Our DNA is under constant threat from endogenous and exogenous damaging agents. Our cells have evolved a network of signaling pathways and repair mechanisms that detect and counteract this threat, collectively referred to as the DNA damage response. Cells that lose the ability to cope with DNA damage risk the acquisition of deleterious changes to DNA sequence or structure. I initially set out to identify and characterize candidate proteins that interact with Mus81-Eme1, an endonuclease that processes DNA intermediates that arise from aberrant or stalled DNA replication. I focused on one interesting candidate protein known as Nuclear protein 95 (Np95) which now is called UHRF1 (Ubiquitin-like, containing PHD and RING finger domains, 1). Although previous studies demonstrate the importance of Mus81-Eme1 enzyme in DNA repair, genome integrity, and tumor suppression, little is known about how the enzyme acts together with other components of signaling pathways that comprise the DNA damage response. My findings in chapter two characterized this interaction and linked Mus81-Eme1 with UHRF1 in the cellular response to DNA damage. Although UHRF1 levels have been linked with sensitivity to antineoplastic agents, a direct role for UHRF1 in the DNA damage response had not been elucidated or reported. Accordingly, the third chapter of my thesis focuses on investigating the role of UHRF1 in the cellular response to DNA damage caused by exposure to γ-irradiation. Our findings for chapter three indicate that (i) UHRF1 is crucial for the cellular iii response to double strand breaks caused by γ-irradiation and that (ii) UHRF1 is critical for maintenance of chromosome integrity. Recent studies have now implicated UHRF1 in processes required for heterochromatin replication. This protein has been shown to play a role in the replication of heterochromatin by helping to replicate DNA methylation patterns and playing a role in propagating the epigenetic mark known as histone 3 lysine 9 trimethylation (H3K9me3). H3K9me3 has been shown to play a role in a signaling pathway involved in the repair of DNA damage in heterochromatic regions. In the fourth chapter of my thesis, we hypothesize that UHRF1 is playing a role in a pathway that responds to DSB damage in heterochromatic regions of chromatin. Our results indicate that a loss of UHRF1 results in a loss of heterochromatic H3K9me3 and heterochromatin associated HP1β. Our findings support the idea that epigenetic alterations maintained by UHRF1 contribute to signals that relax heterochromatin to facilitate access for repair factors. In summary, findings presented in this thesis shed light on processes that protect cells from DNA damage caused by radiation and chemotherapy and safeguard genome integrity.

Our DNA is under constant threat from endogenous and exogenous damaging agents. Our cells have evolved a network of signaling pathways and repair mechanisms that detect and counteract this threat, collectively referred to as the DNA damage response. Cells that lose the ability to cope with DNA damage risk the acquisition of deleterious changes to DNA sequence or structure. I initially set out to identify and characterize candidate proteins that interact with Mus81-Eme1, an endonuclease that processes DNA intermediates that arise from aberrant or stalled DNA replication. I focused on one interesting candidate protein known as Nuclear protein 95 (Np95) which now is called UHRF1 (Ubiquitin-like, containing PHD and RING finger domains, 1). Although previous studies demonstrate the importance of Mus81-Eme1 enzyme in DNA repair, genome integrity, and tumor suppression, little is known about how the enzyme acts together with other components of signaling pathways that comprise the DNA damage response. My findings in chapter two characterized this interaction and linked Mus81-Eme1 with UHRF1 in the cellular response to DNA damage. Although UHRF1 levels have been linked with sensitivity to antineoplastic agents, a direct role for UHRF1 in the DNA damage response had not been elucidated or reported. Accordingly, the third chapter of my thesis focuses on investigating the role of UHRF1 in the cellular response to DNA damage caused by exposure to γ-irradiation. Our findings for chapter three indicate that (i) UHRF1 is crucial for the cellular iii response to double strand breaks caused by γ-irradiation and that (ii) UHRF1 is critical for maintenance of chromosome integrity. Recent studies have now implicated UHRF1 in processes required for heterochromatin replication. This protein has been shown to play a role in the replication of heterochromatin by helping to replicate DNA methylation patterns and playing a role in propagating the epigenetic mark known as histone 3 lysine 9 trimethylation (H3K9me3). H3K9me3 has been shown to play a role in a signaling pathway involved in the repair of DNA damage in heterochromatic regions. In the fourth chapter of my thesis, we hypothesize that UHRF1 is playing a role in a pathway that responds to DSB damage in heterochromatic regions of chromatin. Our results indicate that a loss of UHRF1 results in a loss of heterochromatic H3K9me3 and heterochromatin associated HP1β. Our findings support the idea that epigenetic alterations maintained by UHRF1 contribute to signals that relax heterochromatin to facilitate access for repair factors. In summary, findings presented in this thesis shed light on processes that protect cells from DNA damage caused by radiation and chemotherapy and safeguard genome integrity.

