Thesis (PhD (Biochemistry))--Stellenbosch University, 2008. / This study describes: • the cloning and sequencing of cytochrome P450 17 -hydroxylase/17,20 lyase (CYP17), 3 -hydroxysteroid dehydrogenase (3 HSD) and cytochrome b5 from the South African Angora goat; • the identification of two CYP17 genes encoding two unique CYP17 isoforms in the Angora goat; • the development of a UPLC-APCI-LC method for the separation and quantification of seven adrenal steroids; • the characterisation of the enzymatic activity of the two Angora CYP17 isoforms expressed in non-steroidogenic COS-1 cells. The Km and Vvalues for the metabolism of pregnenolone and progesterone were determined; • the development of a rapid and accurate real-time PCR genotyping test for CYP17 in Angora goats. Three unique genotypes were identified; • the determination of blood cortisol levels upon the stimulation of the HPAaxis by intravenous insulin injection in the three Angora goat genotypes.

Thesis (PhD (Botany and Zoology))--Stellenbosch University, 2008. / Endocrine disruption is an alteration of the chemical messaging processes in the body. The value of studies‐ and monitoring of endocrine disruption using techniques included in the field of toxicogenomics is undoubtedly supported by scientific literature over the past four decades, as is demonstrated in Chapter 1 where I review relevant literature on the topic. Clearly, well sustained bio‐monitoring will include studies both in vitro and in vivo, and very well on transcriptional and translational levels. Animals are providing good models for in vivo studies to report or monitor endocrine disruption. It is imperative though to first understand such an animal’s biology, especially its endocrine system, and characterize what is considered “normal” for a species before engaging in endocrine disrupting exposures. A multitude of studies report endocrine disruption in relation to reproductive systems, with more recent work illustrating alteration of metabolism related to thyroidogenic disruption within the last decade. It is therefore essential to consider sex determination and ‐differentiation when studying sentinel species. Apart from the obvious academic interest in the matter of sex differentiation, altered patterns of sex differentiation in certain appropriate species provide for a very convincing endpoint in monitoring estrogenic endocrine disruption. As I approach to study a potential sentinel species for the southern African subcontinent, I set forward to study aspects of endocrine disruption influencing the reproductive system in a piece‐meal manner, starting with estrogenic endocrine disruption as this is the best studied facet of the endocrine disruption hypothesis to date. Yet, one learn from vast amounts of literature that in cases where sex is not exclusively determined by the genetic fraction of an individual, a number other characteristics may very well be used to determine estrogenic disruption in ecosystems. Quantitative production of the egg yolk precursor protein (vitellogenin) resides under these characteristics, and in the proposed sentinel, South African tilapiine, Oreochromis mossambicus phenotypic sex can be altered by environmental sex determination. The present study therefore targeted firstly the product most often used in tier I screening processes, vitellogenin (VTG). Specimens of O. mossambicus were cultured for this purpose from wild breeding stock, sampled at 5 day intervals and the transcription levels of vitellogenin gene (vtg) studied in those. Hereby, Chapter 2 describes the cloning of partial vtg gene and subsequent temporal expression of vtg quantitatively in O. mossambicus. To shed light on the state of gonadal differentiation sub‐samples were subjected to histology, illustrated in Chapter 3. In addition the quantitative vtg responses has been described in this study at a transcriptional level, both of adult males and juveniles subjected to low and very high levels of natural estrogens. In addition, a 3 kb 5’ flanking region of vtg was cloned and sequenced, and several putative binding sites identified for transcription factors of vtg, including several estrogen responsive elements (EREs). These indicate the expected regulational process of vtg by estrogens. Subsequently I measured the transcription levels of the only enzyme capable of aromatizing androgens into estrogens, Cytochrome P450 19 (cyp19) as has been characterized in Chapter 3. For stable binding of an estrogen to an ERE, binding of the ligand to its specific nuclear receptor (Estrogen receptor, ESR) is required. Since E2 is known to have different mechanisms of action in vertebrates, the expression levels of the ESRs were evaluated in our sample set after cloning 3 different homologues of ESR in O. mossambicus. The results on this matter is discussed in Chapter 4 and provides in addition to data on vtg and cyp19 a platform of “normal” transcription levels of these candidate genes involved in estrogenic endocrine disruption of O. mossambicus. Ultimately, characterization of those candidate genes involved extensively in phenotypic sex, contribute to our understanding of sex determination and differentiation in this species in a small way.

