The cytochromes P450 (P450s) are important enzymes involved in metabolic pathways, which use exogenous and endogenous substances as their substrate for various enzymatic reactions. These enzymes can also use precarcinogens as their substrate and activate them into carcinogens, which leads to a cancer development. If the P450s are induced, the cancer risk increases. Some chemopreventive compounds may induce the P450s and thus be harmful to the human body. Therefore it is necessary to pay enough attention to a study of the mechanism of action of P450s and the influence of the chemopreventive compounds on the activity of cytochromes P450. mRNA expression of most of the P450s isoforms is detected in a number of healthy (nontransformed) tissues, viz. liver, brain, heart, colon, kidney or placenta. Nevertheless there are a few P450s isoforms which mRNAs are expressed at relatively low levels in the nontransformed tissues, whereas the expression in the transformed tissues is significantly higher. One of these P450s is CYP2W1, which can be used as a prognostic marker for colorectal cancer - therefore it is useful to be able to detect a presence of this enzyme in various tissues. A detection of P450s can be accomplished by using a method Western blot. In this method, the immunodetection is achieved by using...

by Cheng Fat-chi. / Bibliography: leaves 85-86 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1984

Chan Pat Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 153-177). / Acknowledgments --- p.i / Presentations Derived from the Present thesis Work --- p.ii / Abstract --- p.iii / Abbreviations --- p.v / Abbreviation Table for Amino Acids --- p.viii / List of Figures --- p.ix / List of Tables --- p.xi / Contents --- p.xii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Environmental Pollutants --- p.1 / Chapter 1.2 --- Pollutant Inducible Genes (PIGs) --- p.1 / Chapter 1.2.1 --- Classification of PIGS --- p.2 / Chapter 1.2.1.1 --- Drug Metabolizing Enzymes/Proteins --- p.2 / Chapter 1.2.1.2 --- Stress Proteins --- p.5 / Chapter 1.2.1.3 --- Antioxidant Enzymes --- p.6 / Chapter 1.2.1.4 --- "Hormones, Growth Factors and Their Receptors" --- p.6 / Chapter 1.2.1.5 --- Enzymes/Proteins Involved in Bioenergetics --- p.6 / Chapter 1.2.2 --- PIGs as a Field of Study --- p.8 / Chapter 1.2.2.1 --- Study of the Mechanism of Detoxification and Toxication --- p.8 / Chapter 1.2.2.2 --- Biomarker Study --- p.9 / Chapter 1.2.2.3 --- Study of Regulation of Gene Expression --- p.11 / Chapter 1.2.2.4 --- Study of Evolution --- p.12 / Chapter 1.3 --- Aims and Rational of the Present Study --- p.12 / Chapter 2 --- General Methodology --- p.15 / Chapter 2.1 --- Materials --- p.15 / Chapter 2.2.1 --- Reagents --- p.15 / Chapter 2.1.1.1 --- Preparation of Plasmid DNA --- p.15 / Chapter 2.1.1.2 --- Preparation of Genomic DNA --- p.15 / Chapter 2.1.1.3 --- Purification of Total RNA --- p.16 / Chapter 2.1.1.4 --- Restriction Enzyme Digestion --- p.16 / Chapter 2.1.1.5 --- Capillary Blotting of DNA (Southern Blotting) --- p.16 / Chapter 2.1.1.6 --- Capillary Blotting of Total RNA (Northern Blotting) --- p.17 / Chapter 2.1.1.7 --- Hybridization --- p.17 / Chapter 2.1.1.8 --- Library Screening --- p.18 / Chapter 2.1.1.9 --- Polymerase Chain Reaction --- p.18 / Chapter 2.1.1.10 --- Transformation of E. coli Competent Cells --- p.19 / Chapter 2.1.1.11 --- Nucleotide Sequence Determination --- p.19 / Chapter 2.1.2 --- List of Primers --- p.20 / Chapter 2.1.2.1 --- Primers used for Nucleotide Sequence Determination --- p.20 / Chapter 2.1.2.2 --- Primer Used for First Strand cDNA Synthesis --- p.20 / Chapter 2.1.2.3 --- Primers for Amplifying Actin cDNA Fragment --- p.20 / Chapter 2.1.2.4 --- Common Carp MT Specific Primers --- p.20 / Chapter 2.1.2.5 --- Teleost CYP1A Specific Primers --- p.21 / Chapter 2.1.2.6 --- Common Carp CYP1A Specific