An animal model has implicated elevated levels of tissue factor (TF), and resultant hypercoagulability and inflammation, as key factors in recurrent pregnancy loss (RPL) and has demonstrated that pravastatin is effective in treating this condition. In this study, we have re-evaluated the contraindication of statins during pregnancy. Evaluation has shown that while animal testing (at maternally toxic doses) and case reports of birth defects have led to the contraindication of statins during pregnancy, our controlled study, similar to previously published controlled studies, has failed to demonstrate increased fetal risks. As well, we demonstrated that transfer of pravastatin across the placenta is likely limited. While short term suspension of therapy during gestation is considered safe, extended time without therapy is detrimental to cardiovascular health. Coupled with a trend of elevated TF levels in women with RPL, reconsideration of the contraindication of statins is warranted based on appropriate risk: benefit assessment.

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.

An animal model has implicated elevated levels of tissue factor (TF), and resultant hypercoagulability and inflammation, as key factors in recurrent pregnancy loss (RPL) and has demonstrated that pravastatin is effective in treating this condition. In this study, we have re-evaluated the contraindication of statins during pregnancy. Evaluation has shown that while animal testing (at maternally toxic doses) and case reports of birth defects have led to the contraindication of statins during pregnancy, our controlled study, similar to previously published controlled studies, has failed to demonstrate increased fetal risks. As well, we demonstrated that transfer of pravastatin across the placenta is likely limited. While short term suspension of therapy during gestation is considered safe, extended time without therapy is detrimental to cardiovascular health. Coupled with a trend of elevated TF levels in women with RPL, reconsideration of the contraindication of statins is warranted based on appropriate risk: benefit assessment.

Populations of Black-African descent have slower rates of nicotine and cotinine metabolism, smoke fewer cigarettes (~10 cigarettes/day), and have higher incidences of tobacco-related illnesses compared to Caucasians. Cytochrome P450 2A6 (CYP2A6) is the main enzyme involved in the metabolism of nicotine and its proximal metabolite cotinine, as well as tobacco-specific nitrosamines. Genetic polymorphisms in CYP2A6 contribute to the large variability observed in rates of nicotine metabolism. Reduced CYP2A6 activity has been associated with fewer cigarettes smoked, higher quit rates, and lower lung cancer risk in predominantly moderate to heavy-smoking (~20–30 cigarettes/day) Caucasians. CYP2A6 genetic variants and their impact on smoking behaviours have not been well studied among individuals of Black-African descent. The main objectives herein were to identify and characterize new CYP2A6 variants that may explain the slower rates of metabolism, and determine whether CYP2A6 variation is a predictor of smoking phenotypes in this population. Furthermore, we examined whether previously validated biomarkers of tobacco exposure have limitations among individuals of Black-African descent given their low and sporadic smoking patterns. A new CYP2A6 variant (CYP2A6*23) was found in individuals of Black-African descent recruited for a nicotine pharmacogenetic-pharmacokinetic study. CYP2A6*23 reduced activity towards nicotine and coumarin in vitro and was associated with slower rates of CYP2A6 kinetics in vivo. In a clinical trial of African-American light smokers, CYP2A6 slow metabolizers were more successful at smoking cessation compared to normal metabolizers, although no differences in cigarette consumption were found. Two common biochemical markers of tobacco smoke exposure, cotinine and exhaled carbon monoxide, were weakly correlated with self-reported cigarette consumption. These biomarkers were not substantially affected by variables previously shown to alter amount smoked and/or rates of cotinine metabolism such as gender, age, body mass index or smoking menthol cigarettes. However, CYP2A6 slow metabolizers had significantly higher cotinine without smoking more cigarettes. Identification and characterization of novel variants adds to our understanding of nicotine pharmacokinetic differences between racial/ethnic minority groups and improves accuracy of CYP2A6 genotype groupings for genetic association studies. Furthermore, better insight into the biological factors associated with smoking behaviours will aid in the development of more efficacious targeted treatments for this understudied population.

