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Antioxidant And Cytotoxic Properties Of Salvia Absconditiflora And Effects On Cyp1a1, Cyp1b1 Gene Expressions In Breast Cancer Cell LinesYilmaz, Selis 01 January 2013 (has links) (PDF)
Salvia genus is a widely cultivated genus and used in medicine for various purposes as having
antimicrobial, antioxidant, anticarcinogen and anti-inflammatory features. In this study the aim
was to investigate phenolic composition of Salvia absconditiflora and understand the possible
effects of those constituents in cancer related drug metabolizing enzymes. Salvia absconditiflora
showed 80,43 % Radical Scavenging Activity against DPPH radical. Total flavonoid content was
found as one third of total phenolic content. Presence of important phenolic acids and flavonoids
such as caffeic acid, luteolin, coumaric acid are validated with LC-MS/MS analysis. Cytotoxicity of
Salvia absconditiflora treatment on MCF-7 and MDA-MB-231 breast cancer cell lines were
investigated through XTT and TBE assays both dose and time dependent manner. Cell
proliferation was inhibited 50 % by different IC50 values calculated in different assays and different
time intervals. This suggests that two breast cancer cell lines response in a different way to
cytotoxic treatments. Cancer related drug metabolizing enzyme gene modulations were
investigated with qRT-PCR. CYP1A1 and CYP1B1 were up-regulated in MCF-7 but down-regulated
in MDA-MB-231 cells in response to Salvia absconditiflora treatment.
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Timing Matters: The Role of Circadian Clock Genes In Development and Toxin ResponsesQu, Xiaoyu 15 May 2009 (has links)
Most members of the PAS (PER-ARNT-SIM) protein family are transcription factors, mediating development and adaptive responses to the environment, such as circadian rhythms and toxin responses. Because the PAS domain mediates protein-protein interactions and functional cross-talk between distinct biological processes, we hypothesized that PAS genes in the circadian clockworks, namely Per1 and Per2, may be involved in development and toxin responses, which are modulated by other PAS members. To explore the possible role of clock genes in development, we examined mammary epithelial cells in vitro and the mouse mammary gland in vivo for evidences of changes in clock gene expression during different stages of development and differentiation. Our results showed that Per1 and Bmal1 expression were up-regulated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. A similar differentiation-dependent profile of clock gene expression was observed in mouse mammary glands; Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. These data suggest that circadian clock genes may play a role in mouse mammary gland development. To examine clock gene function in toxin responses, we evaluated whether disruption or inhibition of Per1 and/or Per2 alters toxin-induced activity of the AhR signaling pathway in the mouse mammary gland and liver. We assessed the activation of the AhR signaling pathway in response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypical AhR agonist, by analyzing the mRNA abundance of its two target genes, cytochrome P450, subfamily I, polypeptide 1 (Cyp1A1) and Cyp1B1. Our results showed that the targeted disruption of Per1, but not Per2, significantly increases the TCDD-induced p450 expression in the mammary gland and liver in vivo. Similar changes in TCDD-mediated p450 expression were observed in vitro using mammary primary cultures of mammary cells derived from from Per1ldc, Per2ldc and Per1ldc/Per2ldc mutant mice and Hepa1c1c7 cells subjected to siRNA-mediated inhibition of Per1 or Per2. These discoveries suggest that the clock gene Per1 may modulate toxin responses perhaps by functioning as a negative regulator for TCDD-mediated activation of the AhR signaling pathway.
