Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 138-152).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 177-200).

Orientador: Carmen Silvia Bertuzzo, Luis Alberto Magna / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T09:41:40Z (GMT). No. of bitstreams: 1 Rueda_LidianeCamila_M.pdf: 2492977 bytes, checksum: d9d2a855bfb9585fb574d275dcfe03a7 (MD5) Previous issue date: 2008 / Resumo: O câncer de mama (CM) é uma doença heterogênea e complexa, compreendendo múltiplas formas de apresentação clínica e morfológica, com diferentes graus de agressividade tumoral e potencial metastático. Acredita-se que 90% a 95% de todos os CM sejam esporádicos e decorram de mutações somáticas que se verificam durante a vida e que 5% a 10% sejam hereditários em virtude de uma mutação germinativa ao nascimento, que confere a estes indivíduos suscetibilidade ao CM. Entre os polimorfismos mais conhecidos de metabolização de drogas, estão os do sistema do citocromo P450. Os genes CYP1A1, CYP2D6 e CYP19 estão sendo estudados e em algumas populações mostraram uma associação positiva com a maior suscetibilidade ao CM. Este trabalho teve como objetivo investigar a presença dos polimorfismos T6235C (m1) e A4889G (m2) no gene CYP1A1, a presença dos polimorfismos A2637del (*3) e G1934A (*4) no gene CYP2D6 e a presença de alterações [TTTAn] no gene CYP19, por meio de um estudo de associação com uma amostra de 170 indivíduos, sendo 45 pacientes portadores de Adenocarcinoma e 120 controles normais. Foram utilizadas as técnicas extração de DNA, PCR e digestão enzimática. Não foi demonstrada, no presente estudo, associação entre os polimorfismos estudados e CM esporádico: (?2(2) = 1,12; p= 0,57) para o polimorfismo m1, (?2(2) = 0,83; p= 0,65) para o polimorfismo m2 do gene CYP1A1; (?2(2) = 0,15; p = 0,69) para o polimorfismo *3, (?2(2) = 2,41; p = 0,30) para o polimorfismo *4 do gene CYP2D6 e para as repetições em tandem [TTTA]n do gene CYP19. Os resultados sugerem que os polimorfismos gênicos estudados não estariam associados ao CM na amostra de indivíduos da região metropolitana de Campinas. Palavras-chave: CYP1A1, CYP2D6, CYP19, câncer de mama esporádico. / Abstract: Breast cancer (BC) is a complex disease, with heterogeneous clinical and morphologic presentation, that has with different degrees of tumoral aggressiveness and metastatic potential. It is known that 90% to 95% of all the BC are sporadical and happens because of somatic mutations that occurs during life and that 5% to 10% are hereditary because of germinate mutation due to births, which makes these individuals susceptibly to the BC. Among the best known polymorphism of metabolism of drugs, are the system cytochrome P450. The genes CYP1A1, CYP2D6 and CYP19 are being studied and in some populations because they had shown a positive association with the biggest susceptibility to the BC. This work had as objective to investigate the presence of polymorphisms T6235C (m1) and A4889G (m2) in gene CYP1A1, polymorphisms A2637del (*3) and G1934A (*4) in gene CYP2D6 and the presence of alterations [TTTAn] in gene CYP19, through a study of association with a sample of 170 individuals: 45 carrying patients of adenocarcinoma and 120 normal controls. The techniques had been used extraction of DNA, PCR and enzymatic digestion. It was not demonstrated, in this study the association between the polymorphisms studied and sporadic BC (?2(2) = 1,12; p= 0,57) for the polymorphism m1, (?2(2) = 0,83; p= 0,65) for the polymorphism m2 of gene CYP1A1; (?2(2) = 0,15; p= 0,69) for polymorphism *3, (?2(2) = 2,41; p= 0,30) for polymorphism *4 of gene CYP2D6 and for gene CYP19. The results suggest that the genic polymorphisms studied would not be associated with the BC in the sample of individuals in the metropolitan area of Campinas. key words: CYP1A1, CYP2D6, CYP19, sporadic breast cancer. / Mestrado / Mestre em Farmacologia

