Plants have evolved a variety of constitutive and induced chemical defense mechanisms against biotic stress. Emission of volatile compounds from plants facilitates interactions with both beneficial and pathogenic organisms. However, knowledge of the chemical defense in roots is still limited. In this study, we have examined the root-specific biosynthesis and function of volatile terpenes in the model plant Arabidopsis. When infected with the root rot pathogen Pythium irregulare, Arabidopsis roots release the acyclic C11-homoterpene (E)-4,8-dimethylnona-1,3,7-triene (DMNT), which is a common constituent of volatile blends emitted from insect-damaged foliage. We have identified a single cytochrome P450 monooxygenase of the CYP705 family that catalyzes a root-specific oxidative degradation of the C30-triterpene precursor arabidiol thereby causing the release of DMNT and a C19-degradation product named arabidonol. We found that DMNT shows inhibitory effects on P. irregulare mycelium growth and oospore germination in vitro, and that DMNT biosynthetic mutant plants were more susceptible to P. irregulare infection. We provide evidence based on genome synteny and phylogenetic analysis that the arabidiol biosynthetic gene cluster containing the arabidiol synthase (ABDS) and CYP705A1 genes possibly emerged via local gene duplication followed by de novo neofunctionalization. Together, our studies demonstrate differences and plasticity in the metabolic organization and function of terpenes in roots in comparison to aboveground plant tissues. Additionally, we demonstrated that the arabidiol cleavage product, arabidonol, is further modified by yet unknown enzymatic reactions into three products, which are found in root exudates. We suggested a pathway for their biosynthesis based on precursor feeding experiments and NMR analysis. Although DMNT biosynthetic genes are clustered on chromosome 4 along with several potential modification genes, we did not find a possible role of these genes in the derivatization of arabidonol. Preliminary experimental results using genetic and biochemical approaches for identifying genes involved in the modification steps are also presented. In summary, this study demonstrates an alternative route for volatile terpene formation belowground different from aboveground plant tissues via triterpene degradation and provides evidence for an unexplored triterpene catabolism pathway in Arabidopsis. / Ph. D.

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

No / The cytochrome P450 family of enzymes is involved in the Phase I metabolism of a wide variety of compounds. Although generally involved with detoxification, overexpression of one family member, cytochrome P450 1B1 (CYP1B1), has been associated with human epithelial tumors. As such, CYP1B1 was hypothesized to be a novel target for the development of anticancer therapies. We investigated expression of CYP1B1 protein in 61 human colorectal adenocarcinomas and compared this to that observed in 14 histologically normal human large bowel samples removed from patients undergoing surgery for large bowel tumors. Although we confirmed that CYP1B1 was expressed at high levels in human colorectal tumor epithelia, we also found that CYP1B1 was not absent from normal colonic epithelia but was expressed at low levels. The expression of CYP1B1 in colon tumors does not correlate with tumor stage or degree of lymph node invasion in this study. Furthermore, in addition to expression in colon epithelia, CYP1B1 is also observed in blood vessels within the colon. As with the epithelia, levels of CYP1B1 were higher in tumor vasculature than that of the normal colon. Although these observations greatly support the development of CYP1B1 targeted anticancer therapies, they also indicate the caution that should be observed when developing such drugs.

Yes / Structural features from the anticancer prodrug nemorubicin (MMDX) and the DNA-binding molecule DRAQ5™ were used to prepare anthraquinone-based compounds, which were assessed for their potential to interrogate cytochrome P450 (CYP) functional activity and localisation. 1,4-disubstituted anthraquinone 8 was shown to be 5-fold more potent in EJ138 bladder cancer cells after CYP1A2 bioactivation. In contrast, 1,5-bis((2-morpholinoethyl)amino) substituted anthraquinone 10 was not CYP-bioactivated but was shown to be fluorescent and subsequently photo-activated by a light pulse (at a bandwidth 532–587 nm), resulting in punctuated foci accumulation in the cytoplasm. It also showed low toxicity in human osteosarcoma cells. These combined properties provide an interesting prospective approach for opto-tagging single or a sub-population of cells and seeking their location without the need for continuous monitoring.

