Methylmercury (MeHg) is an environmental toxin to which we are exposed through the consumption of seafood. Reactive oxygen species (ROS) have been implicated in the mechanism of toxicity, and in vitro studies in our laboratory have implicated DNA oxidation, particularly the DNA repair enzyme oxoguanine glycosylase 1 (OGG1). My studies determined the effects of in utero exposure to MeHg on fetal brain DNA oxidation and postnatal neurodevelopmental deficits, and the role of ROS-mediated oxidative DNA damage using the free radical spin trap, α-phenyl-N-tert-butylnitrone (PBN), and DNA repair-deficient ogg1 knockout mice. While neither MeHg nor PBN altered DNA oxidation in fetal brain, MeHg caused cognitive deficits in passive avoidance and novel object recognition, the latter of which was blocked by PBN pretreatment, suggesting ROS involvement. Preliminary longevity studies following one litter from each treatment group to 16 months suggest that in utero MeHg treatment may shorten lifespan. Endogenous DNA oxidation was increased in the brains of ogg1 knockout fetuses compared to wild-type littermates, although this was not enhanced by MeHg. However, OGG1-deficient animals exhibited cognitive deficits in passive avoidance after MeHg treatment, suggesting a role for DNA damage. Furthermore, ogg1 knockout female mice exhibited a passive avoidance deficit compared to wild-type females regardless of treatment, corroborating a role for oxidative DNA damage in neurodevelopmental deficits. MeHg increased apoptosis in the hippocampal region of fetal brain, and may cause DNA double-strand breaks (DSBs), evidenced by enhanced phosphorylation of histone 2AX (γH2AX). Ogg1 knockout progeny exhibited increased cellular proliferation or migration in the developing hippocampal region, which was blocked by MeHg. My results provide the first evidence that: (1) MeHg may decrease lifespan; (2) PBN protects against some postnatal neurodevelopmental deficits caused by in utero exposure to MeHg; and (3) DNA repair-deficient progeny are more susceptible to postnatal cognitive deficits caused by in utero MeHg exposure, suggesting that ROS-mediated DNA oxidation plays a role in MeHg-initiated neurodevelopmental deficits.

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.

Clinical characteristics of idiosyncratic drug reactions (IDRs) suggest that they are immune mediated. Penicillamine-induced autoimmunity in Brown Norway rats was used as a tool for mechanistic studies of this type of IDR. It has been shown that T helper 17 (Th17) cells play a central role in many types of autoimmune diseases. This study was designed to test whether Th17 cells are involved in the pathogenesis of penicillamine-induced autoimmunity. In sick animals, interleukin (IL) 6 and transforming growth factor-β1, known to be driving forces of Th17 differentiation, were consistently increased following penicillamine treatment. IL-17 and IL-22, characteristic cytokines produced by Th17 cells, were increased in sick animals. Furthermore, the percentage of IL-17-producing CD4 T cells was significantly increased, but only in sick animals. Retinoic acid, which has been reported to inhibit Th17 cell development, made the autoimmunity worse, increased IL-6 production, and did not decrease the number of Th17 cells. An infiltration of CD8 cytotoxic T cells in the liver suggests that they may be the key player in causing liver toxicity induced by D-penicillamine. Drug-induced liver injury (DILI) is one of the major causes of morbidity, mortality, and drug candidate failure. Recently, it has been suggested that Th17 cells may play an active role in inflammatory human liver diseases. In a study of patients being treated with isoniazid, some patients developed mild liver injury. The percentage of Th17 cells in the blood of these patients significantly increased when the ALT increased, and this suggests that they play a role in the mechanism of this liver injury. Furthermore, IL-10-producing T cells also increased and this may have prevented the development of severe liver injury. In another study, two hours after treatment of mice with acetaminophen there was a significant increase in Th17 cells in the liver. This rapid response suggests that Th17 cells can be part of the innate immune response to liver injury. Our data provided evidence that Th17 cells are involved in both “toxic” and idiosyncratic liver toxicity. This pathway could be a new target for the therapeutic interventions to treat DILI.

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.