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.

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.

Novel Regulatory Mechanisms of D1 Dopamine Receptor Maturation and Internalization Michael Ming Chuen Kong Degree of Doctor of Philosophy, 2008 Department of Pharmacology University of Toronto ABSTRACT Dopamine is the most abundant catecholamine neurotransmitter in the mammalian brain and controls various physiological processes. The D1 dopamine receptor (D1DR) is the predominant dopamine receptor in the brain and traditionally couples to stimulatory G proteins, such as Gs, to activate adenylyl cyclase and generate cAMP. Although the trafficking itinerary of ER/Golgi maturation, agonist-induced internalization, and recycling/degradation are features common to many G protein-coupled receptors (GPCRs), the molecular regulation of these individual processes for the D1DR is not fully elucidated. Many GPCRs have been shown to form homo-oligomers; the work presented in this thesis explores how multimerization of D1DR has a role in regulating how these receptors are trafficked to the plasma membrane. In addition, the regulation of D1DR internalization is investigated in the context of emerging evidence highlighting the importance of lipid rafts. Using strategically designed point mutations of the D1DR, specific receptor mutants were found to intracellularly sequester the wild-type receptor by oligomerization. This level of scrutiny by the quality control machinery in the cell could be circumvented by treatment with cell permeable dopaminergic agonists, but not antagonists or inverse agonists. This finding suggests that specific conformational requirements must be achieved before full maturation and anterograde trafficking of the D1DR can proceed. Furthermore, it was determined that cell surface bound D1DRs could internalize through a novel clathrin independent pathway that required binding to the scaffolding protein, caveolin-1. This interaction with caveolin-1 was identified in whole rat brain and was found to require a putative caveolin binding motif in transmembrane domain 7. Palmitoylation of D1DR was found to regulate the rate of agonist-induced caveolae mediated internalization. Finally, we determined that the integrity of caveolae was important in regulating cAMP signaling through D1DR. These findings provide novel insight into the trafficking requirements of newly synthesized D1DRs as well as alternative mechanisms of regulation of receptors after agonist activation. The oligomerization of GPCRs and the localization of GPCRs in lipid rafts represent two emerging concepts important to many aspects of GPCR function. Future work aimed at integrating these overlapping processes will further our understanding of this important group of cell surface receptors.

The aryl hydrocarbon receptor (AHR) and estrogen receptor alpha (ERα) are ligand-activated transcription factors. Reciprocal crosstalk between these two receptor systems has been previously established but the exact molecular mechanisms of their interactions remain incompletely understood. Using chromatin immunoprecipitation followed by DNA microarrays (ChIP-chip), I assessed the role of ERα in AHR signalling after dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) treatment in the T-47D human breast cancer cell line. I determined that ERα is recruited to a subset of AHR target genes suggesting that it is a gene-specific modulator of AHR activity. Transcription factor binding site analysis of our data set also revealed that forkhead motifs were over-represented, implying that they may be important in AHR signalling. To address this, I focused on the regulation of cyclin G2 (CCNG2) to determine the importance of FOXA1 (forkhead box A1) in AHR signalling. CCNG2 is a negative regulator of cell cycle and known to be repressed by ERα. Using ChIP, Co-IP, CCNG2 reporter gene constructs and RNA interference targeting FOXA1, I demonstrated that FOXA1 was important for the AHR-mediated and TCDD-dependent induction of CCNG2. Another finding from the ChIP-chip study was that AHR was recruited to estrogen target genes. To determine the importance of this I used zinc-finger nuclease mediated knockout of AHR and studied ERα signalling as well as the role of AHR in the cell cycle using breast cancer cell lines. Focusing on the regulatory regions of trefoil factor 1 (TFF-1) and gene upregulated in breast cancer 1 (GREB1) I determined that AHR had an inhibitory effect. Cell cycle analysis indicated that AHR facilitated cell cycle progression with cells accumulating in both the G¬1 and G2/M phases in the absence of AHR. My novel findings demonstrated the complexity of AHR-ERα crosstalk, its importance in the cell cycle, and the need for further study.

