of cohesion and renewal of the ideal of the Roman soldiers, which the new sacramentum provides important evidence. Keywords: Roman

Advisors/Committee Members: Rodrigues, Nuno Simões,1968-, Dias, Paula Cristina Barata,1971-.

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Mercury is an environmental contaminant of global concern. The distribution of mercury in freshwater systems is poorly characterized in Mexico, despite widespread contamination from industrial and urban effluents. The land use, geology, and hydrology of the Lake Zapotlán basin, Mexico are conducive to the delivery of elevated mercury in water to the lake due to untreated wastewater discharge, deforestation, and local volcanic history. To assess a mercury exposure risk to fish consumers, the concentrations of total Hg (THg) in water inputs, surface waters, sediments, and the commercial catch of tilapia and carp were investigated. Results indicate that despite high particle-bound inputs of THg to the lake in runoff and wastewater, THg in sediments and surface waters were low. Dense Typha latifolia dominated wetlands are believed to retain THg inflow from water inputs. Concentrations of THg in tilapia and carp were low, suggesting low mercury bioavailability in this system.

MAST

Advisors/Committee Members: Branfireun, Brian, Geography.