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CYP1A1 and CYP1B1 expression and free zinc levels in endothelial cells are differentially regulated by pro-atherogenic versus anti-atherogenic shear stressConway, Daniel Elridge 12 March 2009 (has links)
It is hypothesized that exposing endothelial cells to steady or non-reversing pulsatile shear stress produces a healthy, anti-atherogenic endothelium, whereas a reversing pulsatile shear stress promotes an unhealthy, pro-atherogenic endothelium. To further investigate this hypothesis, a novel parallel plate flow chamber system was used to expose human endothelial cells to a pro-atherogenic reversing shear stress waveform designed to simulate the wall shear stress at the carotid sinus, a region prone to atherosclerosis. Cells exposed to this reversing shear stress were compared to cells exposed to high levels of steady shear stress (15 dynes/cm²), low steady shear stress (1 dyne/cm², the time-average of the carotid shear stress), and static culture conditions. Functional analysis confirmed previous findings that reversing shear stress increases cell proliferation and monocyte adhesion. Microarray results indicate that although there are unique sets of genes controlled by both low average shear stress and by reversing flow, more genes were controlled by low average shear stress. We propose that low-time average shear stress, and not fluid reversal/oscillation, may be the more significant mechanical force. The reversing shear stress system was also used to investigate two shear stress-responsive genes, CYP1A1 and CYP1B1. Both were maximally up-regulated at arterial steady shear stresses of at least 15 dynes/cm² and reversing pulsatile shear stress attenuated expression of both genes. Furthermore, AhR nuclear localization and CYP1A1 protein expression correlate with the flow patterns in the mouse aortic arch. The data strongly suggest that the AhR/CYP1 pathway promotes an anti-atherogenic phenotype in the endothelium. Changes in free zinc were measured under different shear stresses. High steady shear stress dramatically increases the levels of free zinc in endothelial cells as compared to cells grown in static culture. This increase in free zinc is attenuated under reversing shear stress and low steady shear stress, which correlates with an increase in zinc-binding metallothinein proteins and zinc exporter Znt-1. Overall, the findings provide further insight into endothelial responses to mechanical forces and may be important in understanding mechanisms of atherosclerotic development and localization to regions of disturbed flow.
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Effets d'un mélange d'ingrédients actifs de pesticides sur l'activation de la voie du récepteur aux hydrocarbures d'aryleBergeron, Sandra January 2017 (has links)
Depuis bon nombre d’années déjà, la question ne se pose plus ; l’utilisation des pesticides en agriculture est nécessaire puisque sans ces derniers les chances que les terres soient ravagées par les insectes, champignons ou petits rongeurs représenteraient un trop grand risque pour les agriculteurs. Cependant, cette utilisation massive de pesticides en agriculture apporte son lot de questionnements et d’inquiétudes face aux effets néfastes qu’ils pourraient avoir tant sur les humains que sur la faune et la flore. Bien que tous les pesticides utilisés au Canada soient homologués par Santé Canada, et sont donc jugés comme ne représentant pas de risques élevés ni pour l’environnement ni les humains, nul ne connait les effets d’un mélange de pesticides sur nos cellules, notre organisme. Ce projet de recherche vise donc à évaluer les effets d’un mélange de pesticides composé de cinq ingrédients actifs communément utilisés en agriculture sur les niveaux d’expression du gène CYP1A1, un gène cible du récepteur aux hydrocarbures d'aryle (AhR).
Ce récepteur, bien qu’impliqué dans bon nombre d’autres réponses cellulaires, joue un rôle important dans la détoxification de l’organisme en activant la transcription de certains gènes dans la famille du cytochrome P450, dont le gène CYP1A1. Tel que mentionné précédemment, le but du présent projet de recherche est d’évaluer les effets d’un mélange composé d’au moins deux ingrédients actifs de pesticides sur l’activation de la voie de AhR. Les résultats du projet de recherche ont démontré que certaines combinaisons donnent lieu à une activation synergique de la voie AhR alors que d’autres donnent plutôt lieu à une activation additive. Dans le cas où la concentration des ingrédients actifs est élevée, on obtient plutôt un effet inhibiteur. N’est-ce pas paradoxal qu’à faibles doses, il y a un effet soit additif ou synergique alors qu’à de hautes concentrations, l’effet est plutôt inhibiteur ? Il ne faut alors pas croire que de fortes doses de pesticides sont bénéfiques puisque les effets sur les niveaux d’expression des gènes cibles de AhR ne signifient pas qu’il en sera de même pour les tous les autres gènes. Les résultats ont également démontré qu’en présence d’œstrogène, les ingrédients actifs seuls ou en combinaison ont le même effet que le 2,3,7,8-Tétrachlorodibenzo-para-dioxine (TCDD) sur l’interaction croisée entre AhR et le récepteur aux œstrogènes ; l’expression du gène CYP1A1 est réprimée alors que l’expression de CYP1B1 demeure inchangée.