Sustained nitroglycerin (GTN) therapy impairs endothelial function in healthy volunteers and patients with cardiovascular disease, caused by an increase in vascular oxidative stress. This study aims to estimate the effect of rosiglitazone on vascular endothelial function in healthy volunteers continuously dosed to transdermal GTN (0.6mg/hr) for 7 days. To assess endothelial function, forearm blood flow was measured by venous occlusion strain-gauge plethysmography in response to intra-brachial infusions of acetylcholine. GTN-treated subjects experienced significant attenuation of endothelium-dependent responses to acetylcholine (p<0.05; compared to placebo), but was reversed with vitamin C (p=ns; compared to placebo). Endothelium-dependent responses to acetylcholine were blunted in groups randomized to rosiglitazone alone (p<0.05; compared to placebo) and rosiglitazone + GTN (p<0.05 compared to placebo). Interestingly, this effect was not modified by vitamin C. In conclusion, rosiglitazone impairs endothelial function and concurrent therapy with rosiglitazone does not attenuate the adverse effects of transdermal GTN on the vasculature.

There are increasing concerns that exposure to unmetabolized folic acid, which results from folic acid intakes that overwhelm the liver’s metabolic capacity, may be associated with adverse effects. In this study, we examined the folic acid status of women of reproductive age in relation to dietary intake and the effect of folic acid supplementation (1.1 mg or 5 mg). Plasma unmetabolized folic acid was not significantly correlated with folate intake estimated by food frequency questionnaire or biomarkers. The proportion of women with detectable levels of unmetabolized folic acid increased from 65% to 100% after twelve weeks of supplementation (p < 0.05), however, the increase in concentrations did not reach statistical significance and the effect was not sustained. Moreover, there were no significant differences between the two doses. This suggests that there are mechanisms by which the body adapts to high folic acid intakes to limit exposure to unmetabolized folic acid.

3-Methylcholanthrene (MC) is a model polycyclic aromatic hydrocarbon that induces cytochrome P450 1A1 (CYP1A1) expression. This laboratory has shown previously that aromatic hydrocarbons, which are important environmental toxicants, down-regulate the expression of rat liver CYP2C11. Recent observations also suggested that CYP2C8, a human enzyme that metabolizes antineoplastic and antidiabetic drugs, among others, is down-regulated in response to aromatic hydrocarbon exposure in primary human hepatocytes. I examined the regulation of CYP2C8 at the mRNA level by MC in two human hepatocellular carcinoma cell lines, HepG2 and HepaRG. MC down-regulated CYP2C8 mRNA levels in HepG2 cells at 24 hours and in HepaRG cells at 48 hours. CYP1A1 mRNA was induced by MC in both cell lines and HepaRG cells appeared to be more sensitive than HepG2 cells to MC-induced cytotoxicity. Further studies are warranted to define the mechanisms and functional impacts of the modulation of this important human CYP by environmental toxicants.

Alcohol consumption during pregnancy can lead to Fetal Alcohol Spectrum Disorder, and because maternal self-reports are often unreliable, a biomarker of alcohol use during pregnancy is needed to accurately determine fetal exposure. Ethyl glucuronide (EtG) is a direct metabolite of ethanol that has been detected in the meconium of infants born to mothers who consumed alcohol during pregnancy. In the current study, a method was developed and validated for EtG detection in placental perfusate and tissue using gas chromatography-mass spectrometry. Subsequently, the ex vivo human placental perfusion model was used to investigate whether EtG crosses the human placenta. The validated GC-MS method showed sufficient sensitivity in detecting EtG in placental perfusate and tissue. EtG crossed the placenta slowly and transfer was incomplete after 3 hours of perfusion. EtG appears to cross the human placenta and, hence, to represent both maternal and fetal exposure to alcohol.