Yang Lei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 141-148). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract (Chinese Version) --- p.iv / Table of Contents --- p.vi / List of Abbreviations --- p.xii / List of Figures --- p.xiii / List of Tables --- p.xviii / Chapter Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Carbon tetrachloride (CC14) --- p.1 / Chapter 1.2 --- Major uses of CC14 --- p.1 / Chapter 1.3 --- Potential human exposure pathways to CC14 --- p.2 / Chapter 1.4 --- Toxicity of CC14 --- p.3 / Chapter 1.5 --- Mechanism of CCl4-induced hepatotoxicity --- p.5 / Chapter 1.6 --- Role of CYP2E1 involved in CCl4-induced hepatotoxicity --- p.7 / Chapter 1.7 --- Definite proof of the involvement of CYP2E1 in CCl4-induced hepatotoxicity by CYP2El-null mouse in vivo model --- p.10 / Chapter 1.8 --- Identification of CYP2E1 -dependent genes involved in CCl4-induced hepatotoxicity by fluorescent differential display (FDD) --- p.11 / Chapter 1.9 --- Objectives of the study --- p.14 / Chapter Chapter 2 --- Materials and methods --- p.16 / Chapter 2.1 --- Animals and treatments --- p.16 / Chapter 2.1.1 --- Materials --- p.16 / Chapter 2.1.2 --- Methods --- p.16 / Chapter 2.2 --- Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) analyses --- p.17 / Chapter 2.2.1 --- Materials --- p.17 / Chapter 2.2.2 --- Methods --- p.17 / Chapter 2.2.2.1 --- Serum preparation --- p.17 / Chapter 2.2.2.2 --- Activity determination --- p.18 / Chapter 2.3 --- Tail-genotyping by PCR --- p.18 / Chapter 2.3.1 --- Materials --- p.18 / Chapter 2.3.2 --- Methods --- p.20 / Chapter 2.3.2.1 --- Preparation of genomic DNA from mouse tail --- p.20 / Chapter 2.3.2.2 --- PCR reaction --- p.20 / Chapter 2.4 --- Total RNA isolation --- p.21 / Chapter 2.4.1 --- Materials --- p.21 / Chapter 2.4.2 --- Methods --- p.21 / Chapter 2.5 --- DNase I treatment --- p.23 / Chapter 2.5.1 --- Materials --- p.23 / Chapter 2.5.2 --- Methods --- p.23 / Chapter 2.6 --- Reverse transcnption of mRNA and amplification by fluorescent PCR amplification --- p.26 / Chapter 2.6.1 --- Materials --- p.27 / Chapter 2.6.2 --- Methods --- p.27 / Chapter 2.7 --- Fluorescent differential display (FDD) --- p.28 / Chapter 2.7.1 --- Materials --- p.28 / Chapter 2.7.2 --- Methods --- p.28 / Chapter 2.8 --- Excision of differentially expressed cDNA fragments --- p.29 / Chapter 2.8.1 --- Materials --- p.29 / Chapter 2.8.2 --- Methods --- p.29 / Chapter 2.9 --- Reamplification of differentially expressed cDNA fragments --- p.34 / Chapter 2.9.1 --- Materials --- p.34 / Chapter 2.9.2 --- Methods --- p.34 / Chapter 2.10 --- Subcloning of reamplified cDNA fragments --- p.36 / Chapter 2.10.1 --- Materials --- p.36 / Chapter 2.10.2 --- Methods --- p.37 / Chapter 2.11 --- Purification of plasmid DNA from recombinant clones --- p.39 / Chapter 2.11.1 --- Materials --- p.39 / Chapter 2.11.2 --- Methods --- p.39 / Chapter 2.12 --- DNA sequencing of differentially expressed cDNA fragments --- p.40 / Chapter 2.12.1 --- Materials --- p.40 / Chapter 2.12.2 --- Methods --- p.40 / Chapter 2.12.3 --- BLAST search against the GenBank DNA databases --- p.41 / Chapter 2.13 --- Northern blot analysis of differentially expressed cDNA fragments --- p.41 / Chapter 2.13.1 --- Formaldehyde gel electrophoresis of total RNA --- p.41 / Chapter 2.13.1.1 --- Materials --- p.42 / Chapter 2.13.1.2 --- Methods --- p.42 / Chapter 2.13.2 --- Preparation of cDNA probes for hybridization --- p.42 / Chapter 2.13.2.1 --- EcoRI digestion of cDNA inserts from plasmid DNA --- p.42 / Chapter 2.13.2.1.1 --- Materials --- p.42 / Chapter 2.13.2.1.2 --- Methods --- p.43 / Chapter 2.13.2.2 --- Purification of DNA from agarose gel --- p.43 / Chapter 2.13.2.2.1 --- Materials --- p.43 / Chapter 2.13.2.2.2 --- Methods --- p.43 / Chapter 2.13.2.3 --- DIG labeling of cDNA --- p.44 / Chapter 2.13.2.3.1 --- Materials --- p.44 / Chapter 2.13.2.3.2 --- Methods --- p.44 / Chapter 2.13.3 --- Hybridization --- p.45 / Chapter 2.13.3.1 --- Materials --- p.45 / Chapter 2.13.3.2 --- Methods --- p.45 / Chapter Chapter 3 --- Results --- p.47 / Chapter 3.1 --- Liver morphology --- p.47 / Chapter 3.2 --- Serum ALT and AST activities --- p.47 / Chapter 3.3 --- Tail-genotyping by PCR --- p.51 / Chapter 3.4 --- DNase I treatment --- p.51 / Chapter 3.5 --- FDD RT-PCR and excision of differentially expressed cDNA fragments --- p.51 / Chapter 3.6 --- Reamplification of excised cDNA fragments --- p.61 / Chapter 3.7 --- Subcloning of reamplified cDNA fragments --- p.61 / Chapter 3.8 --- DNA sequencing of