Wang Huan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 148-158). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.I / LIST OF FIGURES AND TABLES --- p.VIII / ABSTRACT --- p.1 / 摘要 --- p.3 / Chapter CHAPTER 1 --- GENERAL INTRODUCTION / Chapter I. --- The essential factors related to cancer --- p.5 / Chapter a. --- Carcinogens --- p.5 / Chapter b. --- Carcinogenesis pathways --- p.7 / Chapter c. --- DNA adducts formation and breast cancer --- p.7 / Chapter II. --- Cytochrome P450 I enzyme family --- p.8 / Chapter a. --- CYP450 superfamily --- p.8 / Chapter b. --- CYP1A1 --- p.10 / Chapter c. --- CYP1B1 --- p.11 / Chapter III. --- Transactivation of CYP1 enzymes by aryl hydrocarbon receptor (AhR) --- p.12 / Chapter IV. --- Phase II enzyme UGT and cancer prevention --- p.13 / Chapter V. --- Estrogen metabolism and the hormone-dependent breast cancer --- p.15 / Chapter a. --- Estrogen and breast cancer initiation --- p.15 / Chapter b. --- Estrogen Receptor (ER) --- p.15 / Chapter c. --- Estradiol hydroxylation pathways --- p.15 / Chapter VI. --- Phytochemicals and cancer prevention --- p.18 / Chapter VII. --- Outline of this study --- p.20 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter I. --- Chemicals --- p.21 / Chapter II. --- Cell culture and treatments --- p.21 / Chapter 1. --- Maintenance of cells --- p.21 / Chapter 2. --- Preparation of cell stock --- p.22 / Chapter 3. --- Cell recovery from liquid nitrogen stock --- p.22 / Chapter 4. --- Measurement of cell viability --- p.22 / Chapter 5. --- Preparation of cell lysates --- p.23 / Chapter 6. --- XRE-luciferase gene reporter assay --- p.23 / Chapter a. --- Transient transfection of cell using lipofectamine PLUS reagent --- p.23 / Chapter b. --- Dual Luciferase Assay --- p.24 / Chapter III. --- Enzyme Activities --- p.24 / Chapter 1. --- Isolation of microsomes --- p.24 / Chapter 2. --- EROD activities in intact cells --- p.24 / Chapter 3. --- EROD inhibition assay --- p.25 / Chapter IV. --- Manipulation of Nuclear Acid --- p.26 / Chapter 1. --- Preparation of transfected DNA --- p.26 / Chapter a. --- Separation and purification of DNA from agarose gel --- p.26 / Chapter b. --- Restriction digestion --- p.26 / Chapter c. --- Ligation of DNA fragments --- p.27 / Chapter d. --- Transformation of DH5a --- p.27 / Chapter e. --- Small scale plasmid purification from DH5a (mini prep) --- p.28 / Chapter f. --- Large scale plasmid isolation from DH5a (maxi-prep) --- p.28 / Chapter g. --- Construction of XRE activated luciferase reporter gene --- p.29 / Chapter 2. --- Measurement of DMBA-DNA adduct formation --- p.29 / Chapter 3. --- Semi-quantitative RT-PCR Assay --- p.30 / Chapter a. --- Isolation of RNA using TRIzol® Reagent --- p.30 / Chapter b. --- RT-PCR --- p.31 / Chapter V. --- Phase II enzyme-UGT activity assay --- p.32 / Chapter VI. --- HPLC for estradiol-hydroxylation analysis --- p.33 / Chapter 1. --- HPLC condition for hydroxyestradiol separation and measurement --- p.33 / Chapter 2. --- Determination of microsomal estradiol hydroxylase activity --- p.34 / Chapter 3. --- Assay of estradiol