Primers --- p.21 / Chapter 2.1.2.7 --- Primers and Cassettes for the Cloning of5' Upstream Regions of MT Genes --- p.21 / Chapter 2.1.3 --- Accession Numbers of Selected P450 and MT Nucleotide and Amino Acid Sequences in the Genebank --- p.21 / Chapter 2.1.3.1 --- MTs of Different Teleost Species --- p.21 / Chapter 2.1.3.2 --- MTs of Other Vertebrate Species' --- p.22 / Chapter 2.1.3.3 --- P450s of Different Teleost Species --- p.22 / Chapter 2.1.3.4 --- CYP1s of Different Mammalian Species --- p.22 / Chapter 2.2 --- Methods --- p.23 / Chapter 2.2.1 --- Preparation of Plasmid --- p.23 / Chapter 2.2.2 --- Preparation of Genomic DNA --- p.23 / Chapter 2.2.3 --- Purification of Total RNA --- p.24 / Chapter 2.2.4 --- Restriction Enzyme Digestion --- p.25 / Chapter 2.2.5 --- Capillary Blotting of DNA (Southern Blotting) --- p.25 / Chapter 2.2.5.1 --- Semi-dry Capillary Blotting --- p.25 / Chapter 2.2.5.2 --- Alkaline Transfer --- p.25 / Chapter 2.2.5.3 --- Transfer of Digested Genomic DNA on to Nylon Membrane --- p.26 / Chapter 2.2.6 --- Capillary Blotting of Total RNA (Northern Blotting) --- p.26 / Chapter 2.2.7 --- Radioactive Labeling of Nucleic Acid Probes --- p.26 / Chapter 2.2.8 --- Hybridization --- p.27 / Chapter 2.2.9 --- Library Screening --- p.27 / Chapter 2.2.9.1 --- Construction of Liver cDNA Library of Adult Common Carp --- p.27 / Chapter 2.2.9.2 --- Preparation of Plating Cells --- p.27 / Chapter 2.2.9.3 --- Phage Tittering --- p.27 / Chapter 2.2.9.4 --- Primary Screening --- p.28 / Chapter 2.2.9.5 --- Secondary Screening / Chapter 2.2.9.6 --- Conversion of Phage DNA to Phagemid by invivo Excision --- p.28 / Chapter 2.2.10 --- First Strand cDNA Synthesis --- p.29 / Chapter 2.2.11 --- Polymerase Chain Reaction --- p.29 / Chapter 2.2.12 --- Ligation of DNA with Linearized Plasmid --- p.30 / Chapter 2.2.13 --- Transformation of E. coli Competent Cell --- p.30 / Chapter 2.2.14 --- Nucleotide Sequence Determination --- p.31 / Chapter 2.2.15 --- Densitometric Analysis --- p.31 / Chapter 3 --- "Cloning of Common Carp MT cDNA and Gene, and Induction of MT mRNA Expression" --- p.32 / Chapter 3.1 --- Introduction --- p.32 / Chapter 3.1.1 --- Metals in Biological System --- p.32 / Chapter 3.1.2 --- Metallothionein --- p.33 / Chapter 3.1.2.1 --- Functions of MT --- p.26 / Chapter 3.1.2.2 --- Regulation of MT Expression --- p.39 / Chapter 3.1.3 --- Fish MTs --- p.44 / Chapter 3.1.3.1 --- Detection of MT in Teleost --- p.46 / Chapter 3.1.3.2 --- MT Studies in Common Carp --- p.47 / Chapter 3.1.4 --- Specific Aims of This Chapter --- p.49 / Chapter 3.2 --- Strategies --- p.50 / Chapter 3.3 --- Specific Methods --- p.50 / Chapter 3.3.1 --- Cloning of MT cDNAs of Common Carp --- p.50 / Chapter 3.3.2 --- Analysis of MT cDNA Sequences --- p.51 / Chapter 3.3.3 --- Southern Blot Analysis of Common Carp Genomic DNA --- p.52 / Chapter 3.3.4 --- Amplification of MT Gene Fragments Using PCR --- p.52 / Chapter 3.3.5 --- Amplification of the 5' Upstream Regions of MT Genes Using PCR --- p.52 / Chapter 3.3.6 --- Endogenous MT mRNA Expression of Juvenile and Adult Common Carp --- p.54 / Chapter 3.3.7 --- Induction of MT mRNA of Juvenile Common Carp Injected with Cadmium --- p.55 / Chapter 3.4 --- Results --- p.56 / Chapter 3.4.1 --- Cloning of Common Carp MT cDNAs --- p.56 / Chapter 3.4.2 --- Analysis of the MT cDNA Sequences --- p.57 / Chapter 3.4.3 --- Southern Blot Analysis of the Common Carp Genomic DNA --- p.59 / Chapter 3.4.4 --- Amplification of the MT Gene Fragments of Common Carp Using PCR --- p.62 / Chapter 3.4.5 --- Amplification of the 5' Upstream Regions of MT Genes using PCR --- p.65 / Chapter 3.4.6 --- Endogenous MT mRNA Expression of Juvenile and Adult Common Carp --- p.67 / Chapter 3.4.7 --- Induction of MT mRNA of Juvenile Common Carp Injected with Cadmium --- p.68 / Chapter 3.5 --- Discussion --- p.72 / Chapter 3.5.1 --- MT cDNAs of Common Carp --- p.72 / Chapter 3.5.1.1 --- Coding Region --- p.72 / Chapter 3.5.1.2 --- The 3' Untranslated Region --- p.75 / Chapter 3.5.1.3 --- The 5' Untranslated Region --- p.76 / Chapter 3.5.2 --- MT Genes of Common Carp --- p.77 / Chapter 3.5.3 --- MT mRNA Expression of Common Carp --- p.82 / Chapter 3.5.4 --- Normalization of the Signals of Northern Blot Analysis --- p.85 / Chapter 3.5.5 --- Common Carp MT mRNA as Biomarker of Heavy Metal Exposure? --- p.87 / Chapter 3.6 --- Conclusion --- p.89 / Chapter 4 --- Cloning of Common Carp CYP1A cDNAs and Induction of CYP1A mRNA Expression --- p.90 / Chapter 4.1 --- Introduction --- p.90 / Chapter 4.1.1 --- Cytochrome P450s --- p.90 / Chapter 4.1.2 --- Cytochrome P450 1 (CYP1) --- p.93 / Chapter 4.1.3 --- AhR Mediated CYP1A1 Gene Induction --- p.94 / Chapter 4.1.3.1 --- Anthropogenic Sources of AhR Ligands --- p.95 / Chapter 4.1.3.2 --- Natural Sources of AhR Ligands --- p.97 / Chapter 4.1.3.3 --- Potency of Inducibility --- p.97 / Chapter 4.1.3.4 --- Induction of CYP1A1 Gene Transcription by AhR --- p.98 / Chapter 4.1.3.5 --- Non-AhR Mediated CYP1A1 Gene Transcription? --- p.105 / Chapter 4.1.4 --- CYP1A Studies in Teleost Species --- p.107 / Chapter 4.1.4.1 --- Regulation of CYP1A in Teleost --- p.109 / Chapter 4.1.4.2 --- Detection of CYP1A in Teleost --- p.111 / Chapter 4.1.4.3 --- CYP1A Studies of Common Carp --- p.113 / Chapter 4.1.5 --- Specific Aims of This Chapter --- p.114 / Chapter 4.2 --- Strategies --- p.115 / Chapter 4.3 --- Specific Methods --- p.119 / Chapter 4.3.1 --- RT-PCR of CYP1A cDNAs of Common Carp --- p.119 / Chapter 4.3.2 --- Determination of the Nucleotide Sequences of the CYP1A cDNAs of Common Carp --- p.119 / Chapter 4.3.3 --- Library Screening --- p.119 / Chapter 4.3.4 --- Analysis of the CYP1A Genes of Common Carp --- p.121 / Chapter 4.3.5 --- Induction of CYP1A mRNA of Common Carp Injected with 3-MC --- p.122 / Chapter 4.4 --- Results --- p.123 / Chapter 4.4.1 --- RT-PCR of CYP1A cDNAs of Common Carp --- p.123 / Chapter 4.4.2 --- Determination of the Nucleotide Sequences of the CYP1A cDNAs of Common Carp --- p.124 / Chapter 4.4.3 --- Library Screening --- p.124 / Chapter 4.4.4 --- Analysis of the CYP1A Genes of Common Carp --- p.128 / Chapter 4.4.5 --- Induction of CYP1A mRNA of Common Carp Injected with 3-MC --- p.131 / Chapter 4.5 --- Discussion --- p.134 / Chapter 4.5.1 --- On the Use of Rainbow Trout CYP1A1 cDNA Probe --- p.134 / Chapter 4.5.2 --- CYP1A cDNAs of Common Carp --- p.134 / Chapter 4.5.3 --- CYP1A Genes of Common Carp --- p.138 / Chapter 4.5.4 --- CYP1A Expression in Uninduced and Induced Tissues --- p.142 / Chapter 4.5.5 --- The Use of CYP1A cDNAs As Biomarkers --- p.146 / Chapter 4.6 --- Conclusion --- p.148 / Chapter 5 --- General Conclusion --- p.149 / Chapter 6 --- References --- p.153

Cu+-ATPases are transmembrane enzymes that couple the efflux of cytoplasmic Cu+ to the hydrolysis of ATP. It is well established that Cu+-ATPases control cytoplasmic Cu+ levels. However, bacterial genomes, particularly those of symbiotic/pathogenic organisms, contain multiple copies of genes encoding Cu+-ATPases, challenging the idea of a singular role for these enzymes. Our lab has demonstrated that one of the two Cu+-ATPases in Pseudomonas aeruginosa, a FixI-type ATPase, has an alternative role, most likely Cu+ loading of cytochrome c oxidase (Cox). To further study alternative roles of Cu+-ATPases, we study the symbiont Sinorhizobium meliloti. Rhizobia are soil-dwelling bacteria that interact with legumes, forming plant root nodules that actively fix N2. The S. meliloti genome contains five Cu+-ATPases, two of which are FixI-type. Both of these enzymes, termed FixI1 and FixI2, are downstream of Cox operons. We hypothesized that the presence of multiple FixI-type ATPases was not an example of redundancy, but rather