Maintenant qu’on comprend bien les effets que peuvent avoir une combinaison d’ingrédients actifs de pesticides sur l’activation AhR, il ne reste plus qu’à comprendre pourquoi certains mélanges donnent lieu à une activation synergique et d’autres additive. Une question bien simple, mais à laquelle il est si difficile de répondre.
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An investigation into the metabolic activation of novel chloromethylindolines by isoforms of cytochrome P450. Targeting drug metabolising enzymes in cancer: analysis of the role and function of selected cytochrome P450 oxidising novel cancer prodrugsAlandas, Mohammed N. January 2012 (has links)
Introduction Cytochromes P450 (CYPs) are the major family of enzymes responsible for detoxification and metabolism of a wide range of both endogenous and xenobiotics chemicals in living organisms. The use of CYPs to activate prodrugs to cytotoxins selectively in tumours has been explored including AQ4N, Phortress and Aminoflavone. CYP1A1, CYP1B1, CYP2W1, and CYP4F11 have been identified as expressed in tumour tissue and surrounding stroma at high frequency compared to most normal tissues.
Aim is to investigate the differential metabolism of novel chloromethylindoline by high frequency expressed CYPs in tumours. This differential may be exploited to elicit a selective chemotherapeutic effect by metabolising inert small molecules to potent cytotoxins within the tumour environment.
Materials and Methods Sensitive and specific LC/MS/MS techniques have been developed to investigate the metabolism of chloromethylindolines. Recombinant enzymes and transfected cell lines were used to investigate the metabolic profiles with a focus on production of the cytotoxic derivatives of chloromethylindolines. Results Detailed metabolic studies show that (1-(Chloromethyl)-1,2-dihydropyrrolo
[3,2-e]indol-3(6H)-yl)(5-methoxy-1H-indol-2-yl) methanone (ICT2700) and other chloromethylindolines are converted by CYP1A1 mediated hydroxylation at the C-5 position leading to highly potent metabolites. In vitro cytotoxicity studies showed differentials of up to 1000-fold was achieved between CYP1A1 activated compared to the non-metabolised parent molecules. The reactivity of metabolites of ICT2700 was also explored using glutathione as a nucleophile. The metabolites were identified by a combination of LC/MS and LC MS/MS techniques. Investigations using mouse and human liver microsomes show that a large number of metabolites are created though none were shown to be associated with a potential anticancer effect. Studies focused on CYP2W1 show that this isoform metabolised ICT2706 to a cytotoxic species and a pharmacokinetic study showed a good distribution of ICT2706 into mouse tissues including tumour. However metabolism of ICT2726 by CYP2W1 resulted only in a non-toxic metabolite profile and may have potential as a biomarker for functional CYP2W1 in tissues. Preliminary studies show that palmitic acid hydroxylation is a useful marker of functional CYP4F11. Summary and conclusion The in vitro results show that the chloromethylindolines are a novel class of agent with potential as prodrugs that following specific hydroxylation by CYP1A1 and CYP2W1 are converted to ultra-potent cytotoxins. Other metabolites are also evident which are not cytotoxic. Studies in vivo show that selected chloromethylindolines possess a good pharmacokinetic profile and show potential as prodrug anticancer agents that require activation by CYP1A1 or CYP2W1. The methods, results, progress and suggestions for future work are presented in this thesis.
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ROLE OF THE AHR IN POLYBROMINATED BIPHENYL-INDUCED DEVELOPMENTAL TOXICITYMILLER, KEVIN ANTHONY January 2003 (has links)
No description available.