metabolism in MCF-7 cells --- p.34 / Chapter VII. --- Statistical Analysis --- p.35 / Chapter CHAPTER 3 --- CHALCONES ANTAGONIZE DMBA-INDUCED CARCINOGENESIS BY MODULATION OF CYP1A1/1B1 AND UGT ACTIVITIES / Chapter Part One --- Introduction --- p.36 / Chapter Part Two --- Results --- p.40 / Chapter Section One --- Chalcones antagonize DMBA carcinogenesis by inhibiting CYP1A1 and CYP1B1 activities --- p.40 / Chapter I. --- Chalcones inhibited DMBA-induced EROD activities in MCF-7 cells --- p.40 / Chapter II. --- Inhibition of chalcones on microsomal CYP1A1 & 1B1 enzyme activities --- p.43 / Chapter III. --- Reduction of DMBA-induced DNA adduct by chalcones --- p.52 / Chapter IV. --- Chalcones antagonized CYP1A1 XRE transactivation --- p.54 / Chapter V. --- Chalcones suppressed DMBA-induced CYP1 gene expression --- p.56 / Chapter Section Two --- Chalcones modulate DMBA carcinogenesis by regulating UGT activities --- p.63 / Chapter I . --- Chalcones regulated UGT1A1 gene expression in MCF-7 cells --- p.63 / Chapter II. --- Chalcones affected UGT enzyme activity in HepG2 cells --- p.70 / Chapter III. --- Chalcones regulated UGT1A1 gene expression in HepG2 cells --- p.73 / Chapter Part Three --- Discussion --- p.80 / Chapter I . --- Chalcones are potential chemopreventive agents --- p.80 / Chapter II. --- Chalcones modulated Phase I enzyme activities --- p.80 / Chapter III. --- Chalcones regulated Phase II enzyme activities --- p.82 / Chapter IV. --- Chalcones suppressed DMBA-induced DNA-adduct formation in MCF-7 cells --- p.82 / Chapter V. --- The anti-carcinogenic properties of chalcones and their structures --- p.83 / Chapter CHAPTER 4 --- EFFECTS OF PERILLYL ALCOHOL AND LIMONENE ON CYP1 AND UGT ENZYMES / Chapter Part One --- Introduction --- p.85 / Chapter Part Two --- Results --- p.87 / Chapter I. --- Perillyl alcohol and limonene modulated DMBA-induced CYP1A1/1B1 activities in MCF-7 cells --- p.87 / Chapter II. --- Perillyl alcohol and limonene regulated microsomal CYP1A1/1B1 activities --- p.89 / Chapter III. --- Perillyl alcohol and limonene regulated DMBA-induced DNA adduct formation in MCF-7 cells --- p.93 / Chapter IV. --- Perillyl alcohol and limonene regulated CYP1A1 & CYP1B1 gene expressions in MCF-7 cells --- p.95 / Chapter V. --- Effect of perillyl alcohol on CYP1A1 XRE transactivation --- p.97 / Chapter VI. --- Cytotoxic effect of perillyl alcohol and limonene on MCF-7 cells --- p.98 / Chapter VII. --- Perillyl alcohol and limonene modulated UGT1A1 gene expression in MCF-7 cells --- p.99 / Chapter VIII. --- Perillyl alcohol and limonene modulated UGT enzyme in HepG2 cells --- p.101 / Chapter Part Three --- Discussion --- p.106 / Chapter CHAPTER 5 --- LYCOPENE MEDIATED DMBA-INDUCED PHASE I & PHASE II ENZYME ACTIVITIES AND GENE EXPRESSIONS / Chapter Part Three --- Introduction --- p.109 / Chapter I. --- Biochemical properties of lycopene --- p.109 / Chapter II. --- Bioavailability of lycopene --- p.110 / Chapter III. --- Lycopene and cancers in hormonal sensitive tissues --- p.110 / Chapter Part Two --- Results --- p.111 / Chapter I . --- Lycopene modulated DMBA-induced CYP1A1/1B1 activities in MCF-7 cells --- p.111 / Chapter II. --- Lycopene competitively inhibited microsomal CYP1A1 & CYP1B1 activities --- p.113 / Chapter III. --- Lycopene suppressed DMBA-induced DNA adduct formation in MCF-7 cells --- p.115 / Chapter IV. --- Lycopene regulated CYP1A1 & CYP1B1 gene expression in MCF-7 cells --- p.116 / Chapter V. --- Effect of lycopene on CYP1A1 XRE