Yes / Epidemiological studies have shown that head and neck cancer (HNC) is a complex multistage process that in part involves exposure to a combination of carcinogens and the capacity of certain drug-metabolising enzymes including cytochrome P450 (CYP) to detoxify or activate such carcinogens. In this study, CYP1A1, CYP1B1 and CYP2W1 expression in HNC was correlated with potential as target for duocarmycin prodrug activation and selective therapy. In the HNC cell lines, elevated expression was shown at the gene level for CYP1A1 and CYP1B1 whereas CYP2W1 was hardly detected. However, CYP2W1 was expressed in FaDu and Detroit-562 xenografts and in a cohort of human HNC samples. Functional activity was measured in Fadu and Detroit-562 cells using P450-Glo™ assay. Antiproliferative results of duocarmycin prodrugs ICT2700 and ICT2706 revealed FaDu and Detroit-562 as the most sensitive HNC cell lines. Administration of ICT2700 in vivo using a single dose of ICT2700 (150 mg/kg) showed preferential inhibition of small tumour growth (mean size of 60 mm3) in mice bearing FaDu xenografts. Significantly, our findings suggest a potential targeted therapeutic approach to manage HNCs by exploiting intratumoural CYP expression for metabolic activation of duocarmycin-based prodrugs such as ICT2700. / The authors would like to thank Bradford Institute for Health Research for funding a PhD studentship to DP through a competitive scheme and Yorkshire Cancer Research programme Grant (B381PA) for supporting our cytochrome P450-focused drug discovery research.

Cadmium (Cd), is a non-essential metal with no known physiological function. It is known to alter redox state by disrupting the mitochondrial electron transport chain, as well as inactivating protein and non-protein thiols. It is thus believed that oxidative stress may comprise an important part of the mechanism of Cd toxicity. Accordingly, the initial cellular response to acute Cd exposure is defensive, where various anti-oxidant defence systems are triggered. One of the induced systems is the haem oxygenase-1 (HO-1). Its activation is mediated by the transcription factor Nrf2, which is the general regulator of cellular defence against oxidative stress. The protective effects of HO-1 are mediated, in part, through the generation of potent anti-oxidant bilirubin (BR) and its metabolites, which exploit the intrinsic antioxidant properties of these species at a cellular level. The oxidative metabolism of BR is an important route of detoxification in addition to glucuronidation. However, the major enzyme(s) involved in this oxidative degradation are not known. This thesis presents evidence for a major role of the hepatic cytochrome P450 2a5 (Cyp2a5) in BR degradation during Cd intoxication, where the BR levels are elevated following induction of HO-1. Treatment of DBA/2J male mice with CdCl2 induced both the Cyp2a5 and HO-1, and increased the microsomal BR degradation activity. By way of contrast, the total cytochrome P450 (CYP) content and the expression of Cyp1a2 were down-regulated by the treatment. The induction of the HO-1 and Cyp2a5 was significant at the mRNA, protein and enzyme activity levels. In each case, the up-regulation of the HO-1 preceded that of the Cyp2a5 with a 5-10 hr interval. In addition, BR totally inhibited the microsomal coumarin hydroxylase activity (a Cyp2a5-catalysed reaction) with an IC50 approximately equal to the substrate concentration. The MROD activity, catalysed mainly by the Cyp1a2, was inhibited up to 36% by BR. The microsomal BR degradation was inhibited by coumarin and by a monoclonal antibody against the Cyp2a5 by about 90%. In addition, 7-methoxyresorufin, a substrate for Cyp1a2, inhibited BR degradation activity by approximately 20%. A study using Nrf2 null mutant mice suggests that Cd-mediated induction of Cyp2a5 is dependent on the transcription factor Nrf2. Additionally, acute exposure to Cd activated localisation of Nrf2 from the cytoplasm to the nucleus. Furthermore, electrophoretic mobility shift assay (EMSA) analysis suggests that Cd induced sequence-specific binding of various species of the StRE-binding proteins on the 5’-flanking region of the Cyp2a5 gene. Collectively, these observations strongly suggest that BR may act as a substrate for the hepatic Cyp2a5, a major catalyst for BR degradation under conditions of substantial elevation of BR levels following induction of HO-1 by Cd. Secondly, the concurrent up-regulation of the HO-1 and Cyp2a5 during Cd-mediated injury implicates a coordinated regulation of two enzyme systems in the maintenance of balancing BR production and elimination. Finally, StRE-binding proteins, in particular Nrf2, may be involved in the regulation of the Cyp2a5 gene, which leads to the oxidation of BR. However, the respective roles of these factors in the regulation of the Cyp2a5 gene, as well as the coordinated regulation of ho-1 and Cyp2a5 genes remain an open question, requiring further investigations.