Master of Science / Department of Chemical Engineering / Larry E. Erickson / The damage that can be inflicted by ultraviolet radiation has gained widespread interest. Traditionally sunscreens are made of organic and inorganic components that block two of the three types of ultraviolet radiation, UVA and UVB. This report is a literature review of several articles that have investigated the effects of inorganic UV filters; specifically titanium dioxide and cerium dioxide. There are concerns about absorption of titanium dioxide into the skin and the adverse reactions that could occur, but it was found that there is little to no absorption. Similarly the photostability of titanium dioxide is a concern; this was found to be remedied in part by a surface treatment to the titanium dioxide. The combination of titanium dioxide and carnauba wax was also studied and found to enhance the properties of both the organic and inorganic filters. Ceria was studied as a possible replacement for titanium dioxide. It was found to have similar ultraviolet shielding properties while minimizing the photocatalytic activity and photocytotoxicity seen in titanium dioxide.

Master of Science / Department of Entomology / Kun Yan Zhu / 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 regulating the expression of CYP4J16B in Ae. aegypti. However, different regulatory mechanism (s) may be involved in the up-regulation of other P450 genes examined. Model structure of CYP6AA5 was created by homology modeling and insecticides substrates were docked into the active site of this protein. Our results indicate that all three insecticides can fit into the catalytic pocket. The interaction distances between the heme iron and the putative aromatic hydroxylation site were 9.2, 9.4 and 7.2 Å for permethrin, cypermethrin and deltamethrin, respectively, whereas for aliphatic hydroxylation site these distances were 5.3, 2.8 and 2.9 Å. These results showed that CYP6AA5 may be able to metabolize cypermethrin and deltamethrin preferentially by aliphatic hydroxylation as indicated by the close interaction with the heme iron.

Master of Veterinary Bioscience / Department of Diagnostic Medicine/Pathobiology / Deon Van Der Merwe / Every year thousands of domestic animals are poisoned by ethylene glycol. Exposure is normally orally, but may be dermal, and poisonings are usually accidental and not malicious. Antifreeze, overwhelmingly the source of the ethylene glycol poisoning, is responsible for over 99% of reported cases. Storage, handling and proper disposal of ethylene glycol is extremely important in limiting access to this deadly product. Ethylene glycol exposures were involved in 1737 calls made to the American Society for the Prevention of Cruelty to Animals call center between 2006 and 2011. Dogs were involved in approximately 87% of exposures and cats in 13%. There were no seasonal or breed patterns. The most common clinical signs reported were neurological and gastrointestinal for both cats and dogs. Urinary calcium oxalate crystals were reported in 28.6% of exposed cats, and 21% of dogs. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used to detect calcium oxalate crystals in wax-mounted kidneys from twenty total cases, ten of which were suspected ethylene glycol poisoning submitted to the Kansas State Veterinary Diagnostic Laboratory, and ten samples deemed negative by a pathologist using light microscopy. Pure calcium oxalate monohydrate was used as a reference, and a unique absorption peak was detected between wavenumbers 1290 cm[superscript]-1 and 1320 cm[superscript]-1. The drying of kidney tissues resulted in increased sensitivity for calcium oxalate. Crystal detection by the ATR-FTIR was compared to light microscopy. Bi-fringence of crystals allowed microscopic detection, but the ATR-FTIR specificity for the test was 100%, and sensitivity was 80% compared to traditional microscopy for ca-oxalate crystal identification. ATR-FTIR was also used to detect un-metabolized ethylene glycol in vomitus using wavenumbers 1084 cm[superscript]−1, 1039 cm[superscript]−1, and 882 cm[superscript]−1, but ethylene glycol was not detectable. Ethylene glycol concentrations in samples were much too low to be detected as ethylene glycol on the ATR-FTIR, as the limit of detection was not distinguishable until 5000 ppm using a serial dilution. These methods presented simple, reliable, quick, sensitive, stable, and highly adaptable tests for detection, diagnosis and treatment of ethylene glycol poisoning.