NADPH-cytochrome P450 reductase is the electron transfer partner for the cytochromes P450, heme oxygenase, and squalene monooxygenase, and is a component of the nitric oxide synthases and methionine synthase reductase. P450 reductase shows very high selectivity for NADPH and uses NADH only poorly. Substitution of tryptophan 677 with alanine (W677A) has been shown by others to yield a 3-fold increase in turnover with NADH, but profound inhibition by NADP+ makes the enzyme unsuitable for in vivo applications. In the present study site-directed mutagenesis of amino acids in the 2'-phosphate-binding site of the NADPH domain, coupled with the W677A substitution, was used to generate a reductase that was able to use NADH efficiently in vivo without inhibition by NADP+. Of 11 single, double, and triple mutant proteins, two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold increase in catalytic efficiency (kcat/Km) with NADH. Inhibition by NADP+ was reduced by up to four orders of magnitude relative to the W677A protein and was equal to or less than that of the wild-type reductase. Both proteins were 2- to 3-fold more active than wild-type reductase with NADH in reconstitution assays with cytochrome P450 1A2 and with squalene monooxygenase. In a recombinant cytochrome P450 2E1 Ames bacterial mutagenicity assay the R597M/W677A protein increased the sensitivity to dimethylnitrosamine by approximately 2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay. In addition to providing a valuable tool for understanding the determinants of nucleotide cofactor specificity in this and related enzymes, these mutants might also lend themselves to creation of bioremediation schemes with increased enzymatic activity and robustness in situ, as well as cost-effective reconstitution of enzyme systems in vitro that do not require the use of expensive reducing equivalents from NADPH.

In pharmacotherapeutics, the term "correct dosing" is based on the concept that too high a systemic concentration will lead to drug toxicity, while drug levels that are too low may not produce the intended therapeutic effect. Often, the factors determining the ability of a patient to manage a given dose rely on their capacity to efficiently metabolize and eliminate drugs from the body. The liver plays a crucial role in the processing of many clinically relevant drugs via three stages of hepatic drug management. Drugs must first be taken into hepatocytes by uptake transporters. Drugs are then metabolized by phase I and phase II enzymes to make them more manageable. Finally, metabolites are removed from the hepatocyte by efflux transporters either into the bile for elimination or reintroduction to systemic blood. Alterations in one or more of the hepatic drug management stages increase the potential for adverse drug reactions (ADRs).In the United States, ADRs account for between 3%-12% of admissions to hospitals, and approximately 5% of deaths each year. While less than 20% of these cases are due to genetic polymorphisms, the vast majority of ADRs are due to environmental factors including disease. Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of conditions progressing from steatosis to non-alcoholic steatohepatitis (NASH) and often leading to cirrhosis. Presently, NASH patients represent the greatest population of candidates for liver transplant, illustrating the severity as well as the incidence of this disease. Patients with NAFLD are typically treated for co-existing conditions of the metabolic syndrome (i.e. hyperlipidemina or type II diabetes) and therefore represent a distinct population at risk for adverse drug reactions.The following studies show that experimental NAFLD affects both the signal transduction pathways regulating hepatic drug management genes as well as the hepatic uptake transporter function. Additionally, patient livers diagnosed with progressive stages of NAFLD, display altered CYP activity and efflux transporter expression similar to those previously reported in experimental NAFLD. Given that changes observed in experimental NAFLD result in functional changes in hepatic drug management, similar changes observed in patients with this disease suggest an increased risk for ADRs.