subcloned cDNA fragments --- p.69 / Chapter 3.9 --- Confirmation of differentially expressed patterns by Northern blot analysis --- p.106 / Chapter 3.10 --- Temporal expression of differentially expressed genes --- p.113 / Chapter 3.11 --- Tissue distribution of differentially expressed genes --- p.117 / Chapter Chapter 4 --- Discussion --- p.125 / Chapter 4.1 --- Liver morphology and serum ALT and AST activities --- p.126 / Chapter 4.2 --- Identification of CYP2E1 -dependent genes involved in CCl4-induced hepatotoxicity --- p.127 / Chapter 4.3 --- Functional roles of the identified differentially expressed genes --- p.129 / Chapter 4.3.1 --- Fragment B4 --- p.129 / Chapter 4.3.2 --- Fragment C12 --- p.130 / Chapter 4.3.3 --- Fragment B13 --- p.131 / Chapter 4.3.4 --- Fragment A5 --- p.132 / Chapter 4.4 --- Temporal expression of differentially expressed genes --- p.133 / Chapter 4.4.1 --- Fragment B4 --- p.133 / Chapter 4.4.2 --- Fragment C12 --- p.134 / Chapter 4.4.3 --- Fragment B13 --- p.134 / Chapter 4.4.4 --- Fragment A5 --- p.135 / Chapter 4.5 --- Tissue distribution of differentially expressed genes --- p.136 / Chapter 4.5.1 --- Fragment B4 --- p.136 / Chapter 4.5.2 --- Fragment C12 --- p.136 / Chapter 4.5.3 --- Fragment B13 --- p.137 / Chapter 4.5.4 --- Fragment A5 --- p.137 / Chapter 4.5.5 --- Roles of the identified genes involved in CCl4-induced hepatotoxicity --- p.138 / Chapter 4.6 --- Normalization of Northern blot analysis --- p.13 8 / Chapter 4.7 --- Limitations of FDD technique to identify differentially expressed genes --- p.138 / Chapter 4.8 --- Future studies --- p.139 / Chapter 4.8.1 --- Investigation of the differential expression patterns of the identified genes in acetaminophen-induced liver injury --- p.139 / Chapter 4.8.2 --- Dot blot analysis --- p.140 / Chapter 4.8.3 --- DNA microarray --- p.140 / References --- p.141

Minimizing anti-cancer drug toxicity is a major challenge for the pharmaceutical industry. Toxicity is most frequently due to either the direct interaction of the drug on previously unidentified targets or its conversion to metabolites by drug metabolizing enzymes (e.g. CYP450 enzymes) that cause cellular, tissue or organ damage. Pharmacoproteomics is beginning to take a central role in studying changes in protein expression corresponding to drug administration, the results of which, inform about the mode of action, toxicity, and resistance in pre-clinical and clinical stages of drug development. The main aim of this research is to apply comparative proteomics studies on livers from male and female mice xenograft models treated with major anti-cancer drugs (5-flourouracil, paclitaxel, cisplatin, and doxorubicin) and CYP inducer, TCPOBOP, to investigate their effect on protein expression profiles (proteome). Within this thesis, an attention is paid to optimise a highly validated proteomics workflow for biomarker identification. Proteins were extracted from liver microsomes of mice treated in two separate sets; Set A – male (5-fluoruracil, doxorubicin, cisplatin and untreated) or Set B – female (5-fluoruracil, paclitaxel, TCPOBOP and untreated) using cryo-pulverization and sonication method. The extracts were digested with trypsin ii and the resulting peptides labelled with 4-plex iTRAQ reagents. The labelled peptides were subjected for separation in two-dimensions by iso-electric focusing (IEF) and RP-HPLC techniques before analysis by mass spectrometry and database searching for protein identification. Set A and Set B resulted in identification and quantification of 1146 and 1743 proteins, respectively. Moreover, Set A and Set B recovered 26 and 34 cytochrome P450 isoforms, respectively. The microsomal changes after drug treatments were quite similar. However, more changes were observed in the male set. Up-regulation of MUPs showed the greatest distinction in the protein expression patterns in the treated samples comparing to the untreated controls. In Set A, 5-fluoruracil and cisplatin increased the expression of three isoforms (MUP1, 2, and 6), whereas doxorubicin has increased the expression of four isoforms (MUP1, 2, 3, and 6). On the other side, only TCPOBOP in Set B has increased the expression of two isoforms (MUP1 and 6). Our findings showed that the expression of MUP, normally involved in binding and excretion of pheromones, have drug- and sex-specific differences. The mechanism and significance of MUP up-regulation are ambiguous. Therefore, the impact of each therapeutic agent on MUP and xenobiotic enzymes will be discussed.