Une analyse du transcriptome d’Arabidopsis thaliana soumis à différents stress biotiques a révélé l’activation de certains membres de la famille CYP76, particulièrement celle de CYP76C2 (≈ 50 fois). La caractérisation fonctionnelle de la famille CYP76, et plus particulièrement celle de CYP76C2 a donc fait l’objet de cette thèse. Après confirmation de l’activation sélective de CYP76C2 en réponse aux pathogènes par qRT-PCR, le phénotype de ses mutants d’insertion et de surexpression a été caractérisé sous différentes conditions d’infection par: Pseudomonas syringae pv. tomato DC3000, P. syringae pv. tomato DC3000 avrRpm1 et par Botrytis cinerea. Afin d’identifier la voie métabolique faisant intervenir CYP76C2, un profilage métabolique ciblé et non ciblé a été entrepris, centré sur le(s) métabolite(s) différentiellement accumulés dans les différents mutants en condition d’infection. Alors que des différences subtiles de sensibilité des mutants de CYP76C2 aux pathogènes semblent confirmer son rôle dans la réponse aux pathogènes, les lignées affectées dans son expression ne présentent pas de phénotypes clairement différents de ceux des plantes sauvages. Une analyse non–ciblée en UPLC-MS (Orbitrap) a permis d’identifier un composé absent dans le mutant cyp76c2 qui pourrait correspondre à un dérivé conjugué en C11, sans que sa structure ne puisse pour l’instant être identifiée (formule brute C17H28O9). CYP76C2 ne semble pas impliqué directement dans la synthèse d’une molécule cruciale pour la mise en place du processus de défense, mais exerce plus probablement une fonction spécialisée ou partiellement redondante de défense ou de détoxication. / A transcriptome analysis of Arabidopsis thaliana subjected to biotic stresses has revealed the activation of members of the CYP76 family, especially of CYP76C2 (≈ 50 times). The functional characterization of CYP76C2, has been the objective of this thesis. After confirmation of the selective activation of CYP76C2 by pathogens, the phenotype of its insertion and overexpressor mutants was characterized under infection by Pseudomonas syringae pv. tomato DC3000, P. syringae pv. tomato DC3000 avrRpm1 and Botrytis cinerea. In order to identify the metabolic pathway involving CYP76C2, targeted and non-targeted metabolic profiling was focused on differentially accumulated compounds in the different mutants after infection. Whereas subtle differences of response of the CYP76C2 mutant lines in response to pathogens seemed to confirm its involvement in response to biotic stress, phenotypes strikingly different from those of wild-type plants were not observed. A non-targeted analysis by UPLC-MS (Orbitrap) identified a compound absent in the cyp76c2 line that may correspond to an oxygenated C11 conjugate (raw formula C17H28O9), but its structure was not identified. CYP76C2 thus does not seem directly involved in the synthesis of a molecule crucial for defense responses, but more likely has a role in the synthesis of a potentially redundant specialized defense compound or in a detoxification process.

