Abstract Risk assessment of chemicals needs reliable scientific information and one source of information is the characterization of the metabolic fate and toxicokinetics of a chemical. Metabolism is often the most important factor contributing to toxicokinetics. Cytochrome P450 (CYP) enzymes are a superfamily of microsomal proteins playing a pivotal role in xenobiotic metabolism. In the present study, pesticides were used as representative xenobiotics since exposure to pesticides is a global challenge to risk assessment. Human and animal in vitro hepatic models were applied with the advantage of novel analytical techniques (LC/TOF-MS and LC/MS-MS) to elucidate the in vitro metabolism and interaction of selected pesticides. The results of these studies demonstrate that CYP enzymes catalyze the bioactivation of profenofos, diuron and carbosulfan into their more toxic metabolites desthiopropylprofenofos, N-demethyldiuron and carbofuran, respectively. The suspected carcinogenic metabolite of metalaxyl, 2,6-dimethylaniline, was not detected. CYP3A4 and CYP2C19 activities may be important in determining the toxicity arising from exposure to profenofos and carbosulfan. Individuals with high CYP1A2 and CYP2C19 activities might be more susceptible to diuron toxicity. Qualitative results of in vitro metabolism were generally in agreement with the results obtained from the published in vivo data, at least for the active chemical moiety and major metabolites. Considerable differences in the quantities of the metabolites produced within the species, as well as in the ratios of the metabolites among the species, were observed. These findings illustrate that in vitro screening of qualitative and quantitative differences are needed to provide a firm basis for interspecies and in vitro-in vivo extrapolations. Based on our findings, in vitro-in vivo extrapolation based on the elucidation of the in vitro metabolic pattern of pesticides in human and animal hepatic models could be a good model for understanding and extending the results of pesticides metabolism studies to human health risk assessment.

Mixed-function oxygenase systém (MFO systém) plays a vital role in the metabolism of a variety of both endogenous substrates and xenobiotics. This membrane systém consists of cytochrome P450s, NADPH:cytochrome P450 oxidoreductase (POR), cytochrome b5 and NADH:cytochrome b5 oxidoreductase (b5R). Cytochrome P450 catalyzes a monooxygenation of a substrate, while POR and cytochrome b5 represent its redox partners. Cytochrome b5, itself having a redox partner in b5R, effects the reactions catalyzed by the MFO system in various ways, through mechanisms that are not fully understood. This paper focuses on the purification of b5R and POR from rabbit liver. The microsomal fraction obtained by differential centrifugation contained 42 mg of protein per ml. From a portion of the microsomal fraction, b5R was obtained using chromatography on DEAE-Sepharose, CM-Sepharose and 5'-ADP agarose columns. The yield was 0,3 % of ferricynide-reductase activity and the product contained several contaminants in the molecular weight range of 50-70 kDa. A second purification of b5R from the microsomal fraction was carried out using a column of DEAE-Sepharose directly connected to a 5'-ADP agarose column. The b5R product was purified with a yield of 10,9 % and it once again contained several contaminants in the molecular...

Abstract Metabolism is a major determinant of the pharmacokinetic properties of most drugs and is often behind bioavailability problems, drug-drug interactions, and metabolic idiosyncrasies. Cytochrome P450 (CYP) enzymes are a superfamily of microsomal hemoproteins catalysing the metabolic reactions of several exogenous compounds. The majority of crucial steps within drug metabolism are in connection with CYP enzymes. In the present study, in vivo, in vitro, and in silico approaches were applied and characterised to evaluate the effects of chemical entities on CYP-mediated metabolism. CYP2B6 was used as a target enzyme for these studies. For evaluation of the CYP inhibition potential of new chemical entities, a novel in vitro test system utilising the n-in-one approach was developed. This method proved to be robust and applicable to screening purposes. Validation of the n-in-one assay was done by comparing its performance to commonly used in vitro techniques using six structurally diverse drugs. All assay types yield remarkably similar results with the majority of the CYP forms tested. Several chemicals were screened in vitro and in silico in order to find potent and selective chemical inhibitors for CYP2B6. Ticlopidine, thioTEPA and 4-(4-chlorobenzylpyridine) were found to be highly effective inhibitors of CYP2B6. The selectivity of thioTEPA proved to be very high, whereas ticlopidine and 4-(4-chlorobenzylpyridine) also inhibited other CYPs. At a concentration level of 1 μM for ticlopidine and 0.1 μM for 4-(4-chlorobenzylpyridine), the inhibitory effect towards other CYPs was negligible. Due to wide clinical use and relevance, clopidogrel and ticlopidine were selected for further in vivo interaction studies. Both clopidogrel and ticlopidine significantly inhibited the CYP2B6-catalysed bupropion hydroxylation and patients receiving either clopidogrel or ticlopidine are likely to need dose adjustments when treated with drugs primarily metabolised by CYP2B6. The effect of impaired kidney function on CYP2B6 activity and on bupropion pharmacokinetics was also explored. In patients with kidney disease, the bupropion AUC and Cmax were significantly higher and the apparent oral clearance of bupropion was notably lower compared to healthy controls. The present results indicate that the in silico and in vitro methods used are helpful in predicting in vivo drug-drug interactions. The effective utilisation of these models in the early phases of drug discovery could therefore help to target the in vivo studies and to eliminate metabolically unfavourable drug candidates.