Fish may be exposed to an array of pharmaceuticals that are discharged into the aquatic environment, paralleling advances in medical knowledge, research and technology. Pharmaceuticals by their nature are designed to target specific receptors, transporters, or enzymes. Nuclear receptors (NRs) are often a key component of the therapeutic mechanism at play, and many of these are conserved among vertebrates. Consequently, fish may be affected by environmental pharmaceutical exposure, however there has been relatively little characterisation of NRs in fish compared with in mammals. In this thesis common carp (C. carpio) were exposed to selected pharmaceuticals in vitro and in vivo to investigate effects centred on the pregnane X receptor (PXR) and peroxisome proliferator-activated receptor alpha (PPARα), two key NRs involved in organism responses to pharmaceutical exposure. The PXR acts as a xenosensor, modulating expression of a number of xenobiotic metabolising enzymes (XMEs) in mammals. In a primary carp hepatocyte model it was shown that expression of a number of XMEs was altered on exposure to rifampicin (RIF), as occurs in mammals. This response was repressed by addition of ketoconaozle (KET; PXR-antagonist), indicating possible PXR involvement. The genes analysed showed up-regulation on exposure to ibuprofen (IBU) and clofibric acid (CFA), but not clotrimazole (CTZ) or propranolol (PRP). The lack of response to mammalian PXR-agonist CTZ was unexpected. In contrast, the same XME genes were found to be up-regulated in vivo after 10 days of exposure of carp to CTZ, although this response occurred only for a relatively high exposure concentration. CTZ was found to concentrate in the plasma (with levels up to 40 times higher than the water). Development and application of a reporter gene assay to measure PXR activation in carp (cPXR) and human PXR showed CTZ activation of cPXR, supporting data from the in vivo studies. Furthermore, activation was seen at concentrations as low as 0.01 μM. Interestingly RIF did not induce a response in the cPXR reporter gene assay, contrasting with the hepatocyte culture work. Taken together, the data presented here suggests divergence in the PXR pathway between mammals and fish in terms of ligand activation and downstream gene targets. PPARα was investigated in carp in vivo using CFA as a mammalian PPARα-agonist. Overall the resulting data suggested a broadly similar role for this NR in lipid homeostasis in fish as for mammals, with a number of PPARα-associated genes and acyl-coA oxidase (ACOX1) activity up-regulated in response to CFA exposure. A number of XMEs were also up-regulated by CFA (in vivo and in vitro), potentially extending the role of PPARα in fish (carp) to regulation of xenobiotic metabolism. The work presented has provided further characterisation of PXR and PPARα in fish. Elucidation of these pathways is vital to provide meaningful data in terms of establishing toxicity and mechanism-of-action data for pharmaceuticals and other compounds in fish, to allow validation of read-across approaches and ultimately aid in their environmental risk assessment. In vitro approaches are attractive ethically, financially and can provide useful mechanistic characterisation of compounds and the primary hepatocyte model and reporter gene assays used here show potential for the screening of pharmaceutical compounds in fish. However, further understanding of the metabolism of drugs and chemicals in fish is required to establish the true value of these methods for informing on possible effects in fish, in vivo.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Les isoenzymes du CYP450 sont grandement impliquées dans le métabolisme oxydatif des médicaments et des variations dans leur activité (interactions médicamenteuses, polymorphismes génétiques) peuvent occasionner des changements importants dans les concentrations plasmatiques et tissulaires des médicaments. Le foie régit de façon prédominante les concentrations plasmatiques des médicaments en raison d’une forte concentration des CYP450s dans cet organe. En moyenne, l’élimination d’un médicament par métabolisme représente environ 75% des mécanismes de clairance par rapport à 20% par voie rénale et 10% par la bile sous forme inchangée. Les CYP450s contribuent à environ 75% du métabolisme des médicaments. Il est ainsi logique de s’attarder au comportement et facteurs influençant l’expression et l’activité des CYP450s. L’abondance et la variété des CYP450s exprimés diffèrent entre le foie et les tissus extra-hépatiques. Le rôle de ces isoenzymes dans le devenir extra-hépatique des composés endogènes et exogènes demeure peu étudié malgré leur implication et association reconnue dans certains échecs thérapeutiques, interactions médicamenteuses ou toxicités tissulaires. Le ventricule de cœur humain exprime (ARNmessager) de façon variable certaines isoenzymes dont le CYP1A1, 2B6, 2C8, 2C9, 2D6, 2E1, 2J2, 3A5 et 4A11. La participation de ces isoenzymes exprimées dans la paroi ventriculaire cardiaque dans le métabolisme des médicaments reste à ce jour inconnue. Établir l’existence d’une participation active de certains CYP450s, étudier leur profil métabolique et leur contribution dans la clairance locale tout en déterminant les facteurs responsables de leur variabilité en terme d’activité et d’expression (ARNm) dans le tissu ventriculaire cardiaque humain représente l’objectif principal des travaux de recherche présentés dans cette thèse. Le premier volet fût le développement de cocktails médicamenteux afin d’optimiser la quantité de résultats à travers l’usage unique d’un tissu avec des substrats sélectifs jumelés à des méthodes analytiques ultra-sensibles et spécifiques (manuscrit 1). Parmi les 9 composés testés, les substrats ébastine et chlorzoxazone, substrats-cibles reconnus des isoenzymes CYP2J2 et CYP2E1 respectivement, ont démontré des activités menant à l’étude approfondie de leur profil métabolique généré par la fraction microsomale de ventricules de cœurs humains explantés (MCH). Ces travaux ont démontré une cinétique préférentielle et de très haute affinité par les MCH envers la voie d’hydroxylation de l’ébastine décrite par un modèle Michaelis-Menten. Jumelé à des essais d’inhibition, un lien fût établi, pour la toute première fois, entre l’hydroxylation de l’ébastine et l’isoenzyme CYP2J2 (manuscrit 2). L’hydroxylation de l’ébastine par les MCH fût aussi reconnue comme étant hautement variable entre