Over 220 million people worldwide are diagnosed with diabetes and rising prevalence is reported in nearly all surveyed populations. Accordingly, the percentage of pregnancies affected by pre-existing type 1 or 2 diabetes or by diabetes that develops during pregnancy, called gestational diabetes mellitus (GDM), is also on the rise. Today, approximately 8% of all pregnancies are complicated by diabetes. Diabetes alters drug disposition mechanisms in non-pregnant subjects but the impact of diabetes on drug disposition in pregnancy has not been properly evaluated. Atypical drug disposition in pregnancy has implications for maternal and fetal health. Because liver tissue from pregnant women is not readily available, this thesis investigated drug disposition mechanisms primarily in a rat model of experimental GDM. This model consisted of administering streptozotocin, a diabetogenic toxin, to pregnant rats on gestational day 6. One key finding was that elevated circulating lipids in GDM rats competed with drugs (e.g., glyburide and saquinavir) for plasma protein binding so as to increase free drug concentrations. Another key finding was that important hepatic drug efflux transporters (e.g., Mdr1a/b) and metabolic enzymes (e.g., Cyp3a2 and Ugt1a1) were upregulated in GDM as a consequence of, most likely, enhanced nuclear receptor activity (e.g., pregnane X receptor upregulation). Upregulation of hepatic drug efflux transporters and metabolic enzymes, coupled with larger unbound drug fractions, would be expected to increase the hepatic clearance of many drugs. Consistent with this, in GDM, maternal and fetal exposure to the Mdr1 and Cyp3a2 substrate lopinavir was substantially lower than controls post-administration and data supporting enhanced lopinavir metabolite formation were obtained. Placental drug efflux transporters were also examined in this lopinavir study. Elevated placental Mdr1b and Bcrp expression was observed in GDM, which was associated with decreased fetal exposure to lopinavir (even after correcting for maternal unbound concentrations). Taken together, this thesis demonstrates that experimental GDM can significantly impact drug disposition by altering key drug disposition mechanisms. If confirmed in humans, this drug-disease interaction would need to be considered when atypical therapeutic outcomes occur in diabetic pregnancies. Data from experiments with human placentas, obtained from pregnancies complicated by insulin-managed diabetes, is included/discussed.

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.

Populations of Black-African descent have slower rates of nicotine and cotinine metabolism, smoke fewer cigarettes (~10 cigarettes/day), and have higher incidences of tobacco-related illnesses compared to Caucasians. Cytochrome P450 2A6 (CYP2A6) is the main enzyme involved in the metabolism of nicotine and its proximal metabolite cotinine, as well as tobacco-specific nitrosamines. Genetic polymorphisms in CYP2A6 contribute to the large variability observed in rates of nicotine metabolism. Reduced CYP2A6 activity has been associated with fewer cigarettes smoked, higher quit rates, and lower lung cancer risk in predominantly moderate to heavy-smoking (~20–30 cigarettes/day) Caucasians. CYP2A6 genetic variants and their impact on smoking behaviours have not been well studied among individuals of Black-African descent. The main objectives herein were to identify and characterize new CYP2A6 variants that may explain the slower rates of metabolism, and determine whether CYP2A6 variation is a predictor of smoking phenotypes in this population. Furthermore, we examined whether previously validated biomarkers of tobacco exposure have limitations among individuals of Black-African descent given their low and sporadic smoking patterns. A new CYP2A6 variant (CYP2A6*23) was found in individuals of Black-African descent recruited for a nicotine pharmacogenetic-pharmacokinetic study. CYP2A6*23 reduced activity towards nicotine and coumarin in vitro and was associated with slower rates of CYP2A6 kinetics in vivo. In a clinical trial of African-American light smokers, CYP2A6 slow metabolizers were more successful at smoking cessation compared to normal metabolizers, although no differences in cigarette consumption were found. Two common biochemical markers of tobacco smoke exposure, cotinine and exhaled carbon monoxide, were weakly correlated with self-reported cigarette consumption. These biomarkers were not substantially affected by variables previously shown to alter amount smoked and/or rates of cotinine metabolism such as gender, age, body mass index or smoking menthol cigarettes. However, CYP2A6 slow metabolizers had significantly higher cotinine without smoking more cigarettes. Identification and characterization of novel variants adds to our understanding of nicotine pharmacokinetic differences between racial/ethnic minority groups and improves accuracy of CYP2A6 genotype groupings for genetic association studies. Furthermore, better insight into the biological factors associated with smoking behaviours will aid in the development of more efficacious targeted treatments for this understudied population.