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Modulation of cytochrome P450 1 activity and DMBA-DNA adduct formation by baicalein, isoflavones and theaflavins.January 2002 (has links)
Chan Ho Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 121-138). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.II / ABSTRACT --- p.III / 摘要 --- p.V / ABBREVIATIONS --- p.VI / "TABLE OF CONTENTS, " --- p.VII / LIST OF FIGURES AND TABLES --- p.XI / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Xenobiotic-metabolizing enzymes --- p.1 / Cytochrome P450 1 family --- p.4 / CYP1A1 --- p.5 / CYP1A2 --- p.5 / CYP1B1 --- p.5 / Transactivation of CYP1 enzymes by Aryl hydrocarbon receptor (AhR) --- p.8 / Implication of PAHs and CYP1 family in breast cancer --- p.10 / Potential role of phytochemicals on cancer prevention --- p.11 / Significance of this project --- p.13 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.14 / Chemicals --- p.14 / Maintenance of cells --- p.14 / Preparation of cell stock --- p.14 / Cell recovery from liquid nitrogen stock --- p.15 / Measurement of cell viability --- p.15 / Preparation of cell lysates (NP-40 cell lysis buffer) --- p.15 / XRE-luciferase gene reporter assay --- p.16 / Manipulation of DNA and RNA --- p.17 / Separation and purification of DNA from agarose gel --- p.17 / Separation of DNA from acrylamide gel --- p.17 / Restriction digestion --- p.18 / Ligation of DNA fragments --- p.18 / Transformation of DH5 a --- p.19 / Small scale plasmid purification from DH5a (mini prep) --- p.19 / Large scale plasmid isolation from DH5a (maxi-prep) --- p.20 / Construction of XRE activated luciferase reporter gene --- p.21 / Measurement of DMBA-DNA adduct formation --- p.21 / Semi-quantitative RT-PCR Assay --- p.22 / ENZYME ACTIVITIES --- p.23 / Isolation of microsomes --- p.23 / EROD activities in intact cells --- p.23 / EROD inhibition assay --- p.24 / Stattstical Analysis --- p.24 / Chapter CHAPTER 3 --- BAICALEIN INHIBITS DMBA-DNA ADDUCT FORMATION BY MODULATING CYP1A1 AND 1B1 ACTIVITIES --- p.26 / Introduction --- p.26 / Results --- p.28 / EROD activities in MCF-7 cells and inhibition assay --- p.28 / Baicalein suppressed DMBA-induced XRE-driven luciferase activities --- p.31 / Baicalein inhibited DMBA-induced CYP1A1 and CYP1B1 mRNA expression --- p.31 / The cytotoxic effect of DMBA was reduced by baicalein --- p.35 / Inhibition of DMBA-DNA adduct formation after baicalein treatment --- p.35 / Discussion --- p.39 / Chapter CHAPTER 4 --- INHIBITION OF DMBA-DNA ADDUCT FORMATION BY (-)-EPIGALLOCATECHIN GALLATE AND THEAFLAVINS --- p.41 / Introduction --- p.41 / Results --- p.45 / Persistence of DMBA-induced DNA adducts --- p.45 / Inhibition of theaflavins and EGCG on human recombinant CYP1A1 and CYP1B1 enzyme activities --- p.48 / EGCG suppressed DMBA-induced EROD activity while thealfavin had no significant effect on this --- p.48 / Kinetic analysis of EGCG on CYP1A1 and CYP1B1 activities --- p.53 / Modulation of DMBA-induced XRE-driven luciferase activities by theaflavins and EGCG --- p.56 / The influence of theaflavins and EGCG on CYP1A1 and CYP1B1 abundance --- p.56 / Discussion --- p.65 / Chapter CHAPTER 5 --- ISOFLAVONES PREVENT DMBA-INDUCED CARCINOGENESIS BY INHIBITING CYP1A1 AND CYP1B1 ACTIVITIES --- p.67 / Introduction --- p.67 / Results --- p.70 / Isoflavones inhibited DMBA-induced EROD activity in MCF-7 cells --- p.70 / Inhibition of MCF-7 microsomal EROD activities by isoflavones --- p.70 / Kinetic analysis of the inhibition of human recombinant CYP1 enzymes by isoflavones --- p.74 / XRE-driven Luciferase activities --- p.83 / Both biochanin A and genistein suppressed DMBA-induced CYP1 mRNA expression --- p.83 / Cytotoxicity of DMBA and isoflavones co-treatment --- p.88 / Isoflavones reduced the binding of activated DMBA to DNA --- p.89 / Discussion --- p.93 / Chapter CHAPTER 6 --- IN VITRO EFFECTS OF BAICALEIN AND THEAFLAVINS ON RAT HEPATIC P450 ACTIVITIES --- p.96 / Introduction --- p.96 / Results --- p.98 / Inhibition of EROD and MROD activities in rat liver microsomes by baicalein --- p.98 / Effects of theaflavins on EROD and MROD activities in rat liver microsomes --- p.102 / Kinetic studies for EROD and MROD activities of theaflavins --- p.104 / DISCUSSION --- p.114 / Chapter CHAPTER 7 --- CONCLUSION --- p.116 / APPENDIX 1 PRIMER LISTS --- p.118 / APPENDIX 2 REAGENTS --- p.119 / BIBLIOGRAPHY --- p.121