Keung Hoi Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 151-174). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract --- p.II / 摘要 --- p.V / Content --- p.VII / List of tables --- p.XIII / List of figures --- p.XIV / Abbreviations --- p.XVII / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Biology of Pleurotus tuber-regiun (Ptr) --- p.1 / Chapter 1.1.1 --- Ptr grown in the wild --- p.1 / Chapter 1.1.2 --- Cultivation of Ptr --- p.2 / Chapter 1.2 --- Functional food and pharmaceutical application of Ptr sclerotium --- p.3 / Chapter 1.2.1 --- Traditional food and medicinal uses of Ptr sclerotium --- p.3 / Chapter 1.2.2 --- Nutritional value and chemical composition --- p.4 / Chapter 1.2.3 --- Anti-tumor activity --- p.7 / Chapter 1.2.4 --- Anti-viral activity --- p.8 / Chapter 1.2.5 --- Immunologic function --- p.8 / Chapter 1.2.6 --- Pharmaceutical application --- p.9 / Chapter Chapter 2 --- Toxicological evaluation on Ptr sclerotium --- p.11 / Chapter 2.1 --- Introduction --- p.11 / Chapter 2.1.1 --- Toxicological concern of Ptr sclerotium --- p.11 / Chapter 2.1.2 --- Toxicological study --- p.12 / Chapter 2.1.3 --- Biochemical methods for toxicological evaluation --- p.14 / Chapter 2.1.3.1 --- Serum enzyme activities --- p.15 / Chapter 2.1.3.2 --- Other serum analytes --- p.17 / Chapter 2.1.4 --- Histopathological study --- p.20 / Chapter 2.1.5 --- Acute toxicity --- p.21 / Chapter 2.1.6 --- Sub-acute and sub-chronic toxicity --- p.23 / Chapter 2.1.7 --- Objectives --- p.26 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Sample materials and chemicals --- p.27 / Chapter 2.2.2 --- Acute toxicity test --- p.27 / Chapter 2.2.2.1 --- Diet and animals --- p.27 / Chapter 2.2.2.2 --- Experimental design --- p.28 / Chapter 2.2.2.3 --- Calculation of sclerotium intake dose --- p.29 / Chapter 2.2.2.4 --- Biochemical assays --- p.30 / Chapter 2.2.2.5 --- Histopathological examination --- p.31 / Chapter 2.2.3 --- Sub-acute and sub-chronic toxicity tests --- p.32 / Chapter 2.2.3.1 --- Diet Preparation --- p.32 / Chapter 2.2.3.2 --- Experimental design --- p.32 / Chapter 2.2.3.3 --- Biochemical assays --- p.36 / Chapter 2.2.3.4 --- Organ weight --- p.40 / Chapter 2.2.3.5 --- Histopathological examination --- p.41 / Chapter 2.2.4 --- Statistical analyses --- p.41 / Chapter 2.3 --- Results and Discussion --- p.42 / Chapter 2.3.1 --- Acute toxicity test --- p.42 / Chapter 2.3.1.1 --- Food consumption --- p.43 / Chapter 2.3.1.2 --- Serum transaminase activities --- p.44 / Chapter 2.3.1.3 --- Histopathology --- p.45 / Chapter 2.3.1.4 --- NOAEL --- p.45 / Chapter 2.3.2 --- Sub-acute toxicity test --- p.50 / Chapter 2.3.2.1 --- Body weight gain --- p.50 / Chapter 2.3.2.2 --- Biochemical assays --- p.51 / Chapter 2.3.2.3 --- Organ per body weight and histopathology --- p.52 / Chapter 2.3.2.4 --- Effects of Ptr sclerotial diets --- p.53 / Chapter 2.3.3 --- Sub-chronic toxicity test --- p.59 / Chapter 2.3.3.1 --- Food and energy consumption --- p.59 / Chapter 2.3.3.2 --- Biochemical assays --- p.63 / Chapter 2.3.3.3 --- Organ per body weight --- p.67 / Chapter 2.3.3.4 --- Body weight increase --- p.75 / Chapter 2.3.3.5 --- NOAEL --- p.80 / Chapter 2.4 --- Summary --- p.81 / Chapter Chapter 3 --- Hepatoprotection of Ptr sclerotium --- p.82 / Chapter 3.1 --- Introduction --- p.82 / Chapter 3.1.1 --- Hepatotoxicity --- p.82 / Chapter 3.1.2 --- Potential hepatoprotection effect of Ptr sclerotium --- p.83 / Chapter 3.1.3 --- Toxicity of CC14 --- p.85 / Chapter 3.1.4 --- Toxicity of AFB! --- p.89 / Chapter 3.1.5 --- Bioactivity of chlorophyllin --- p.92 / Chapter 3.1.6 --- Comet assay --- p.93 / Chapter 3.1.7 --- Objectives --- p.98 / Chapter 3.2 --- Materials and Methods --- p.99 / Chapter 3.2.1 --- Sample materials and chemicals --- p.99 / Chapter 3.2.2 --- Curative and preventive tests of Ptr sclerotium against CCl4-induced hepatotoxicity --- p.99 / Chapter 3.2.2.1 --- Animal and diets --- p.99 / Chapter 3.2.2.2 --- Dose-response of CCl4 on rat model --- p.100 / Chapter 3.2.2.3 --- Biochemical assays --- p.100 / Chapter 3.2.2.4 --- Curative hepatoprotection test on Ptr --- p.101 / Chapter 3.2.2.5 --- Preventive hepatoprotection test on Ptr --- p.101 / Chapter 3.2.3 --- Preventive tests of Ptr sclerotium against AFB1-induced hepato- and geno-toxicity --- p.103 / Chapter 3.2.3.1 --- Dose-response of AFB1 on rat model --- p.103 / Chapter 3.2.3.2 --- Preventive test of Ptr against AFB1 --- p.103 / Chapter 3.2.3.3 --- Biochemical assays --- p.105 / Chapter 3.2.3.4 --- Histopathological examination --- p.105 / Chapter 3.2.4 --- Comet assay --- p.106 / Chapter 3.2.4.1 --- Reagent preparations --- p.106 / Chapter 3.2.4.2 --- Procedures --- p.107 / Chapter 3.2.5 --- Statistical analyses --- p.110 / Chapter 3.3 --- Results