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

This study focuses on the characterisation of three novel cytochrome P450-partner (P450-fusion) enzymes of unknown structure and function. Despite several well-established P450 functions, new structures of P450s are published frequently, with the P450-redox partner fusion systems being among the most discussed, due to their enhanced activity and biotechnological potential. Other, more intriguing, P450-fusions involve partners with functions distinct from electron transfer. Understanding why evolution drove the ‘partner’ proteins to evolve into a single unit is often unclear, but provides an important challenge for the understanding of the breadth of biochemical reactions mediated by P450s. The first P450-fusion analysed (Chapter 3) is CYP116B1 from a soil bacterium, Cupriavidus metallidurans, that displays important environmental implications. The enzyme was characterised as a functional fusion, composed of three domains: a P450 from the CYP116B family, and a phthalate dioxygenase reductase (PDOR)-like reductase binding FMN and a 2Fe-2S cluster. CYP116B1 is a stable, cytosolic enzyme but can undergo FMN cofactor loss. Studies included redox potentiometry of the intact fusion and its individual domains using spectro-electrochemical and EPR methods to enable the determination of midpoint redox potentials for individual cofactors. The CYP116B1 EPR signature was shown to be typical of P450s, and changed upon binding heme-coordinating inhibitors of the azole class. Extensive compound library screening did not reveal a substrate-like physiological “hit”. However, catalytic activity was detected towards selected thiocarbamate herbicides. GC-MS data revealed the enzymatic mechanism of herbicide degradation. The second system studied (Chapter 4) is P450-CAD, an atypical fusion of an uncharacterised soluble P450 and a cinnamyl alcohol dehydrogenase (ADH) module from Streptomyces ghanaensis; a member of the major antibiotic producing genus of bacteria. The CAD module appears unlikely to be a redox partner, but instead possibly mediates substrate/product exchange with the P450. The intact fusion was shown to aggregate during extraction. Genetic dissection of domains revealed that this was due to the highly insoluble ADH moiety. The heme domain (HD) was soluble and was characterised extensively. The enzyme displays an unusual spectrum when in the FeII-CO complex (Amax = 445 nm). The P450-CAD HD catalytic activity is supported by heterologous redox partners (E. coli flavodoxin reductase [FldR] and flavodoxin [FldA], and spinach ferredoxin reductase [FdR] and ferredoxin [Fdx]). The CAD-HD binds fatty acid substrates of carbon chain length C8-14, with the highest affinity for 12-methylmyristic acid (12M14C acid), the C12 lauric acid, its aldehyde and alcohol, indicating that the terminal methyl group is important for binding to the enzyme. Unusually, the CAD-HD also binds a range of detergent compounds. Further analysis included SEC-MALLS, thermostability and structural studies. The final enzyme studied (Chapter 5) is the P450-BDOR (a P450 linked to a benzoate dioxygenase reductase) redox-partner fusion. The unconventional trait of this enzyme is the inclusion of an FCD (a fatty acid metabolism regulator protein [FadR] C-terminal DNA-binding domain). From the point of view of P450s, DNA interaction would represent an unprecedented function, suggesting novel functions for a P450 enzyme. Thus, this enzyme requires extensive research with the expectations that new information will contribute to an expansion of knowledge of P450 diversity. This study provides initial analytical insights into the P450-BDOR system, supported with functional and kinetic data on the P450 and its reductase domain.

Cytochromes P450 (P450s) are a superfamily of heme-thiolate monooxygenases. They catalyse a wide variety of reactions on a vast number of substrates and are of particular interest for biocatalyst development due to their ability to oxidise non-activated C-H bonds. Fusion of a P450 to a suitable redox partner protein produces a catalytically self-sufficient enzyme and removes the need to produce electron transfer proteins separately. The well-studied bacterial protein P450cam (Pseudomonas putida) has been fused to the reductase (RhFRed) from the natural fusion protein P450-RhF (Rhodococcus sp.). The P450cam-RhFRed system catalyses the oxidation of camphor and several non-natural substrates and served as the basis for P450cam re-engineering in this current project, with the aim of expanding the substrate scope towards a more mammalian-like activity. The P450cam active site was partitioned into seven paired amino acids and each pair randomised in turn to generate seven sub-libraries of P450cam variants. These were screened for activity using a specially developed colony screen for detection of the blue pigment indigo. In total 94 new variants were identified and then pooled for secondary screening on a number of new substrates, identifying potentially novel activities within the ‘indigo positive’ population. In a separate ‘chimeragenesis’ approach substrate recognition sites (SRSs) within P450cam were targeted for exchange with equivalent portions from a number of human P450s. The B’ helix and F-G loop regions from CYPs 1A2, 2C8, 2D6 and 3A4 were grafted onto the P450cam structure and several of the B’ helix swaps were produced as soluble proteins. The P450cam-2C8-B’-RhFRed chimera gave a Soret peak at 420 nm in the Fe(II)-CO state although an additional substitution next to the proximal cysteine appeared to restore a P450-like state. SRS-exchange therefore offered some insight into structural modularity in P450s, providing a basis for further biocatalyst development.