trasactivation --- p.117 / Chapter VI. --- Cytotoxic effect of lycopene on MCF-7 cells --- p.118 / Chapter VII. --- Lycopene modulated UGT enzyme in MCF-7 cells --- p.119 / Chapter VIII. --- Lycopene modulated UGT enzyme in HepG2 cells --- p.121 / Chapter Part Three --- Discussion --- p.123 / Chapter CHAPTER 6 --- CHALCONES AND PERILLYL ALCOHOL REGULATEDCYP1A1 & CYP1B1 MEDIATED ESTRADIOL METABOLIZING PATHWAYS / Chapter Part One --- Introduction --- p.125 / Chapter I . --- Estrogen hydroxylation and human breast cancer risk --- p.125 / Chapter II. --- CYP1 enzymes catalyze estradiol-hydroxylation in human breast cancer cells --- p.126 / Chapter III. --- Phytochemicals mediate estrogen-hydroxylation pathways --- p.126 / Chapter Part Two --- Estrogen metabolite detection and separation by HPLC --- p.127 / Chapter Part Three --- Results --- p.129 / Chapter I . --- Perillyl alcohol modulated CYP1A1 & CYP1B1-mediated Estradiol hydroxylation --- p.129 / Chapter II. --- Kinetics assays of chalcones on CYP1A1 & CYP1B1 microsomes induced estradiol hydroxylation --- p.131 / Chapter III. --- Chalcones suppressed Estradiol-hydroxylase activities in MCF-7 cells --- p.137 / Chapter Part Four --- Discussion --- p.140 / Chapter CHAPTER 7 --- SUMMARY / Chapter I . --- Chalcones displayed inhibitory effects on DMBA-induced carcinogenesis --- p.142 / Chapter II. --- Perillyl alcohol and limonene modulated DMBA-induced carcinogenesis --- p.143 / Chapter III. --- Lycopene also possessed chemoproventive properties --- p.143 / APPENDIX 1 ABBREVIATIONS --- p.144 / APPENDIX 2 REAGENTS --- p.145 / APPENDIX 3 PRIMER LISTS --- p.147 / REFERENCE --- p.148

Danshen, the dried root of Salvia miltiorrhiza Bunge, is a herb listed in the Chinese Pharmacopoeia for the treatment of cardiovascular and cerebrovascular diseases. Danshen has been reported to have antiplatelet, cardioprotective, anti-inflammatory, hepatoprotective, and anti-HIV effects in preclinical studies. However, exaggerated anticoagulation and bleeding complications have also been observed during concurrent use of Danshen and warfarin in patients, although the mechanism(s) of the herb-drug interaction, pharmacodynamic and/or pharmacokinetic interactions, remained uncertain. Characterization of the cytochrome P450 isoforms responsible for the metabolism(s) of drugs and herbal constituents is important for the identification of potential drug-drug or drug-herb interactions. The present study investigated the effects of Danshen on the metabolism of probe substrates of specific CYP isoforms including CYP1A2, CYP3A and CYP2C9, the isoforms that are responsible for the metabolism of warfarin to assess the potential interactions of Danshen with drugs that utilize these isoforms for their biotransformation. / Firstly, the effects of Danshen and its tanshinone components on CYP1A2 activity were investigated in vitro and in vivo in the rat. Formulated Danshen extract, the ethanolic extract and the aqueous extract from Danshen root, and individual tanshinones inhibited phenacetin O-deethylation (CYP1A2) activity in vitro. Enzyme kinetic studies showed that tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone were competitive CYP1A2 inhibitors. Acute, sub-chronic and chronic pretreatments of formulated Danshen extract decreased the clearance (CL) of caffeine, with a concomitant increase in the area under concentration-time curve (AUC), and prolongation of the plasma half-life (T 1/2). These results suggested that Danshen inhibited