and Discussion --- p.111 / Chapter 3.3.1 --- Curative and preventive tests of Ptr sclerotium against CCl4-induced hepatotoxicity --- p.112 / Chapter 3.3.1.1 --- Dose-response of CCl4 on rat model --- p.112 / Chapter 3.3.1.2 --- Curative test of Ptr sclerotium against CCl4-induced hepatotoxicity --- p.116 / Chapter 3.3.1.3 --- Preventive test of Ptr sclerotium against CCl4-induced hepatotoxicity --- p.121 / Chapter 3.3.2 --- Preventive tests of Ptr sclerotium against AFB1-induced hepato- and geno-toxicity --- p.126 / Chapter 3.3.2.1 --- Dose-response of AFB1 on rat model --- p.126 / Chapter 3.3.2.2 --- Preventive test of Ptr sclerotium against AFB1-induced geno- and hepatotoxicity --- p.134 / Chapter 3.3.2.3 --- CHL versus 30% Ptr sclerotial diet --- p.137 / Chapter 3.3.3 --- A comparison of the hepatotoxicity of CC14 and AFB1 --- p.142 / Chapter 3.4 --- Summary --- p.147 / Chapter Chapter 4 --- Conclusions and future work --- p.148 / References --- p.151 / Related publication --- p.175

京都大学 / 新制・課程博士 / 博士(薬学) / 甲第23844号 / 薬博第851号 / 新制||薬||242(附属図書館) / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 山下 富義, 教授 石濱 泰, 教授 金子 周司 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Abstract Various genetic aetiologies — including mitochondrial diseases, chromosomal disorders, and other monogenic diseases — are involved in status epilepticus (SE), a common neurologic emergency occurring in children and adults that exhibits high rates of morbidity and mortality. The exact frequency of mitochondrial SE is currently undefined. Furthermore, patients with pathogenic variants of POLG1 encoding mitochondrial DNA polymerase gamma have an increased risk of acute liver failure (ALF) induced by the common antiepileptic drug, valproic acid (VPA), which is problematic due to these patients also often experiencing drug-resistant seizures. Overall, the role of liver transplantation (LT) in VPA-ALF due to mitochondrial disease has been controversial. In the present work, large retrospective cohort studies were conducted for two main purposes: (1) to determine the genetic aetiologies of SE among Finnish paediatric and adult patients by specifically focusing on the common mitochondrial genetic defects associated with an increased risk of SE and (2) to examine whether common POLG1 p.Q1236H and p.E1143G variants are connected to liver or pancreatic toxicity upon exposure to VPA monotherapy. This thesis also describes the characteristics of VPA-ALF associated with the pathogenic POLG1 variant p.W748S and the prognosis of LT in a retrospective case series. Mitochondrial diseases explained 4.5% of SE cases in the study cohort. Patients with mitochondrial SE suffered from refractory SE significantly more often than patients with other forms of genetic or non-genetic SE. Additionally, mortality rates were higher in patients with mitochondrial or chromosomal disorders compared with the other groups, reflecting the severity of the underlying condition and the higher frequency of refractory SE. POLG1 variants p.Q1236H and p.E1143G could not be identified as risk factors for VHT or pancreatic toxicity, implying that VPA treatment might be suitable for patients harbouring these variants when other pathogenic variants are absent. Finally, the homozygous status of the pathogenic POLG1 variant p.W748S and older age of the patient during the presentation of VPA-ALF seem to be associated with higher survival rates following LT, which should be considered in the management of VPA-ALF. / Tiivistelmä Useita perinnöllisiä syitä, kuten mitokondriotauteja, kromosomihäiriöitä ja muita geenimuutoksia on tunnistettu status epilepticuksen (SE) eli pitkittyneen epileptisen kohtauksen taustalla. SE on yleinen neurologinen hätätilanne, johon liittyy merkittävää oheissairastavuutta ja kuolleisuutta sekä lapsilla että aikuisilla. Mitokondriotauteihin liittyvän SE:n tarkkaa esiintyvyyttä ei tiedetä. Potilailla, joilla on patogeenisia variantteja mitokondrioiden DNA-polymeraasia koodaavassa tuman POLG1-geenissä, on todettu kohonnut riski yleisesti käytetyn epilepsialääkkeen valproaatin (VPA) aiheuttaman akuutin maksavaurion kehittymiselle. Tämä tekee lääkehoidon valinnasta ongelmallista, koska näillä potilailla on usein epilepsialääkkeille resistenttejä kohtauksia. Maksansiirron merkitys akuutin maksavaurion hoidossa mitokondriotauteja sairastavilla potilailla on ollut kiistanalainen. Tutkimuksen tavoitteena oli selvittää SE:n perinnöllisiä syitä suomalaisilla lapsi- ja aikuispotilailla retrospektiivisesti kerätyssä laajassa potilasaineistossa. Tutkimuksessa keskityttiin yleisimpiin mitokondriaalisiin perinnöllisiin muutoksiin, joiden on aiemmin todettu liittyvän SE:n lisääntyneeseen riskiin. Tutkimuksen toisena päätavoitteena oli selvittää väestössä yleisten