des individus adressés à une transplantation cardiaque présentant des cardiomyopathies potentiellement mortelles n’étant plus maîtrisées par les thérapies ou traitements standards. L’objectif de ce dernier volet était d’étudier des facteurs (extrinsèques et intrinsèques, dont le polymorphisme du CYP2J2*7) connus chez ces patients et leur implication dans la variabilité de l’expression du CYP2J2 (ARNm) et de son activité enzymatique, déduite par l’étude du comportement métabolique du marqueur ébastine suite à une analyse des paramètres de cinétique enzymatique (Km, Vmax, CLint). Il a été démontré que le sexe, le côté gauche du ventricule, la prise d’amiodarone et le diagnostic clinique d’ischémie sont associés à la variabilité d’expression (ARNm) et d’activité (Km, Vmax, CLint) du CYP2J2 et expliquent environ 20% de la variabilité observée entre les individus. Il s’agit des premiers travaux expliquant une portion de la variabilité associée à l’expression (ARNm) et l’activité du CYP2J2 dans le ventricule du cœur humain (manuscrit 3). Finalement, nos résultats suggèrent que la capacité métabolique du CYP2J2 au sein des MCH est réelle et importante et pourrait participer aux fluctuations locales des concentrations de médicaments. L’extrapolation des résultats obtenus in vitro à l’échelle in vivo (organe entier) a permis de mettre en perspective la contribution métabolique du CYP2J2 dans le contexte locale physiologique du tissu ventriculaire cardiaque étudié. Nos résultats appuient la proposition que la concentration libre plasmatique ne reflète pas nécessairement la concentration tissulaire efficace puisque des mécanismes locaux de métabolisme enzymatique (CYP2J2) peuvent grandement faire varier la concentration libre au site d’action intracellulaire. Les résultats obtenus répondent à un besoin en recherche de comprendre le comportement et l’ampleur de la participation de différentes isoenzymes du CYP450 connues s’exprimant dans le cœur humain envers le métabolisme de médicaments s’y distribuant et ultimement prédire leurs concentrations intracellulaires. / CYP450 isoenzymes are greatly involved in oxidative drug metabolism and changes in their activity (drug interactions, genetic polymorphisms) can cause significant changes in plasma and tissue concentrations of the drugs. The liver predominantly governs plasma concentrations of drugs because of a high concentration of CYP450s. On average, the elimination of a drug through metabolism accounts for about 75% of clearance mechanisms compared to 20% via renal excretion and 10% via the bile excretion. The CYP450s contribute to about 75% of drug metabolism. It is thus logical to study factors influencing the expression and activity of CYP450s. Abundance and variety of expressed CYP450s differ between the liver and extrahepatic tissues. The role of these enzymes endogenous and drugs concentrations in the extrahepatic tissues is poorly studied despite their involvement and association recognized in some treatment failures, drug interactions or tissue toxicity. The ventricles of the human heart express (messenger RNA) variably some isoenzymes including CYP1A1, 2B6, 2C8, 2C9, 2D6, 2E1, 2J2, 3A5 and 4A11. The participation of these isoenzymes expressed in the cardiac ventricular wall in drug metabolism remains unknown to this day. Establishing the existence of an active participation of some CYP450s, their metabolic profile and contribution to the local clearance and determining factors responsible for variability in activity and expression (mRNA) in the cardiac ventricular tissue human is the main objective of the research presented in this thesis. The first module was the development of drug cocktails to maximize the amount of results through the use of low amount of heart tissue with selective substrates matched with highly sensitive and specific analytical methods (manuscript 1). Of the nine compounds tested 2 substrates, ebastine and chlorzoxazone, probe of CYP2E1 and CYP2J2 respectively, have demonstrated activities leading to a thorough study of their metabolic profile generated by the microsomal fraction of the ventricles of explanted human hearts (MCH). This work demonstrated preferential kinetics, described by a Michaelis-Menten model, and very high affinity towards the MCH hydroxylation pathway for ebastine. Combined with inhibition assays, a link was established, for the first time, between the hydroxylation of ebastine and the isoenzyme CYP2J2 (manuscript 2). Hydroxylation of ebastine by MCH was also recognized as being highly variable between individuals that went through a heart transplant with life-threatening cardiomyopathies that are no longer controlled by standard therapies or treatments. The aim of this last part was to study factors (extrinsic and intrinsic, such as polymorphism of CYP2J2 * 7) known in these patients and their involvement in the variability of expression (mRNA) and enzymatic activity of CYP2J2 studied through the analysis of the enzyme kinetic profile of ebastine (Km, Vmax, CLint). It has been shown that gender, the left ventricle, use of amiodarone and clinical diagnosis of ischemia were associated with the variability of expression (mRNA) and activity (Km, Vmax, CLint) of CYP2J2 and those factors account for about 20% of the observed variability between individuals. This is the first work to explain a portion of the variability associated with the expression (mRNA) and activity of CYP2J2 in the ventricle of the human heart (manuscript 3). Overall, our results suggested that the metabolic capacity of CYP2J2 by MCH is real and important and could participate in local fluctuations of drug levels. The extrapolation of in vitro results to in vivo scale (whole body) helped to put into perspective the contribution of CYP2J2 in the local physiological context of the cardiac ventricular tissue studied. Our results support the proposal that the free plasma concentration does not necessarily reflect the effective tissue concentration as local mechanisms, in this case enzymatic metabolism by CYP2J2, could greatly provoke variation in free intracellular concentration at the site of action. The results obtained meet a need in research to understand the behavior and extent of participation of various CYP450 isoenzymes known to be expressed in the human heart on the local metabolism of drugs that are distributed locally and ultimately developing tools to predict the intracellular drug concentrations in the physiological context.