rat CYP1A2 activity and altered the pharmacokinetics of the CYP1A2 probe substrates in vivo. / In conclusion, these results confirmed that Danshen-inhibited CYP activity, especially CYP1A2, then CYP2C9/11 (CYP2C9 in human, CYP2C11 in rats) in vitro. In vivo studies confirmed the clearance of the probe substrates was also decreased when co-administered with Danshen. Given that CYP1A2, CYP2C9 and CYP3A4 are responsible for the metabolism and disposition of a large number of drugs currently used in man, the concomitant use of Danshen with drugs which are substrates of CYP1A2, 2C9 and 3A4, especially CYP1A2, must be met with great caution. / Secondly, the effects of Danshen and its tanshinone components on CYP3A activity were investigated in vitro and in vivo in the rat. Formulated Danshen extract, the ethanolic extract from Danshen root, and tanshinones inhibited testosterone 6beta-hydroxylation (CYP3A) activity in vitro. Enzyme kinetic studies showed that tanshinone I, tanshinone IIA, and cryptotanshinone were competitive CYP3A inhibitors, whereas dihydrotanshinone was a noncompetitive CYP3A inhibitor. In vivo studies showed the pretreatments of formulated Danshen extract did not significantly change the pharmacokinetics of midazolam. / The effects of Danshen and its tanshinones on human CYP1A2 (phenacetin O-deethylation), CYP3A4 (testosterone 6beta-hydroxylation), and CYP2C9 (tolbutamide 4-hydroxylation) activities were also investigated in vitro using pooled human liver microsomes and human CYP isoforms. The ethanolic fraction of Danshen root was more effective than water-soluble fraction in inhibiting human CYP1A2, CYP3A4 and CYP2C9 activities. Enzyme kinetic studies showed that tanshinone I, tanshinone IIA, and cryptotanshinone were competitive inhibitors of CYP1A2, CYP3A4 and CYP2C9 with varying effectiveness. Dihydrotanshinone was not a competitive inhibitor of CYP1A2 and CYP2C9, but a noncompetitive CYP3A4 inhibitor. CYP1A2 was most affected and CYP3A4 was least affected by Danshen and tanshinones. Compared with the results obtained from rat and human, rat is a good animal model for predicting Danshen-drug interactions in humans, especially drugs which are substrates of CYP1A2. / Thirdly, the effects of Danshen and its tanshinone components on CYP2C11 activity were investigated in vitro and in vivo in the rat. Formulated Danshen extract, the ethanolic extract from Danshen root, and tanshinones inhibited testosterone 2alpha-hydroxylation (CYP2C11) activity in vitro. Enzyme kinetic studies showed that tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone were competitive CYP2C11 inhibitors. Sub-chronic pretreatment of formulated Danshen extract increased the AUC, T1/2 but decreased CL of tolbutamide. These results suggested that Danshen inhibited the CYP2C activity in the rat. In conclusion, these results confirmed the possible mechanism is enzyme inhibition, involved in the interaction of Danshen and warfarin previously observed in rats. / Wang, Xin. / "September 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4699. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 284-302). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Thesis (Ph. D.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains xiv, 180 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (M.S.)--West Virginia University, 2004 / Title from document title page. Document formatted into pages; contains ix, 49 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 41-48).

Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains viii, 141 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Grace S.N. Lau. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 335-362). / List of Abbreviations --- p.i / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction and Study Objectives / Chapter 1.1 --- Metabolic activation - role in drug toxicity and carcinogenesis --- p.5 / Chapter 1.2 --- Hepatocellular carcinoma --- p.12 / Chapter 1.2.1 --- Epidemiology --- p.12 / Chapter 1.2.2 --- Aetiological factors --- p.17 / Chapter 1.2.2.1 --- Hepatitis B virus infection --- p.17 / Chapter 1.2.2.2 --- Cirrhosis --- p.24 / Chapter 1.2.2.3 --- Aflatoxins --- p.25 / Chapter 1.2.2.4 --- Other factors --- p.26 / Chapter 1.2.2.5 --- Summary --- p.29 / Chapter 1.3 --- Study objectives --- p.30 / Chapter Chapter 2 --- The Metabolism of Paracetamol in Healthy Subjects andin Patients with Liver Disease and Hepatocellular Carcinoma / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.1.1. --- History of paracetamol --- p.34 / Chapter 2.1.2 --- Pharmacology of paracetamol --- p.37 / Chapter 2.1.3 --- "Absorption, Distribution, Metabolism and Excretion" --- p.38 / Chapter 2.1.3.1 --- Absorption --- p.38 / Chapter 2.1.3.2 --- Distribution --- p.41 / Chapter 2.1.3.3 --- Metabolism --- p.42 / Chapter 2.1.3.4 --- Excretion --- p.57 / Chapter 2.1.4 --- Toxicity and Overdosage --- p.59 / Chapter 2.2 --- Estimation of paracetamol and its metabolites in plasma and urine by high performance liquid chromatography --- p.72 / Chapter 2.2.1 --- Introduction --- p.72 / Chapter 2.2.2 --- Analytical method --- p.76 / Chapter 2.2.2.1 --- Materials --- p.76 / Chapter 2.2.2.2 --- Instrumentation --- p.77 / Chapter 2.2.2.3 --- Collection and storage of samples --- p.79 / Chapter 2.2.2.4 --- Chromatographic conditions --- p.79 / Chapter 2.2.3 --- Urine assay --- p.79 / Chapter 2.2.3.1 --- Preparation of standards and test samples for urine assay --- p.79 / Chapter 2.2.3.2 --- Calculation of results for urine assay --- p.80 / Chapter 2.2.3.3 --- Results of urine assay --- p.81 / Chapter 2.2.3.4 --- Validation of urine assay --- p.81 / Chapter 2.2.4 --- Plasma assay --- p.83 / Chapter 2.2.4.1 --- Preparation of standards and test samples for plasma assay --- p.83 / Chapter 2.2.4.2 --- Calculation of results for plasma assay --- p.91 / Chapter 2.2.4.3 --- Results of plasma assay --- p.91 / Chapter 2.2.4.4 --- Validation of plasma assay --- p.93 / Chapter 2.2.5 --- Summary --- p.99 / Chapter 2.3 --- The pharmacokinetics of paracetamol in healthy subjects --- p.103 / Chapter 2.3.1 --- Introduction --- p.103 / Chapter 2.3.2 --- Study protocol --- p.103 / Chapter 2.3.3 --- Methods --- p.103 / Chapter 2.3.3.1 --- Subjects --- p.103 / Chapter 2.3.3.2 --- Drug administration and sampling --- p.104 / Chapter 2.3.3.3 --- Drug analysis --- p.108 / Chapter 2.3.3.4 --- Calculations --- p.108 / Chapter 2.3.4 --- Pharmacokinetic analysis --- p.109 / Chapter 2.3.5 --- Statistical analysis --- p.113 / Chapter 2.3.6 --- Results --- p.114 / Chapter 2.3.6.1 --- Plasma Results --- p.114 / Chapter 