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Investigação molecular e epidemiológica de genes do metabolismo de xenobióticos em pacientes com câncer colorretal esporádicoFernandes, Glaucia Maria de Mendonça 12 December 2013 (has links)
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Previous issue date: 2013-12-12 / Fundação de Amparo a Pesquisa do Estado de São Paulo / Introduction: The xenobiotics are exogenous substances to the organism, as N-nitrosamines, heterocyclic amines (HAs) and polycyclic aromatic hydrocarbons (PAHs), can which result in DNA adducts formation. Polymorphisms in genes involved in the metabolism of xenobiotics could contribute to this process and modulate the development of cancer. Objectives: To investigate the CYP1A1*2A (rs4646903), CYP1A1*2C (rs1048943), CYP2E1*5B (rs2031920), CYP1E1*6 (rs6413432), Tyr113His EPHX1 (rs1051740) and His139Arg EPHX1 (rs2234922) polymorphisms related to the metabolism of xenobiotics, the risk of sporadic colorectal (SCRC) cancer, the interaction of these polymorphisms with lifestyle (smoking and drinking) and clinical and histopathological parameters and to evaluate the association of SCRC with socio-demographic factors. Methods: A case-control study was conducted in 641 subjects in the Brazilian population (241 patients with colorectal cancer and 400 controls (individuals without a history of cancer). Real-Time PCR and PCR-RFLP was performed for genotyping. Statistical analysis was performed using the chi-square tet and multiple logistic regression binary. Results: The results showed statistically significant differences between the case and control groups for age greater than 50 years (OR=8.21, 95%CI=5.49-12.28, p<0.01) and male gender (OR=0.50, 95%CI=0.32-0.87, p<0.01) The analysis of polymorphisms revealed an association between the alleles polymorphic CYP2E1*5B (OR=2.84, 95%CI=1.78-4.52, p<0.01, additive model) and CYP2E1*6 (OR=2.78, 95%CI=1.91-4.06, p<0.01, additive model) and the SCRC. Tumor size, lymph node involvement and disease primary site were not associated with polymorphisms. Conclusion: The CYP2E1*5B and CYP2E1*6 polymorphisms are involved in the risk of SCRC and individuals with age ≥ 50 years are more susceptible to this tumor type, of males are less susceptible. / Introducão: Os xenobióticos são substâncias exógenas ao organismo, tais como as N-nitrosaminas, aminas heterocíclicas (HAs) e hidrocarbonetos policíclicos aromáticos (HPAs), que podem formar adutos de DNA. Polimorfismos em genes envolvidos no metabolismo dos xenobióticos podem contribuir com este processo e, consequentemente, modular o desenvolvimento de câncer. Objetivos: Investigar os polimorfismos CYP1A1*2A (rs 4646903), CYP1A1*2C (rs1048943), CYP2E1*5B (rs 2031920), CYP1E1*6 (rs 6413432), EPHX1 Tyr113His (rs1051740) e EPHX1 His139Arg (rs2234922), relacionados com o metabolismo dos xenobióticos, no risco de câncer de colorretal esporádico (CCRE), a interação desses polimorfismos com os hábitos de vida (tabagismo e etilismo) e parâmetros clínico-histopatológicos e avaliar a associação do CCRE com os fatores sócio-demográficos. Os Métodos: Um estudo caso-controle foi realizado em 641 indivíduos da população brasileira (241 pacientes com câncer de coloretal e 400 controles (indivíduos sem histórico de câncer). As técnicas de PCR em Tempo Real e PCR-RFLP foram realizadas para a genotipagem dos polimorfismos. A análise estatística utilizou os testes de Qui-Quadrado e Regressão Logística Múltipla Binária. Resultados: Os resultados mostraram diferenças estatisticamente significantes entre os grupos caso e controle para idade superior a 50 anos (OR=8,21; IC95%=5,49-12,28, p<0,01) e gênero masculino (OR=0,50; IC95%=0,32-0,87, p<0,01). A análise dos polimorfismos revelou associação entre os alelos polimórficos CYP2E1*5B (OR=2,84; IC95%=1,78-4,52; p<0,01, modelo aditivo) e CYP2E1*6 (OR=2,78; IC95%=1,91-4,06, p<0,01, modelo aditivo) e o CCRE. O tamanho do tumor, envolvimento de linfonodos e sítio primário da doença não foram associados com os polimorfismos. Conclusão: Os polimorfismos CYP2E1*5B e
CYP2E1*6 estão envolvidos no risco de CCRE e indivíduos com idade superior ou igual a 50 anos são mais suscetíveis a este tipo tumoral, enquanto aqueles do gênero masculino são menos suscetíveis.