POLG1-geenin muutosten eli varianttien p.Q1236H ja p.E1143G yhteyttä maksatoksisuuteen tai haimatoksisuuteen VPA-monoterapian aikana. Lisäksi tutkittiin VPA:n aiheuttaman maksavaurion kliinisiä erityispiirteitä patogeeniseen POLG1-varianttiin p.W748S liittyen sekä mutaatiostatuksen vaikutusta maksansiirron jälkeiseen ennusteeseen. Mitokondriotaudit selittivät 4,5 % SE-tapauksista tämän väitöskirjatyön potilasaineistossa ja näillä potilailla SE pitkittyi hoitoresistentiksi tai erittäin resistentiksi merkitsevästi muita potilasryhmiä useammin. Kuolleisuus oli suurin potilailla, joilla todettiin mitokondriotauti tai kromosomihäiriö, liittyen todennäköisimmin vakavaan taustasairauteen ja hoitoresistentin SE:n suurempaan esiintyvyyteen. Tutkittuja POLG1-variantteja p.Q1236H ja p.E1143G ei voitu tunnistaa maksa- tai haimatoksisuuden riskitekijöiksi, mikä tarkoittaa, että VPA-hoito voisi sopia näille potilaille, mikäli muita patogeenisiä variantteja ei todeta. Patogeenisen POLG1-variantin p.W748S homotsygoottisuus ja nuoruusikä tai varhainen aikuisikä maksavaurion ajankohtana ovat maksansiirron ennustetta parantavia tekijöitä, mikä tulisi ottaa huomioon hoitopäätöksiä tehtäessä.

Minimizing anti-cancer drug toxicity is a major challenge for the pharmaceutical industry. Toxicity is most frequently due to either the direct interaction of the drug on previously unidentified targets or its conversion to metabolites by drug metabolizing enzymes (e.g. CYP450 enzymes) that cause cellular, tissue or organ damage. Pharmacoproteomics is beginning to take a central role in studying changes in protein expression corresponding to drug administration, the results of which, inform about the mode of action, toxicity, and resistance in pre-clinical and clinical stages of drug development. The main aim of this research is to apply comparative proteomics studies on livers from male and female mice xenograft models treated with major anti-cancer drugs (5-flourouracil, paclitaxel, cisplatin, and doxorubicin) and CYP inducer, TCPOBOP, to investigate their effect on protein expression profiles (proteome). Within this thesis, an attention is paid to optimise a highly validated proteomics workflow for biomarker identification. Proteins were extracted from liver microsomes of mice treated in two separate sets; Set A – male (5-fluoruracil, doxorubicin, cisplatin and untreated) or Set B – female (5-fluoruracil, paclitaxel, TCPOBOP and untreated) using cryo-pulverization and sonication method. The extracts were digested with trypsin ii and the resulting peptides labelled with 4-plex iTRAQ reagents. The labelled peptides were subjected for separation in two-dimensions by iso-electric focusing (IEF) and RP-HPLC techniques before analysis by mass spectrometry and database searching for protein identification. Set A and Set B resulted in identification and quantification of 1146 and 1743 proteins, respectively. Moreover, Set A and Set B recovered 26 and 34 cytochrome P450 isoforms, respectively. The microsomal changes after drug treatments were quite similar. However, more changes were observed in the male set. Up-regulation of MUPs showed the greatest distinction in the protein expression patterns in the treated samples comparing to the untreated controls. In Set A, 5-fluoruracil and cisplatin increased the expression of three isoforms (MUP1, 2, and 6), whereas doxorubicin has increased the expression of four isoforms (MUP1, 2, 3, and 6). On the other side, only TCPOBOP in Set B has increased the expression of two isoforms (MUP1 and 6). Our findings showed that the expression of MUP, normally involved in binding and excretion of pheromones, have drug- and sex-specific differences. The mechanism and significance of MUP up-regulation are ambiguous. Therefore, the impact of each therapeutic agent on MUP and xenobiotic enzymes will be discussed.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第25223号 / 薬科博第185号 / 新制||薬科||21(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 山下 富義, 教授 小野 正博, 教授 寺田 智祐 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