Recreational abuse of the drugs cocaine, methamphetamine, and morphine continues to be prevalent in the United States of America and around the world. While numerous methods of detection exist for each drug, they are generally limited by the lifetime of the parent drug and its metabolites in the body. However, the covalent modification of endogenous proteins by these drugs of abuse may act as biomarkers of exposure and allow for extension of detection windows for these drugs beyond the lifetime of parent molecules or metabolites in the free fraction. Additionally, existence of covalently bound molecules arising from drug ingestion can offer insight into downstream toxicities associated with each of these drugs. This research investigated the metabolism of cocaine, methamphetamine, and morphine in common in vitro assay systems, specifically focusing on the generation of reactive intermediates and metabolites that have the potential to form covalent protein adducts. Results demonstrated the formation of covalent adduction products between biological cysteine thiols and reactive moieties on cocaine and morphine metabolites. Rigorous mass spectrometric analysis in conjunction with in vitro metabolic activation, pharmacogenetic reaction phenotyping, and computational modeling were utilized to characterize structures and mechanisms of formation for each resultant thiol adduction product. For cocaine, data collected demonstrated the formation of adduction products from a reactive arene epoxide intermediate, designating a novel metabolic pathway for cocaine. In the case of morphine, data expanded on known adduct-forming pathways using sensitive and selective analysis techniques, following the known reactive metabolite, morphinone, and a proposed novel metabolite, morphine quinone methide. Data collected in this study describe novel metabolic events for multiple important drugs of abuse, culminating in detection methods and mechanistic descriptors useful to both medical and forensic investigators when examining the toxicology associated with cocaine, methamphetamine, and morphine.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22484号 / 農博第2388号 / 新制||農||1075(附属図書館) / 学位論文||R2||N5264(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 加納 健司, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Philosophiae Doctor - PhD / Tuberculosis (TB) remains a global epidemic despite the fact that treatment has been available since the 1950’s. This disease is highly contagious and spreads via transmission of the Mycobacterium Tuberculosis (MTB) tubercle via coughing, sneezing and spitting. The disease has various side effects including weight loss, fatigue and even death. To date no cure has been found for TB and thus optimisation of treatment is a constant focus in health related research. TB is highly prevalent in South Africa due to the increased level of patients who are co-infected with HIV. Treatment for TB consists of first line drugs including isoniazid (INH), ethambutol (ETH), pyrazinamide (PYR) and rifampicin (RIF). These drugs are highly effective but also produce many adverse drug reactions (ADR’s) over the 6-month course of treatment. These reactions lead to patients not completing the course, losing quality of life and ultimately adding to the development of drug resistant strains of TB. A method of minimising these ADR’s is the development of a phenotype sensor, which is able to determine the metabolic profile of patients. Metabolic profiles play a huge role in the efficacy of treatment by tailoring treatment in order for patients to stay within the therapeutic range of treatment. This would in turn minimise both toxicity and ineffective treatment. Various methods for the quantification of drugs have been developed such as high performance liquid chromatography (HPLC), mass spectrometry (MS) and ultra-violet visible spectroscopy (UV-vis). / 2023-12-01

17α-ethinylestradiol (EE2) is a synthetic hormone, derivative of the natural hormone estradiol. EE2 is one of the the most prescribed drugs in the world. It belongs to the estrogenic endocrine disrupter chemicals. These compounds are able to alter functions of the endocrine system and cause adverse effects in the organism, offspring and (sub)population. In this thesis, there are observed effects of 17α-ethinylestradiol on enzyme activities of main enzymes involved in phase I of xenobiotic biotransformation, i.e. cytochromes P450 (CYP), in vitro. Isoforms of CYP subfamilies 1A, 2B, 2C, 2E and 3A were studied in rats and humans. Each CYP isoform was incubated with EE2 at two concentrations, 10μM EE2 and the concentration corresponding to the substrate concentration in the specific marker reactions of individual CYP isoforms. The results indicate, that in rat liver microsomes the activity of all studied isoforms except CYP1A2 was decreased in the presence of EE2. When EE2 was added to the incubation mixture at the concentration of the reaction substrate, the greatest decrease in enzyme activity was observed for CYP2C6, with the remaining activity only 36%. In human liver microsomes, the activity of CYP2B6, CYP2C9, CYP2E1 and CYP3A4 was also effected by EE2. As in the case of rat model, CYP2C subfamily...