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.

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.

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.

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.

There is a strong association between estrogen exposure and breast cancer risk. Estrogen can activate estrogen receptor alpha (ERalpha) to increase cell proliferation. Estrogen can also be metabolized into genotoxic compounds to induce DNA damage and mutations. Activation of the aryl hydrocarbon receptor (AHR) and nuclear factor erythroid-2 like 2 (NFE2L2; NRF2) can alter the production of genotoxic estrogen. The present thesis investigated the signalling mechanisms of ERalpha, AHR, and NRF2 and how their interaction might modulate breast cancer risk. In Chapter 2, genome-wide, but promoter-focused analysis of ERalpha binding sites in T-47D breast cancer cells identified potential cell line specific differences in estrogen signalling between T-47D and the commonly used MCF-7 breast cancer cells. CYP2B6 was identified to be an ERalpha target gene in T-47D cells but not MCF-7 cells, supporting cell line dependent effect in estrogen signalling. In Chapter 3 and 4, genome-wide analyses of AHR binding sites were performed to investigate the molecular criteria governing genomic AHR transactivation in vivo in mouse and in vitro in MCF-7 breast cancer cells. Our analysis identified 1) the previously established aryl hydrocarbon response element to be an important, but not an absolute requirement in AHR transactivation and 2) key epigenetic modifications that modulate AHR-dependent gene regulation. Lastly, in Chapter 5, interaction among ERalpha, AHR, and NRF2 was presented at the regulatory region of two NRF2 target genes, NADPH Quinone Oxidoreductase 1 (NQO1) and Heme Oxygenase 1 (HMOX1). ERalpha repressed, whereas AHR enhanced NRF2-dependent NQO1 and HMOX1 mRNA expression through altered p300 recruitment and Histone H3 Lysine 9 acetylation. Collectively, this thesis examined novel molecular mechanisms that might alter breast cancer development/progression by modulating ER, AHR, and NRF2 activity.

There is a strong association between estrogen exposure and breast cancer risk. Estrogen can activate estrogen receptor alpha (ERalpha) to increase cell proliferation. Estrogen can also be metabolized into genotoxic compounds to induce DNA damage and mutations. Activation of the aryl hydrocarbon receptor (AHR) and nuclear factor erythroid-2 like 2 (NFE2L2; NRF2) can alter the production of genotoxic estrogen. The present thesis investigated the signalling mechanisms of ERalpha, AHR, and NRF2 and how their interaction might modulate breast cancer risk. In Chapter 2, genome-wide, but promoter-focused analysis of ERalpha binding sites in T-47D breast cancer cells identified potential cell line specific differences in estrogen signalling between T-47D and the commonly used MCF-7 breast cancer cells. CYP2B6 was identified to be an ERalpha target gene in T-47D cells but not MCF-7 cells, supporting cell line dependent effect in estrogen signalling. In Chapter 3 and 4, genome-wide analyses of AHR binding sites were performed to investigate the molecular criteria governing genomic AHR transactivation in vivo in mouse and in vitro in MCF-7 breast cancer cells. Our analysis identified 1) the previously established aryl hydrocarbon response element to be an important, but not an absolute requirement in AHR transactivation and 2) key epigenetic modifications that modulate AHR-dependent gene regulation. Lastly, in Chapter 5, interaction among ERalpha, AHR, and NRF2 was presented at the regulatory region of two NRF2 target genes, NADPH Quinone Oxidoreductase 1 (NQO1) and Heme Oxygenase 1 (HMOX1). ERalpha repressed, whereas AHR enhanced NRF2-dependent NQO1 and HMOX1 mRNA expression through altered p300 recruitment and Histone H3 Lysine 9 acetylation. Collectively, this thesis examined novel molecular mechanisms that might alter breast cancer development/progression by modulating ER, AHR, and NRF2 activity.

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.

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.

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.

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.