by Wong Wing-yee, Felice. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 144-166). / Abstract also in Chinese. / Acknowledgements --- p.i / List of Abbreviations --- p.ii / Abstract --- p.iv / Abstract (Chinese Version) --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiv / List of Appendices --- p.xvi / Chapter Chapter I --- Literature Review / Chapter 1. --- Introduction --- p.1 / Chapter 2. --- Background of Cytochrome P450 --- p.3 / Chapter 2.1 --- Discovery --- p.3 / Chapter 2.2 --- Tissue Distribution --- p.3 / Chapter 2.3 --- Structure and Functions --- p.7 / Chapter 2.4 --- Nomenclature of the P450 Superfamily --- p.10 / Chapter 3. --- Cytochrome P450 2E1 (CYP2E1) --- p.11 / Chapter 3.1 --- Discovery --- p.11 / Chapter 3.2 --- Tissue Distribution --- p.12 / Chapter 3.3 --- Substrates and Inducers --- p.13 / Chapter 3.4 --- Toxicological Role of CYP2E1 --- p.15 / Chapter 4. --- CYP2E1-knockout Mouse Model --- p.17 / Chapter Chapter II --- Carbon Tetrachloride (CC14) Study / Chapter 1. --- Introduction --- p.19 / Chapter 1.1 --- General Properties and Usage of CC14 --- p.19 / Chapter 1.2 --- Toxicological Aspects of CC14 --- p.19 / Chapter 1.3 --- Mechanism of CCl4-induced Hepatotoxicity --- p.20 / Chapter 1.4 --- Role of CYP2E1 in CCl4-induced Hepatotoxicity --- p.23 / Chapter 1.5 --- Objectives of the Study --- p.27 / Chapter 2. --- Materials and Methods --- p.29 / Chapter 2.1 --- Chemicals and Materials --- p.29 / Chapter 2.2 --- Animals --- p.29 / Chapter 2.3 --- Acute CC14 Treatment --- p.29 / Chapter 2.4 --- Preparation of Microsomal Fractions --- p.30 / Chapter 2.5 --- Determination of Microsomal Protein Concentration --- p.31 / Chapter 2.6 --- Determination of Serum Aminotransferase Activities --- p.31 / Chapter 2.7 --- Liver Histology --- p.32 / Chapter 2.8 --- Hepatic Microsomal CYP2E1 Activity -p-nitrophenol Assay --- p.34 / Chapter 2.9 --- SDS-PAGE and Western Blot Analysis --- p.35 / Chapter 2.10 --- Detection of Lipid Peroxidation in vitro and in vivo --- p.35 / Chapter 2.10.1 --- In vitro Lipid Peroxidation - 2-Thiobarbituric Acid (TBA) assay --- p.35 / Chapter 2.10.2 --- In vivo Lipid Peroxidation - Microsomal Conjugated Dienes Detection --- p.36 / Chapter 2.11 --- Hepatic Lipid Fatty Acid Composition Analysis --- p.39 / Chapter 2.11.1 --- Lipid Extraction --- p.39 / Chapter 2.11.2 --- Thin Layer Chromatography --- p.39 / Chapter 2.11.3 --- Methylation --- p.40 / Chapter 2.11.4 --- Gas Chromatography --- p.40 / Chapter 2.12 --- Statistical Analysis --- p.41 / Chapter 3. --- Results --- p.42 / Chapter 3.1 --- "Mortality, Liver Weight and Liver Color" --- p.42 / Chapter 3.2 --- Hepatotoxicity --- p.42 / Chapter 3.2.1 --- Serum ALT and AST activities --- p.42 / Chapter 3.2.2 --- Liver Histology --- p.45 / Chapter 3.3 --- CYP2E1-catalysed PNP Activities and CYP2E1 Protein Levels --- p.49 / Chapter 3.3.1 --- CYP2El-catalyzed PNP Activities --- p.49 / Chapter 3.3.2 --- CYP2E1 Protein Levels --- p.52 / Chapter 3.4 --- Lipid Peroxidation --- p.52 / Chapter 3.4.1 --- In vitro Lipid Peroxidation --- p.52 / Chapter 3.4.2 --- In vivo Lipid Peroxidation --- p.54 / Chapter 3.5 --- Hepatic Lipid Fatty Acid Composition --- p.56 / Chapter 3.5.1 --- Fatty Acid Composition in Hepatic Phospholipid --- p.56 / Chapter 3.5.2 --- Fatty Acid Composition in Hepatic Microsomal Phospholipid --- p.59 / Chapter 3.5.3 --- Fatty Acid Composition in Hepatic Triglyceride --- p.61 / Chapter 4. --- Discussion --- p.63 / Chapter 4.1 --- CYP2E1 is Required in CCl4-mediated Hepatotoxicity --- p.63 / Chapter 4.2 --- CYP2E1 is Degraded following CC14 Exposure --- p.65 / Chapter 4.3 --- CYP2E1 is Required in CCl4-induced Lipid Peroxidation --- p.67 / Chapter 4.4 --- CYP2E1 is Required in CCl4-induced Hepatic Phospholipid Depletion --- p.70 / Chapter 4.5 --- CYP2E1 is Required in CCl4-induced Hepatic Triglyceride Accumulation --- p.72 / Chapter 5. --- Conclusion --- p.76 / Chapter Chapter III --- Chronic Ethanol Consumption Study / Chapter 1. --- Introduction --- p.77 / Chapter 1.1 --- Multiple Metabolic Pathways for Ethanol Metabolism --- p.77 / Chapter 1.2 --- Metabolism of Ethanol by the Microsomal Ethanol Oxidizing System --- p.79 / Chapter 1.3 --- Role of CYP2E1 in Ethanol Metabolism --- p.82 / Chapter 1.4 --- Role of CYP2E1 in Alcoholic Liver Disease and Associated Oxidative Stress --- p.84 / Chapter 1.5 --- Objectives of the Study --- p.89 / Chapter 2. --- Materials and Methods --- p.90 / Chapter 2.1 --- Chemicals and Materials --- p.90 / Chapter 2.2 --- Animals --- p.90 / Chapter 2.3 --- Chronic Ethanol Treatment --- p.90 / Chapter 2.3.1 --- Ethanol Diet Composition --- p.90 / Chapter 2.3.2 --- Ethanol Feeding --- p.90 / Chapter 2.4 --- Monitoring of Blood Ethanol Levels --- p.96 / Chapter 2.5 --- Preparation of Microsomal Fractions --- p.96 / Chapter 2.6 --- Determination of Microsomal Protein Concentration --- p.97 / Chapter 2.7 --- Determination of Serum Aminotransferase Activities --- p.98 / Chapter 2.8 --- Liver Histology --- p.98 / Chapter 2.9 --- SDS-PAGE and Western Blot Analysis --- p.99 / Chapter 2.10 --- Hepatic Fatty Acid Composition Analysis --- p.100 / Chapter 2.10.1 --- Lipid Extraction --- p.100 / Chapter 2.10.2 --- Thin Layer Chromatography --- p.101 / Chapter 2.10.3 --- Methylation --- p.101 / Chapter 2.10.4 --- Gas Chromatography --- p.102 / Chapter 2.11 --- Statistical Analysis --- p.103 / Chapter 3. --- Results --- p.104 / Chapter 3.1 --- Average Food Consumption --- p.104 / Chapter 3.2 --- Average Ethanol Consumption for Ethanol Liquid Diet Feeding Group --- p.104 / Chapter 3.3 --- Body Weight Gain --- p.104 / Chapter 3.4 --- Blood Ethanol Levels --- p.108 / Chapter 3.5 --- "Mortality, Liver Weight and Liver Color" --- p.108 / Chapter 3.6 --- Serum ALT and AST Activities --- p.110 / Chapter 3.7 --- Liver Histology --- p.114 / Chapter 3.8 --- Western Blot Analysis --- p.119 / Chapter 3.9 --- Hepatic Lipid Fatty Acid Composition --- p.119 / Chapter 3.9.1 --- Fatty Acid Composition in Hepatic Phospholipid --- p.119 / Chapter 3.9.2 --- Fatty Acid Composition in Hepatic Triglyceride --- p.123 / Chapter 4. --- Discussion --- p.126 / Chapter 4.1 --- Nutrients Displacement after Chronic Ethanol Consumption --- p.126 / Chapter 4.2 --- Varied Blood Ethanol Levels after Chronic Ethanol Consumption --- p.127 / Chapter 4.3 --- Increase in CYP2E1 Levels after Chronic Feeding of Ethanolin WT mice --- p.127 / Chapter 4.4 --- Lack of Evidence Indicating the Development of Ethanol- Induced Liver Injury --- p.129 / Chapter 4.4.1 --- No Elevations in Serum ALT and AST Activities --- p.129 / Chapter 4.4.2 --- Normal Liver Histology --- p.130 / Chapter 4.4.3 --- Lack of Triglyceride Accumulation --- p.131 / Chapter 4.4.4 --- Elevations in Hepatic PL --- p.132 / Chapter 4.5 --- Possible Reasons for the Absence of Liver Damage after Chronic Ethanol Consumption in our Mouse Model --- p.134 / Chapter 5. --- Conclusion --- p.137 / Chapter Chapter IV --- Concluding Remarks / Chapter 1. --- A Comparison between Acute CC14 Study and Chronic Ethanol Consumption Study --- p.139 / Chapter 1.1 --- Regulation of CYP2E1 Expression --- p.139 / Chapter 1.2 --- Free Radical Production Involved in CC14- and Chronic Ethanol Consumption-Mediated Liver Injury --- p.140 / Chapter 1.3 --- An Overall Comparison between CC14 study and Chronic Ethanol Consumption Study --- p.140 / Chapter 2. --- Future Studies --- p.142 / Chapter 2.1 --- Acute CC14 Study --- p.142 / Chapter 2.1.1 --- Calcium Homeostasis Studies --- p.142 / Chapter 2.1.2 --- Spin Trapping Studies --- p.142 / Chapter 2.2 --- Chronic Ethanol Study --- p.142 / Chapter 2.2.1 --- "Generation of a Heterozygous ""Ethanol-Sensitive"" Mouse Strain (SV/129/ter x C57BL/6)" --- p.143 / Chapter 3. --- Concluding Remarks --- p.143 / References --- p.144 / Appendix --- p.167

La cholestase intrahépatique représente environ 40% des lésions hépatiques induites par les médicaments et se caractérise par une accumulation intracellulaire des acides biliaires (AB). Les mécanismes impliqués sont encore mal connus et sa prédiction reste difficile. Le but de ce travail était de caractériser dans la cholestase d’origine médicamenteuse et de développer des méthodes de screening pour sa prédiction précoce, en utilisant la lignée humaine hépatique HepaRG et les hépatocytes humains. Tout d’abord, nous avons démontré que la motilité des canalicules biliaires (CB) est indispensable à la clairance des AB et requiert une alternance de phosphorylation/déphosphorylation de la chaine légère de la myosine (MLC), contrôlé par la voie Rho-kinase/Myosin Light Chain Kinase (ROCK/MLCK). Nous avons ensuite montré pour la première fois que les médicaments cholestatiques altèrent la voie ROCK/MLCK/MLC et la dynamique des CB. En utilisant la famille des antibiotiques résistant à la pénicillinase, dont fait partie la flucloxacilline, responsable de nombreux cas de cholestase, nous avons observé que la dérégulation de ROCK pouvait se faire par activation de HSP27, associée aux voies de signalisation PKC/P38 et PI3K/AKT. Enfin, nous avons montré une capacité variable des médicaments cholestatiques à moduler les profils des AB. En effet, les médicaments cholestatiques majeurs induisent une accumulation préférentielle des AB hydrophobes toxiques, in vitro, dans les premières 24h, qui résulte d’une inhibition de leur amidation. Au total, l’ensemble du travail a permis de progresser dans la compréhension des mécanismes impliqués dans la cholestase d’origine médicamenteuse et de mettre en évidence de nouveaux biomarqueurs utiles pour sa prédiction. / Intrahepatic cholestasis represents around 40% of drug-induced liver injuries and is characterized by intracellular accumulation of bile acids (BA); mechanisms involved and its accurate prediction remains challenging. The aim of the current work was to characterize the mechanisms involved in drug-induced cholestasis and to develop screening methods for its early prediction, using human differentiated HepaRG and primary human hepatocytes. First, we demonstrated that bile canaliculi (BC) motility is essential for BA clearance and requires alternating phosphorylation/dephosphorylation of myosin light chain (MLC) that is controlled by the Rho-kinase/Myosin Light Chain