Introduction: Cellular membrane proteins, such as the cytochrome P450 enzyme superfamily (P450), have important roles in the physiology of the cell. P450s are important in metabolising endogenous molecules, as well as metabolising xenobiotic substances for detoxification and excretion. P450s are also implicated in cancer as they can act to 'negatively' de-activate or 'positively' activate cancer therapeutics. Identifying specific P450s that are highly up-regulated at the tumour site could be used to predict drug response and formulate targeted cancer therapy to help diminish systemic side-effects. Methods: Previous enrichment strategies have been unable to isolate the full complement of the P450 superfamily. To develop enrichment procedures for the P450s, a proteomic strategy was developed so that compounds could be screened for their effectiveness as general P450 probes. A standardised work-flow was created, encompassing affinity chromatography, protein concentration/desalting, followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography-mass spectrometry (HPLC-MS). A ketoconazole analogue and a 2-EN analogue, with known P450 inhibition, were immobilised on a solid support for comparison to immobilised histamine. Co-factor removal, competitive elution and DTT cleavage of disulfide bonds of probes were utilised to elute bound proteins. Results/Discussion: Inhibitor-beads bound a large range of proteins, including P450's, of which some were eluted by co-factor removal, some by competitive elution. Specificity of binding was improved by optimising buffer conditions and solid supports, however non-specific binding was not totally eradicated. All human P450s from spiked samples and 18 P450s from more complex mouse liver samples were recovered using one or more ligands.

With an increasing population suffering from obesity or Diabetes Mellitus (DM), it is more pertinent than ever to understand how physiological changes impact cellular processes. Patients with DM often suffer from obesity, hyperglycemia, altered fatty acids that contribute to vascular dysfunction, and increased risk to ischemia. Caenorhabditis elegans is a model system used to study the conserved insulin signaling pathway, cellular responses in whole organisms and the impact a glucose diet has on oxygen deprivation (anoxia) responses. RNA-sequencing (RNA-Seq) was used to analyze the expression of genes in the anoxia sensitive populations of N2 (wild-type) fed glucose and hyl-2(tm2031), a mutant with altered ceramide metabolism. Comparison of the altered transcripts in the anoxia sensitive populations revealed 199 common transcripts- 192 upregulated and 7 downregulated. One of the gene families that have altered expression in the anoxia sensitive populations encode for Cytochrome P450 (CYP). CYPs are located both in the mitochondria and endoplasmic reticulum (ER), but the CYPs of interest are all predicted to be mainly subcellularly localized to the ER. Here, I determined that knock-down of specific cyp genes, using RNA interference (RNAi), increased anoxia survival in N2 animals fed a standard diet. Anoxia sensitivity of the hyl-2(tm2031) animals was supressed by RNAi of cyp-25A1 or cyp-33C8 genes. These studies provide evidence that the CYP detoxification system impacts oxygen deprivation responses. using hsp-4::GFP animals, a transcriptional reporter for ER unfolded protein response (UPR), I further investigated the impact of cyp knock-down, glucose, and anoxia on ER UPR due to the prediction of CYP-33C8 localization to the ER. Glucose significantly increased ER UPR and cyp knock-down non-significantly increased ER UPR. Measurements of ER UPR due to anoxia were made difficult, but representative images show an increase in ER stress post 9-hour anoxia exposure. This study provides evidence that glucose affects ER stress and that ER stress is involved in oxygen deprivation responses.