is an evolutionary adaptation that allows rhizobia to survive under the wide variety of adverse conditions faced during early infection and establishment of symbiosis. Towards this goal, this work focused on examining the effects of mutation of each ATPase on both free-living bacteria and on the ability of rhizobia to establish an effective symbiosis with its host legume. Each of these mutants presents a different phenotype at varying points of the nodulation process, and only the fixI2 mutation produces a respiratory-deficient phenotype during aerobic growth. These results are consistent with our hypothesis that the two proteins have non-redundant physiological functions. Understanding the factors that contribute to an effective symbiosis is beneficial, since N2 fixation in legumes is important to both agriculture and industry.

Yung Cheuk Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 89-113). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iii / Acknowledgement --- p.v / Table of content --- p.vii / List of figures --- p.xi / Symbols and abbreviations --- p.xiii / Scientific names --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Structure of ovarian follicles --- p.2 / Chapter 1.2 --- Steroidogenesis in the ovary --- p.3 / Chapter 1.2.1 --- Two-cell-type model --- p.5 / Chapter 1.2.2 --- Steroidogenic shift --- p.8 / Chapter 1.3 --- Aromatase --- p.8 / Chapter 1.3.1 --- Structure --- p.8 / Chapter 1.3.2 --- Function --- p.9 / Chapter 1.3.3 --- Mechanism of aromatase action --- p.11 / Chapter 1.3.4 --- Expression --- p.13 / Chapter 1.3.5 --- Regulation --- p.14 / Chapter 1.3.5.1 --- Gonadotropins --- p.15 / Chapter 1.3.5.2 --- Insulin-like growth factor-I --- p.17 / Chapter 1.3.5.3 --- Activin --- p.19 / Chapter 1.4 --- Objectives of the present study --- p.23 / Chapter Chapter 2 --- Expression profiles of the ovarian aromatase in the zebrafish --- p.25 / Chapter 2.1 --- Introduction --- p.25 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Animals --- p.27 / Chapter 2.2.2 --- Total RNA extraction from intact ovaries and ovarian follicles --- p.27 / Chapter 2.2.3 --- Validation of semi-quantitative RT-PCR assays for aromatase and GAPDH --- p.28 / Chapter 2.2.4 --- Data analysis --- p.29 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- Validation of the semi-quantitative RT-PCR assays for aromatase and GAPDH --- p.30 / Chapter 2.3.2 --- Developmental expression profile of aromatase in the whole ovary during sexual maturation --- p.32 / Chapter 2.3.3 --- Stage-dependent expression of aromatase in the ovarian follicles of mature gravid zebrafish --- p.35 / Chapter 2.4 --- Discussion --- p.37 / Chapter Chapter 3 --- Regulation of aromatase expression in vitro --- p.42 / Chapter 3.1 --- Introduction --- p.42 / Chapter 3.2 --- Materials and Methods --- p.45 / Chapter 3.2.1 --- Animals --- p.45 / Chapter 3.2.2 --- Chemicals and hormones --- p.45 / Chapter 3.2.3 --- Preparation of goldfish pituitary extract --- p.45 / Chapter 3.2.4 --- Primary follicle cell culture --- p.46 / Chapter 3.2.5 --- Preparation of freshly isolated mid-vitellogenic follicles --- p.46 / Chapter 3.2.6 --- Preparation of ovarian fragments --- p.47 / Chapter 3.2.7 --- "Total RNA extraction from cultured follicle cells, ovarian follicles and ovarian fragments" --- p.47 / Chapter 3.2.8 --- Validation of semi-quantitative RT-PCR assays --- p.48 / Chapter 3.2.9 --- Data analysis --- p.48 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- Validation of the semi-quantitative RT-PCR assays for aromatase and GAPDH --- p.49 / Chapter 3.3.2 --- Expression of aromatase in the zebrafish primary follicle cell culture system --- p.52 / Chapter 3.3.3 --- Gonadotropin regulation of aromatase expression in the zebrafish ovarian fragments and freshly isolated intact follicles --- p.54 / Chapter 3.3.4 --- Effects of db-cAMP and forskolin