Kinase (ROCK/MLCK) signaling pathway. Then, we showed, for the first time that cholestatic drugs could alter the ROCK/MLCK/MLC pathway and BC dynamics. Using the penicillinase-resistant antibiotics family, including flucloxacillin that is responsible for many cases of cholestasis, we found that deregulation of ROCK could be modulated by HSP27, associated with PKC/P38 and PI3K/AKT signaling pathways. Finally, we evidenced variable potency of cholestatic drugs to modulate BA profiles. Indeed, the well-known cholestatic drugs induced a preferential accumulation of unconjugated toxic hydrophobic BA in vitro within the first 24h that resulted from inhibition of their amidation. Altogether, these data bring new information on the understanding of the mechanisms involved in drug-induced cholestasis and highlight new morphological and molecular predictive biomarkers of drug-induced cholestasis.

La cholestase est l'une des manifestations les plus courantes des lésions induitespar des médicaments. Dans 40% des cas elle n’est pas prévisible; une meilleure prédictibilité représente donc un défi majeur. Tout d'abord, nous avons démontré que les cellules hépatiques humaines HepaRG différenciées sont un modèle approprié pour étudier la cholestase induite par les médicaments en comparant la localisation et l’activité des transporteurs d'influx et d'efflux avec les hépatocytes humains primaires. Tous les transporteurs d'efflux et d’influx testés ont été correctement localisés au niveau des membranes canaliculaire (BSEP, MRP2, MDR1 et MDR3) et basolatéral (NTCP, MRP3) et sont fonctionnels. En outre, ces cellules expriment également les enzymes qui métabolisent les acides biliaires (ABs) et ont la capacité de les synthétiser et de les conjuguer avec la taurine, la glycine et le sulfate, à un taux similaire à celui des hépatocytes primaires. Des changements ont été observés sur la répartition des ABs totaux après traitements de cellules HepaRG par un médicament cholestatique, la cyclosporine A (CsA), de manière concentration- dépendante. L'inhibition de l'efflux et de l'influx de taurocholate a été observée après 15 min et 1 h respectivement. Ces premiers effets ont été associés à la dérégulation de la voie des cPKC et l'induction d’un stress du réticulum endoplasmique puis d’un stress oxydant. Nous avons également montré pour la première fois une accumulation intracellulaire d’ABs endogènes avec un médicament cholestatique in vitro. En outre, notre travail apporte des preuves que la motilité des canalicules biliaires (BC) est indispensable à la clairance des ABs. La voie ROCK et le complexe actomyosine sont fortement impliqués. Nous avons fourni la première démonstration que la voie ROCK et les dynamiques des BC sont des cibles majeures des composés cholestatiques. Nos données devraient contribuer à l'élaboration de méthodes de screening pour la prédiction précoce des effets secondaires induits par les médicaments cholestatiques. / Cholestasis is one of the most common manifestations of drug-induced liver injury (DILI). Since up to now it is unpredictable in 40% of all cases its accurate prediction represents a major challenge. First, we validated that differentiated HepaRG human liver cells are a suitable in vitro model to study drug-induced cholestasis, by comparing localization of influx and efflux transporters and their functional activity in these cells and primary human hepatocytes. All tested influx and efflux transporters were correctly localized to canalicular (BSEP, MRP2, MDR1, and MDR3) or basolateral (NTCP, MRP3) membrane domains and were functional. In addition, the HepaRG cell line also exhibits bile acids (BAs) metabolizing enzymes and has the capacity to synthesize BAs and to further amidate these BAs with taurine and glycine as well as sulfate, at a rate similar to that of primary hepatocytes. Concentration- dependent changes were observed in total BAs disposition after treatment of HepaRG cells by the cholestatic drug cyclosporine A (CsA). Inhibition of efflux and uptake of taurocholate was evidenced as early as 15 min and 1 h respectively. These early effects were associated with deregulation of cPKC pathway and induction of endoplasmic reticulum stress that preceded generation of oxidative stress. We also showed for the first time intracellular accumulation of endogenous BAs by a cholestatic drug in vitro. In addition, our work brings evidences that motility of bile canaliculi (BC) is essential for BAs clearance where ROCK pathway and actomyosin complex are highly implicated. We provided the first demonstration that ROCK pathway and BC dynamics are major targets of cholestatic compounds. Our data should help in the development of screening methods for early prediction of drug-induced cholestatic side effects.