The rising use of natural products creates an imperative need for an enhanced awareness of the safety of current and new products making their way into the marketplace. An important example is natural products containing lignans as the principal active component. Despite their structural similarity the lignan of creosote bush can cause hepato- and renal toxicity while the lignans of flaxseed have no reported serious toxicity. This dissertation aimed to investigate the oxidative metabolism of such lignans to determine whether reversible, competitive interactions and/or bioactivation may explain the differences in their apparent toxicity.<p> The first objective was to study the metabolism and bioactivation of nordihydroguaiaretic acid (creosote bush) and secoisolariciresinol (flaxseed). Nordihydroguaiaretic acid metabolism in rat liver microsomes led to the production of three glutathione adducts formed via ortho¬-quinone reactive intermediates. This metabolism was independent of NADPH and thus attributed to autoxidation. Secoisolariciresinol metabolism yielded lariciresinol and no glutathione adducts suggesting an absence of bioactivation to reactive quinone intermediates.<p> The second objective was to study the autoxidation of nordihydroguaiaretic acid. The major autoxidation product was a unique, stable schisandrin-like cyclolignan which was the result of nordihydroguaiaretic acid cyclization. The half-life of nordihydroguaiaretic acid in aqueous solution, pH 7.4, 37ºC is 3.14 hours suggesting the cyclolignan may be responsible for some of the biological effects of nordihydroguaiaretic acid.<p> The third objective was to study the inhibition of cytochrome P450 isoforms 1A2, 2B, 2C11 and 3A by lignans derived from creosote bush and flaxseed. None of the lignans caused irreversible inhibition. Both creosote bush and flaxseed lignans caused reversible inhibition of P450 enzyme activity that involved competitive or mixed-type inhibition, however the inhibition was present at nonphysiologically relevant concentrations. Activation of cytochrome P450 isoforms was also observed at low lignan concentrations. The results suggest that P450-mediated bioactivation or reversible inhibition cannot explain the differences in toxicity noted between the lignans of creosote bush and flaxseed.<p> This work suggests a minimal risk for drug-lignan interactions at P450 enzymes. Further studies are warranted to determine the presence and biological and toxicological role of the nordihydroguaiaretic acid cyclolignan in herbal preparations.

Seventeen Cree anti-diabetic herbal medicines and eight Traditional Chinese Medicines have been examined for their potential to cause interactions with drugs, which is considered as a major reason for adverse drug effects. Specifically, the effect of these natural health products was examined on major Phase I drug metabolism enzymes including cytochrome P450, human carboxylesterase-1 and flavin-containing monooxygenases. Several of these natural health products have the potential to cause adverse drug effect through the inhibition of major drug metabolism enzymes. The results indicated that 7 Cree medicines plant extracts inhibited CYP3A4 activity, and 3 of them have been proven to cause potent mechanism-based inactivation of CYP3A4. Seven of eight Traditional Chinese Medicines have been identified as strong CYP3A4 inhibitors; the ethanol extract of Goji has identified as a potent inhibitor for CYP2C9 and 2C19. Goji juice showed universal inhibitory effects on most of the tested enzymes except flavin-containing monooxygenases 3.

The rising use of natural products creates an imperative need for an enhanced awareness of the safety of current and new products making their way into the marketplace. An important example is natural products containing lignans as the principal active component. Despite their structural similarity the lignan of creosote bush can cause hepato- and renal toxicity while the lignans of flaxseed have no reported serious toxicity. This dissertation aimed to investigate the oxidative metabolism of such lignans to determine whether reversible, competitive interactions and/or bioactivation may explain the differences in their apparent toxicity.<p> The first objective was to study the metabolism and bioactivation of nordihydroguaiaretic acid (creosote bush) and secoisolariciresinol (flaxseed). Nordihydroguaiaretic acid metabolism in rat liver microsomes led to the production of three glutathione adducts formed via ortho¬-quinone reactive intermediates. This metabolism was independent of NADPH and thus attributed to autoxidation. Secoisolariciresinol metabolism yielded lariciresinol and no glutathione adducts suggesting an absence of bioactivation to reactive quinone intermediates.<p> The second objective was to study the autoxidation of nordihydroguaiaretic acid. The major autoxidation product was a unique, stable schisandrin-like cyclolignan which was the result of nordihydroguaiaretic acid cyclization. The half-life of nordihydroguaiaretic acid in aqueous solution, pH 7.4, 37ºC is 3.14 hours suggesting the cyclolignan may be responsible for some of the biological effects of nordihydroguaiaretic acid.<p> The third objective was to study the inhibition of cytochrome P450 isoforms 1A2, 2B, 2C11 and 3A by lignans derived from creosote bush and flaxseed. None of the lignans caused irreversible inhibition. Both creosote bush and flaxseed lignans caused reversible inhibition of P450 enzyme activity that involved competitive or mixed-type inhibition, however the inhibition was present at nonphysiologically relevant concentrations. Activation of cytochrome P450 isoforms was also observed at low lignan concentrations. The results suggest that P450-mediated bioactivation or reversible inhibition cannot explain the differences in toxicity noted between the lignans of creosote bush and flaxseed.<p> This work suggests a minimal risk for drug-lignan interactions at P450 enzymes. Further studies are warranted to determine the presence and biological and toxicological role of the nordihydroguaiaretic acid cyclolignan in herbal preparations.