2.3.6.2 --- Urine Results --- p.118 / Chapter 2.3.6.3 --- Pharmacokinetic Results --- p.125 / Chapter 2.3.6.4 --- Statistical Results --- p.134 / Chapter 2.3.7 --- Discussion --- p.145 / Chapter 2.4 --- "The pharmacokinetics of paracetamol in healthy subjects, patients with liver disease and hepatocellular carcinoma" --- p.155 / Chapter 2.4.1 --- Introduction --- p.155 / Chapter 2.4.2 --- Study protocol --- p.156 / Chapter 2.4.3 --- Methods --- p.156 / Chapter 2.4.3.1 --- Subjects --- p.156 / Chapter 2.4.3.2 --- Drug administration and sampling --- p.157 / Chapter 2.4.3.3 --- Drug analysis --- p.160 / Chapter 2.4.3.4 --- Calculations --- p.160 / Chapter 2.4.4 --- Pharmacokinetic analysis --- p.161 / Chapter 2.4.6 --- Results --- p.162 / Chapter 2.4.6.1 --- Plasma Results --- p.162 / Chapter 2.4.6.2 --- Urine Results --- p.162 / Chapter 2.4.6.3 --- Pharmacokinetic Results --- p.179 / Chapter 2.4.7 --- Discussion --- p.194 / Chapter 2.4.8 --- Summary --- p.203 / Chapter Chapter 3 --- Metabolic Activation of Aflatoxin B1 in Healthy Subjects and in Patients with Liver Disease and Hepatocellular Carcinoma / Chapter 3.1 --- General introduction --- p.206 / Chapter 3.1.1 --- Chemical structures and properties --- p.207 / Chapter 3.1.2 --- Contamination of food by aflatoxins --- p.209 / Chapter 3.1.3 --- Metabolism of aflatoxins --- p.210 / Chapter 3.1.4 --- Human diseases possibly related to exposure to aflatoxins --- p.226 / Chapter 3.1.4.1 --- Acute aflatoxicosis --- p.226 / Chapter 3.1.4.2 --- Reye's syndrome --- p.227 / Chapter 3.1.4.3 --- Kwashiorkor --- p.228 / Chapter 3.1.4.4 --- Impaired immune function --- p.229 / Chapter 3.1.4.5 --- Hepatocellular carcinoma --- p.230 / Chapter 3.1.5 --- Biochemical and molecular epidemiology of aflatoxins --- p.232 / Chapter 3.2 --- Development of an ELISA method to monitor AFB1 exposure in human serum --- p.237 / Chapter 3.2.1 --- Introduction --- p.237 / Chapter 3.2.2 --- Preparation of all the components necessary for analysing AFB1-albumin adducts by ELISA --- p.243 / Chapter 3.2.2.1 --- Materials --- p.243 / Chapter 3.2.2.2 --- Preparation of rabbit AFB1 antiserum --- p.244 / Chapter 3.2.2.3 --- Preparation of the rat monoclonal antibody --- p.244 / Chapter 3.2.2.4 --- Concentration of cell culture supernatant by ammonium sulphate precipitation --- p.246 / Chapter 3.2.2.5 --- Preparation of the BSA-AFB1 conjugate --- p.248 / Chapter 3.2.2.6 --- Preparation of the immunoaffinity gel --- p.250 / Chapter 3.2.2.7 --- Preparation of the ELISA plates --- p.251 / Chapter 3.2.3 --- General procedures used in the analysis of AFB1- albumin adducts --- p.252 / Chapter 3.2.3.1 --- Competitive ELISA binding assay --- p.253 / Chapter 3.2.3.2 --- Sep-pak C18 cartridge --- p.254 / Chapter 3.2.3.3 --- Immunoaffinity column --- p.255 / Chapter 3.2.3.4 --- Evaporation process --- p.255 / Chapter 3.2.3.5 --- HPLC --- p.256 / Chapter 3.2.3.6 --- Radioactive counting --- p.256 / Chapter 3.2.3.7 --- Albumin isolation --- p.257 / Chapter 3.2.3.8 --- Digestion of albumin --- p.257 / Chapter 3.2.3.9 --- Animal procedures --- p.258 / Chapter 