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An investigation into the metabolic activation of novel chloromethylindolines by isoforms of cytochrome P450 : targeting drug metabolising enzymes in cancer : analysis of the role and function of selected cytochrome P450 oxidising novel cancer prodrugsAlandas, Mohammed Nasser January 2012 (has links)
Introduction: Cytochromes P450 (CYPs) are the major family of enzymes responsible for detoxification and metabolism of a wide range of both endogenous and xenobiotics chemicals in living organisms. The use of CYPs to activate prodrugs to cytotoxins selectively in tumours has been explored including AQ4N, Phortress and Aminoflavone. CYP1A1, CYP1B1, CYP2W1, and CYP4F11 have been identified as expressed in tumour tissue and surrounding stroma at high frequency compared to most normal tissues. Aim is to investigate the differential metabolism of novel chloromethylindoline by high frequency expressed CYPs in tumours. This differential may be exploited to elicit a selective chemotherapeutic effect by metabolising inert small molecules to potent cytotoxins within the tumour environment. Materials and Methods: Sensitive and specific LC/MS/MS techniques have been developed to investigate the metabolism of chloromethylindolines. Recombinant enzymes and transfected cell lines were used to investigate the metabolic profiles with a focus on production of the cytotoxic derivatives of chloromethylindolines. Results: Detailed metabolic studies show that (1-(Chloromethyl)-1,2-dihydropyrrolo [3,2-e]indol-3(6H)-yl)(5-methoxy-1H-indol-2-yl) methanone (ICT2700) and other chloromethylindolines are converted by CYP1A1 mediated hydroxylation at the C-5 position leading to highly potent metabolites. In vitro cytotoxicity studies showed differentials of up to 1000-fold was achieved between CYP1A1 activated compared to the non-metabolised parent molecules. The reactivity of metabolites of ICT2700 was also explored using glutathione as a nucleophile. The metabolites were identified by a combination of LC/MS and LC MS/MS techniques. Investigations using mouse and human liver microsomes show that a large number of metabolites are created though none were shown to be associated with a potential anticancer effect. Studies focused on CYP2W1 show that this isoform metabolised ICT2706 to a cytotoxic species and a pharmacokinetic study showed a good distribution of ICT2706 into mouse tissues including tumour. However metabolism of ICT2726 by CYP2W1 resulted only in a non-toxic metabolite profile and may have potential as a biomarker for functional CYP2W1 in tissues. Preliminary studies show that palmitic acid hydroxylation is a useful marker of functional CYP4F11. Summary and conclusion: The in vitro results show that the chloromethylindolines are a novel class of agent with potential as prodrugs that following specific hydroxylation by CYP1A1 and CYP2W1 are converted to ultra-potent cytotoxins. Other metabolites are also evident which are not cytotoxic. Studies in vivo show that selected chloromethylindolines possess a good pharmacokinetic profile and show potential as prodrug anticancer agents that require activation by CYP1A1 or CYP2W1. The methods, results, progress and suggestions for future work are presented in this thesis.
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Mechanisms of activation of the aryl hydrocarbon receptor by novel inducers of the CYP1A1 gene /Backlund, Maria, January 1900 (has links)
Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.
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