L’annotation détaillée de la superfamille des cytochromes P450 dans le génome de la vigne nous a permis d’étudier sa structure génétique, sa phylogénie et son expression, mais aussi d’identifier des gènes dont l’expression est activée dans le grain à maturité, lors de la synthèse de nombreux composés aromatiques. La lactone du vin est la molécule dont le seuil de détection olfactive est le plus bas, ce qui en fait un composant essentiel de l’arôme du vin. Nous avons pu démontrer que cette lactone se forme au cours du vieillissement du vin par une réaction lente et non-enzymatique à partir du 8-carboxylinalool. L’accumulation de ce dernier dans la baie est concomitante à l’expression de plusieurs P450s, dont CYP76F14 est le plus fortement exprimé. Trois enzymes catalysent des étapes d’oxydation conduisant du linalool au (E)-8-carboxylinalool, mais seul CYP76F14 catalyse efficacement la formation de l’acide. Tant par son activité catalytique que son profil d’expression, CYP76F14 apparaît donc comme le responsable le plus probable de la formation du précurseur de la lactone du vin. / A thorough annotation of the P450 superfamily in grapevine, revealed its genomic organization, phylogeny and expression. Specifically, we identified genes showing an activated expression in the ripe grape berry, the stage during which the biosynthesis of many aroma compounds takes place. Among the known oxygenated monoterpenols in grapevine, wine lactone has the lowest odor detection threshold and therefore the largest potential impact on wine aroma. We demonstrated that wine lactone is formed during wine ageing via a slow non-enzymatic reaction from the precursor (E)-8-carboxylinalool. We showed that the accumulation of this precursor in grape berries parallels the expression of several cytochrome P450 genes, among which CYP76F14 has the highest expression. While three of them catalyzed some of the oxidative steps from linalool to (E)-8-carboxylinalool, only CYP76F14 efficiently catalyzed the whole pathway. Taken together, CYP76F14 catalytic activity and expression pattern indicate that it is a prime candidate for the formation of the wine lactone precursor in grape berries.

Parmi les 57 cytochromes P450 identifiés lors du séquençage complet du génome humain, on en dénombre environ 15 dont on ne connaît pratiquement rien de leurs rôles physiologiques, de leurs substrats, et de leurs structures, d’où le nom de «P450 orphelins». Le CYP2U1 est l’un des cytochromes P450 les plus fortement exprimé au niveau du cerveau et du cervelet mais c’est aussi l’un des plus conservé parmi les différentes espèces du règne animal. Ce travail de thèse a tout d’abord consisté à optimiser les conditions d’expression du CYP2U1 sous une forme active. Un premier système d’expression dans la levure Saccharomyces Cerevisiae a permis une production d’un complexe CYP2U1-P450 réductase catalytiquement actif permettant des études de recherche de substrat. Un second système d’expression dans Escherichia Coli devrait permettre d’obtenir de plus grandes quantités d’enzyme soluble destinée à des études structurales. Dans un second temps, une recherche de substrats a été effectuée à l’aide d’analyse d’incubats par chromatographie liquide couplée à une détection par spectrométrie de masse. A ce jour, un screening dirigé de plus de soixante-dix molécules, substrats de P450s de la famille 2, a permis d’identifier les premiers substrats exogènes du CYP2U1, les analogues de terfénadone et la débrisoquine. D’autre part, une étude par modélisation moléculaire de la structure du CYP2U1 a été effectuée. Cette étude montre que le CYP2U1 diffère de tous les autres P450s par la présence d’un insert très spécifique dans son domaine N-terminal. Des modèles par homologie basés sur les structures cristallographiques des P450s de la famille 2 ont été construits. Ces modèles ont été validés par dynamique moléculaire et ont permis de proposer un mode d’interaction avec la membrane, d’identifier la position des canaux d’accès ainsi que de déterminer la topologie du site actif. Enfin, un docking des premiers substrats exogènes au sein du site actif du CYP2U1 a permis de confirmer la régioselectivité des hydroxylations catalysées par le CYP2U1. / Among the 57 human cytochrome P450 genes that have been identified; substrates, structure and physiologic role of 15 of them is practically unknown. They are called orphan. One of them, CYP2U1 is one of the most expressed cytochrome P450 in the brain and in the cerebellum but also one of the most conserved isoform in the all animal kingdom. This manuscript first describes the optimization of the heterologous expression of an active form of CYP2U1. Expression in a eukaryotic host, yeast Saccharomyces Cerevisiae first allows the production of a catalytic active CYP2U1-P450 reductase complex needed for substrate screening. Another expression system in a prokaryote host Escherichia Coli will allow higher production rate of a truncated and soluble form of the protein which will permit structural studies. Then a directed substrate screening was performed with the liquid chromatography – mass spectrometry analysis of CYP2U1 incubations. To date, 70 molecules, CYP2 family substrates, were tested that allow the identification of the two first exogenous CYP2U1 substrates: débrisoquine and terfenadone analogs. A structural study was achieved using a homology tridimensional model of the enzyme. We have found that CYP2U1 is longer than the other human CYPs, with an N-terminal 20 amino acids insertion, located after the  helical membrane spanning domain. Structural models were built using six crystallized human CYP2s as templates. Molecular dynamics experiments in membrane suggested a specific interaction with the membrane. The active site topology and the access channels were also determined and a docking of the two first exogenous CYP2U1 substrates was performed in order to confirm the regioselective hydroxylation activities observed in vitro.