Human epithelium-specific ETS transcription factor-1 (ESE-1), which is also known as E74-like factor-3 (Elf3) in mice, is strongly expressed in lung during fetal development and in certain lung cancers. The primary goal of the work presented in this thesis was to investigate whether ESE-1 is involved in regeneration of the injured lung epithelium by administering naphthalene to both wild-type (Elf3 +/+) and Elf3-deficient (Elf3 -/-) mice. However, optimal conditions for proper utilization of the naphthalene-induced lung injury model must first be established. Therefore, dose-response studies were initially conducted by administering three different doses of naphthalene to both male and female mice, as described in chapter 2. Although it is shown that the extent of naphthalene-induced Clara cell injury is dose-dependent in both male and female mice, female mice are more sensitive to naphthalene-induced injury than male mice independent of the dose. Furthermore, it is also demonstrated that these gender-dependent differences in naphthalene injury can subsequently influence downstream lung repair kinetics. In light of these findings, lung regeneration was examined in both sexes of both Elf3 +/+ and Elf3 -/- mice. As reported in chapter 3, the kinetics of bronchiolar epithelial cell proliferation and differentiation is delayed considerably in Elf3 -/- mice following naphthalene injury. Moreover, expression of transforming growth factor-beta type II receptor, which is a well-known transcriptional target gene of ESE-1 and is involved in the induction of epithelial cell differentiation, is significantly lower in the bronchiolar airway epithelium of Elf3 -/- mice as compared to Elf3 +/+ mice under steady-state conditions and during repair of naphthalene-induced damage. Collectively, these findings occur to a similar extent in both sexes of both Elf3 +/+ and Elf3 -/- mice, and suggest that ESE-1 plays an important role in regulating the kinetics of airway epithelial regeneration after acute lung injury.

Nevirapine (NVP) can cause serious skin rashes and hepatotoxicity. It also causes an immune-mediated skin rash in rats but not hepatotoxicity. There is strong evidence that the rash is due to 12-hydroxynevirapine (12-OH-NVP), which is further metabolized to a reactive benzylic sulfate in the skin. This could both act as a hapten and induce a danger signal. In contrast, most of the covalent binding in the liver appears to involve oxidation of the methyl group leading to a reactive quinone methide. In this study we examined the effects of NVP and 12-OH-NVP on gene expression in the liver and skin. Both NVP and 12-OH-NVP induced changes in the liver, but the list of genes was different, presumably reflecting different bioactivation pathways. In contrast, many more genes were up-regulated in the skin by 12-OH-NVP than by NVP, which is consistent with the hypothesis that the 12-hydroxylation pathway is involved in causing the rash. Some genes up-regulated by 12-OH-NVP were Trim63, S100a7a, and IL22ra2, etc. Up-regulation of genes such as S100a7a, which is considered a danger signal, supports the danger hypothesis. Up-regulation of genes such as the ubiquitin ligase and Trim63 are consistent with protein-adduct formation. Up-regulation of IL-22ra2 gene suggests an immune response. These results provide important clues to how NVP causes induction of an immune response, in some cases leading to an idiosyncratic drug reaction.

Human epithelium-specific ETS transcription factor-1 (ESE-1), which is also known as E74-like factor-3 (Elf3) in mice, is strongly expressed in lung during fetal development and in certain lung cancers. The primary goal of the work presented in this thesis was to investigate whether ESE-1 is involved in regeneration of the injured lung epithelium by administering naphthalene to both wild-type (Elf3 +/+) and Elf3-deficient (Elf3 -/-) mice. However, optimal conditions for proper utilization of the naphthalene-induced lung injury model must first be established. Therefore, dose-response studies were initially conducted by administering three different doses of naphthalene to both male and female mice, as described in chapter 2. Although it is shown that the extent of naphthalene-induced Clara cell injury is dose-dependent in both male and female mice, female mice are more sensitive to naphthalene-induced injury than male mice independent of the dose. Furthermore, it is also demonstrated that these gender-dependent differences in naphthalene injury can subsequently influence downstream lung repair kinetics. In light of these findings, lung regeneration was examined in both sexes of both Elf3 +/+ and Elf3 -/- mice. As reported in chapter 3, the kinetics of bronchiolar epithelial cell proliferation and differentiation is delayed considerably in Elf3 -/- mice following naphthalene injury. Moreover, expression of transforming growth factor-beta type II receptor, which is a well-known transcriptional target gene of ESE-1 and is involved in the induction of epithelial cell differentiation, is significantly lower in the bronchiolar airway epithelium of Elf3 -/- mice as compared to Elf3 +/+ mice under steady-state conditions and during repair of naphthalene-induced damage. Collectively, these findings occur to a similar extent in both sexes of both Elf3 +/+ and Elf3 -/- mice, and suggest that ESE-1 plays an important role in regulating the kinetics of airway epithelial regeneration after acute lung injury.