3.2.4 --- Validations --- p.259 / Chapter 3.2.4.1 --- Analysis of standard AFB1 and AFB1- lysine in ELISA --- p.259 / Chapter 3 2.4.2 --- Optimisation of antiserum dilution and concentration of coating antigenin ELISA --- p.259 / Chapter 3 2.4.3 --- Elution characteristics and capacity of the immunoaffinity column --- p.261 / Chapter 3.2.4.4 --- Comparison of immunoaffinity gels prepared with different affinity gels --- p.261 / Chapter 3.2.4.5 --- Immunoaffinity column experiment of AFB1-lysine --- p.263 / Chapter 3.2.4.6 --- HPLC Analysis of fractions from immunoaffinity column --- p.263 / Chapter 3.2.4.8 --- HPLC analysis of fractions from Sep- Pak C18 cartridge --- p.264 / Chapter 3.2.4.9 --- Digestion of serum albumin by proteinase K --- p.264 / Chapter 3.2.4.10 --- Effect of ethanol in samples to be loaded onto Sep-Pak C18 cartridge --- p.265 / Chapter 3.2.4.11 --- Effect of drying in a vacuum concentrator on recovery of radioactivity of 3H-AFB1 --- p.266 / Chapter 3.2.4.12 --- Evaluation of the overall procedure for the analysis of serum albumin adducts of AFB1 --- p.267 / Chapter 3.2.4.13 --- HPLC analysis of samples obtained after digestion and all clean-up procedures --- p.268 / Chapter 3.2.5 --- Results and discussion --- p.268 / Chapter 3.2.5.1 --- BSA-AFB1 conjugate --- p.268 / Chapter 3.2.5.2 --- Treatment of experimental animals with 3H-AFB1 --- p.270 / Chapter 3.2.5.3 --- Optimisation of antiserum dilution and concentration of coating antigenin ELISA --- p.272 / Chapter 3.2.5.4 --- Analysis of standard AFB1 and AFB1- lysine in ELISA --- p.275 / Chapter 3.2.5.5 --- Sep-Pak C18 cartridge - elution characteristics and capacity --- p.279 / Chapter 3.2.5.6 --- Elution characteristics of immunoaffinity columns --- p.282 / Chapter 3.2.5.7 --- Immunoaffinity column experiment of AFB1-lysine --- p.290 / Chapter 3.2.5.8 --- Digestion of serum albumin by proteinase K --- p.295 / Chapter 3.2.5.9 --- Effect of ethanol in samples to be applied onto Sep-Pak C18 cartridges --- p.297 / Chapter 3.2.5.10 --- Recovery of radioactivity after dryingin a vacuum concentrator --- p.300 / Chapter 3.2.5.11 --- Recovery of the overall clean-up procedure for the analysis of serum albumin adducts of AFB1 --- p.300 / Chapter 3.2.5.12 --- HPLC analysis of samples obtained after all clean-up procedures --- p.305 / Chapter 3.2.5.13 --- The use of rabbit anti-AFB1 anti-serum and rat anti-AFB1 monoclonal antibody --- p.308 / Chapter 3.2.6 --- Summary --- p.309 / Chapter 3.3 --- Monitoring of AFBralbumin adducts in plasma of patients with liver disease and hepatocellular carcinoma --- p.311 / Chapter 3.3.1 --- Introduction --- p.311 / Chapter 3.3.2 --- Material and methods --- p.314 / Chapter 3.3.2.1 --- Subject --- p.314 / Chapter 3.3.2.2 --- Sample collections --- p.315 / Chapter 3.3.2.4 --- Assay for AFB1-albumin adducts --- p.315 / Chapter 3.3.2.5 --- Statistical analysis --- p.318 / Chapter 3.3.3 --- Results and discussion --- p.318 / Chapter Chapter 4 --- Summary and Ideas for Further Studies --- p.330 / Acknowledgements --- p.333 / References --- p.335 / Appendices --- p.364