Cytochrome P450 enzymes (P450s) are involved in cancer development and treatment due to their roles in the oxidative metabolism of various endogenous (e.g. oestrogen) and exogenous (e.g. tamoxifen) compounds. It is well-known that intermediate P450 metabolites derived from oestrogen metabolism are associated with breast carcinogenesis. The main aim of this project was to profile the cytochrome P450 and P450-regulatory nuclear receptor mRNAs in a series of breast cancer cell lines (BCCs) and compare this profile with normal breast cells. This study used the qualitative reverse transcriptasepolymerase chain reaction (RT-PCR) to detect mRNA expression of target genes. Results showed CYP1B1, CYP2D6, CYP2J2, CYP2R1, CYP2U1 and CYP4X1 mRNA to be present in all cell lines. CYP2A6, CYP2C8, CYP2C18, CYP2F1 and CYP4Z1 mRNA were expressed in oestrogen receptor (ER)-positiveCaucasian and ER-negative Afro- Caribbean BCCs. Although no differences in P450 mRNA were observed between the different ethnic groups, these preliminary findings suggest potential similarities in the ERpositive Caucasian and ER-negative Afro-Caribbean BCCs which warrant further investigation The CYP4Z1 PCR product was identified as two distinct bands. Specific primer sets were used to demonstrate potential intron retention in CYP4Z1. Using established in vitro models for the study of regulatory mechanisms of CYP4Z1, T47D and ZR-75-1 breast cancer cell lines were used to determine the appropriate nuclear receptors (i.e. progesterone receptor, glucocorticoid receptor or peroxisome proliferator-activated receptor alpha ). These findings suggest that there may be an alternative receptor mechanism involved in CYP4Z1 mRNA induction in these cells. In conjunction, pre-treatment of these two cell lines with the RNA synthesis inhibitor actinomycin D followed by the agonists showed a significant reduction (p < 0.05) of CYP4Z1 mRNA levels and inhibited CYP4Z1 induction by either progesterone, dexamethasone or pirinixic acid, indicating that these agonists have effects on CYP4Z1 mRNA transcription or stability. In contrast, cycloheximide differentially affected the level of CYP4Z1 mRNA induction by these agonists. Taken together, these results suggest that CYP4Z1 mRNA induction in T47D and ZR-75-1 is mediated through differential cell type specific regulatory mechanisms and there is evidence for differential regulation of the splice variants.