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This study firstly reports the development and characterization of PVP-AgNPs, PANSA and PVPAgNPs/ PANSA nanocomposite on gold. AFM and TEM analyses revealed highly electroactive nanocomposites whose morphogy and properties were essential for the immobilization of CYP2E1. Secondly, the development and characterization of Au/PVPAgNPs/ PANSA/CYP2E1, Au/PVP-AgNPs/PANSA/SA-CYP2E1 and Au/PVPAgNPs/ PANSA/EG-CYP2E1 nanobiosensors are reported. AFM studies displayed globular morphologies with large roughness for the enzyme modified electrodes as opposed to those electrodes without enzymes. Finally, the biotransformation of standard solutions of TB drugs (isoniazid, ethambutol, pyrazinamide and rifampicin) in pH 7.4, 0.1 M phosphate buffer solution is reported. The biotransformations of the TB drugs were successfully studied using cyclic voltammetry (CV), square wave voltammetry (SWV), differential voltammetry (DPV) and steady state amperometry under aerobic conditions. Very good detection limits were obtained for the standard solutions of TB drugs and were found to be in the micromolar range. The detection limit values for the individual TB drugs were 0.55 μM (isoniazid), 0.7 μM (ethambutol), 0.054 μM (pyrazinamide) and 0.05 μM (rifampicin). The detection limit results showed that the nanobiosensors were more sensitive and suitable for the determination of the respective drugs in plasma and serum.

L’activité catalytique du cytochrome P450 dépend de la disponibilité d’électrons produits par la NADPH P450 réductase (NPR). Notre étude a pour but de déterminer comment l’expression de la NPR est modulée chez le lapin. Afin de comprendre comment l’expression de la NPR est modulée, des hépatocytes de lapins témoins ont été incubés pendant 2, 4, 24 et 48 heures en présence de plusieurs activateurs de facteurs de transcription connus du cytochrome P450. De plus, des lapins ayant reçu une injection sous-cutanée de térébenthine afin de produire une réaction inflammatoire aseptique sont sacrifiés 48 heures plus tard dans le but d’étudier les effets de l’inflammation sur l’expression de la NPR. La rosiglitazone, le fénofibrate, l’acétate de plomb et le chlorure de cobalt (des inducteurs des PPAR, PPAR, AP-1 et HIF-1), après 48 heures d’incubation, n’ont provoqué aucun changement d’expression ou d’activité de la NPR. Après 48 heures d’incubation, la dexaméthasone (Dexa) a augmenté la quantité d’ARNm (QT-PCR), l’expression et l’activité de la NPR (p<0,05), en plus d’augmenter l’ARNm des récepteurs nucléaires CAR (récepteur constitutif à l’androstane) et PXR (récepteur X prégnane) (p<0.05). Le phénobarbital (PB) a augmenté seulement l’activité de la NPR (p<0.05). Par contre, après 48 heures d’incubation, la combinaison PB et Dexa a augmenté la quantité d’ARNm, ainsi que l’expression et l’activité de la NPR (p<0.05). La combinaison de PB et Dexa a induit une augmentation d’ARNm des récepteurs nucléaires CAR, PXR et RXR (récepteur X du rétinoïde) plus précocement, soit après 2 heures d’incubation (p<0.05). Le PD098059 (PD), un bloqueur de l’activation de MAPK1 (mitogen-activated protein kinase), et l’acide okadaïque (OA), un inhibiteur de la protéine phosphatase 2A (PP2A), ont bloqué l'augmentation d'expression et d'activité de la NPR induite par le PB après 48 heures d’incubation. La réaction inflammatoire aseptique a diminué l’expression et l’activité de la NPR après 48 heures d’incubation (p<0.05). On conclue que la dexaméthasone et le phénobarbital sont des inducteurs potentiels de la NPR et que les voies de signalisation de CAR, PXR et RXR semblent être impliquées dans le contrôle de cette induction. Des études supplémentaires devront être complétées afin de confirmer ces résultats préliminaires. / The catalytic activity of the cytochrome P450 depends on the availability of electrons produced by the NADPH P450 reductase (NPR). Our study aims to determine how the expression of the NPR is modulated in rabbits. In order to understand how the expression of the NPR is modulated, hepatocytes from rabbits in the control group were incubated for 2, 4, 24 and 48 hours in the presence of several cytochrome P450 transcription factor activators. Furthermore, a group of rabbits received a sub-cutaneous injection of turpentine in order to create an aseptic inflammatory response with the aim to assess the effects of inflammation on the expression of the NPR. Rosiglitazone, fenofibrate, lead acetate and cobalt chloride (inducers of PPAR, PPAR, AP-1 and HIF-1) did not produce any change in the expression or the activity of the NPR after a 48 hour incubation period. Dexamethasone (Dexa) increased the amount of mRNA (QT-PCR), and NPR's expression and activity as well as CAR (constitutive androstane receptor) and PXR (pregnane X receptor) nuclear receptors' mRNA after a 48 hour incubation period (p<0.05). Phenobarbital (PB) increased NPR's activity (p<0.05). However, the combination of PB and Dexa increased the amount of mRNA, as well as NPR's expression and activity after a 48 hour incubation period (p<0.05). The combination of PB and Dexa increased CAR, PXR and RXR (retinoid X receptor) nuclear receptors' mRNA after a 2 hour incubation period. PD098059 (PD), a inhibitor of MAPK1 (mitogen-activated protein kinase) activation, and okadaic acid (OA), an inhibitor of the phosphatase 2A protein (PP2A), prevented the increase of NPR expression and activity induced by PB after a 48 hour incubation period. The aseptic inflammatory reaction decreased NPR's expression and activity after a 48 hour incubation period (p<0.05). We conclude that dexamethasone and phenobarbital are potential NPR inductors and that CAR, PXR and RXR signaling pathways appear to be involved in controlling this induction. However, further studies will be needed to confirm these preliminary results.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Seventeen Cree anti-diabetic herbal medicines and eight Traditional Chinese Medicines have been examined for their potential to cause interactions with drugs, which is considered as a major reason for adverse drug effects. Specifically, the effect of these natural health products was examined on major Phase I drug metabolism enzymes including cytochrome P450, human carboxylesterase-1 and flavin-containing monooxygenases. Several of these natural health products have the potential to cause adverse drug effect through the inhibition of major drug metabolism enzymes. The results indicated that 7 Cree medicines plant extracts inhibited CYP3A4 activity, and 3 of them have been proven to cause potent mechanism-based inactivation of CYP3A4. Seven of eight Traditional Chinese Medicines have been identified as strong CYP3A4 inhibitors; the ethanol extract of Goji has identified as a potent inhibitor for CYP2C9 and 2C19. Goji juice showed universal inhibitory effects on most of the tested enzymes except flavin-containing monooxygenases 3.

The enzymes CYP27A1 and CYP7B1 are widely expressed in various human tissues and perform catalytic reactions in cholesterol homeostasis and endocrine signaling. We have investigated the metabolism of a synthetic oxysterol. In this study, we show that CYP27A1 is the enzyme responsible for a 28-hydroxylation of this oxysterol and that the rate of CYP27A1-mediated metabolism is relatively slow. This may give an explanation for the prolonged inhibitory effects on cholesterol biosynthesis that have been shown for this oxysterol. The current study contributes to the knowledge of synthetically produced oxysterols and their potential use as cholesterol lowering drugs. In two studies we investigated CYP7B1-mediated metabolism of different sex hormones. Our data indicate that CYP7B1 may carry out a previously unknown catalytic reaction involving an androgen. Taken together the data suggest that varying steroid concentrations in cells and tissues may be important for CYP7B1-dependent metabolism of sex hormones and sex hormone precursors. CYP7B1-mediated hydroxylation of sex hormones may influence the cellular levels of these steroids and may be a potential pathway for elimination of the steroids from the cell. Some known CYP7B1 substrates are agonists for ERα and ERβ but the reported role(s) of CYP7B1 for ER action are not fully understood. In the last study we investigated the role(s) of CYP7B1-mediated metabolism for ER-mediated action. Our data indicate that CYP7B1-mediated conversion of steroids that affect ER-mediated response into their 7α-hydroxymetabolites will result in loss of action. This indicates that CYP7B1 may have an important role for regulation of ER-mediated processes in the body. In summary, results from this thesis contribute to the knowledge on the metabolism of synthetic oxysterols of potential use as cholesterol lowering drugs and the role(s) of CYP7B1-mediated metabolism for processes related to the functions of sex hormones. / Disputationsordförande;Professor Eva Brittebo, Inst. för Biovetenskap, Avd. för Toxikologi, Uppsala Universitet, UppsalaBetygsnämndens ledamöten; Docent Lena Ekström, Inst. för Laboratoriemedicin, Avd. för Klinisk Kemi, Karolinska Universitetssjukhuset, HuddingeDocent Ulf Diczfaluzy, Inst. för Laboratoriemedicin, Avd. för Klinisk Kemi, Karolinska Universitetssjukhuset, HuddingeProfessor Agneta Oskarsson, Inst. BVF, Avd. för farmakologi och toxikologi, SLU, Uppsala

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 5 uppsatser.