on aromatase expression in cultured zebrafish follicle cells --- p.59 / Chapter 3.3.5 --- Involvement of protein kinase A (PKA) in the regulation of aromatase expression by db-cAMP in cultured zebrafish follicle cells --- p.64 / Chapter 3.3.6 --- Effects of insulin-like growth factor I (IGF-I) on aromatase expression in zebrafish ovarian fragments --- p.66 / Chapter 3.3.7 --- Effects of activin on aromatase expression in zebrafish ovarian fragments --- p.68 / Chapter 3.4 --- Discussion --- p.71 / Chapter Chapter 4 --- General Discussion --- p.78 / Chapter 4.1 --- Expression profiling of aromatase in the zebrafish ovarian and follicle development --- p.81 / Chapter 4.2 --- Mechanisms for the dynamic expression of aromatase --- p.84 / Chapter 4.3 --- Contribution of the present study --- p.87 / Chapter 4.3 --- Future prospects --- p.88 / References --- p.89

The cytochrome P450 (CYP) superfamily of enzymes catalyses the oxidative metabolism of lipophilic xenobiotics such as drugs and environmental chemicals, and also plays an essential role in the biosynthesis and metabolism of endogenous compounds such as cholesterol and bile acids, vitamins, steroids, arachidonic acid and eicosanoids. Cytochrome P450 2J2 (CYP2J2) is a recently identified member of the human CYP protein family that is highly expressed in the heart, vasculature, liver and other tissues. CYP2J2 metabolises arachidonic acid (AA) into epoxyeicosatrienoic acids (EETs), which have a number of potent biological activities including cytoprotective, vasodilatory and anti-inflammatory effects. Given its widespread tissue distribution and the biological actions of EETs, CYP2J2 is likely to play an important role in cellular physiology, and altered expression of CYP2J2 may have pathophysiological consequences. Indeed, recent literature studies have indicated that CYP2J2 protein levels are decreased in vascular endothelial cells exposed to hypoxia and reoxygenation, and that maintenance of CYP2J2 expression enhances cell survival. Thus, CYP2J2 expression may be impaired in diseases or conditions associated with decreased oxygen availability, such as ischaemic heart disease, stroke and atherosclerosis, and this may contribute to their pathogenic consequences. Despite its likely importance in human physiology and pathophysiology, very little is known about the regulation of CYP2J2 gene expression. The aim of this study was to investigate the molecular mechanisms that control expression of the CYP2J2 gene. In particular, this study was designed to identify factors that regulate the expression of the CYP2J2 gene in the liver-derived HepG2 cell line during normoxia and hypoxia. A 2.4 kb fragment of the 5???-flanking region of the CYP2J2 gene (corresponding to nucleotides -2341 to +98, relative to the translation start site) was isolated from a human genomic library. Automated searching of the upstream regulatory region of CYP2J2 identified several putative binding sites for the transcription factor activator protein-1 (AP-1). Because AP-1 activity is altered in hypoxia, the possibility that AP-1 may participate in the regulation of CYP2J2 expression in hypoxia was explored. Cell culture studies examined the relationship between the expression of CYP2J2, and the AP-1 genes c-fos and c-jun, in HepG2 cells cultured in normoxia and hypoxia. Down-regulation of CYP2J2 mRNA and protein in hypoxic HepG2 cells was associated with the pronounced up-regulation of c-Fos protein from an undetectable level in normoxic cells; c-Jun protein levels were readily detectable in normoxia, and were also increased in hypoxia. Transient transfection studies revealed distinct effects of Fos and Jun proteins on CYP2J2 promoter activity. While the CYP2J2 promoter was strongly activated by c-Jun, c-Fos was inactive, and also abolished gene transactivation elicited by c-Jun. These results suggest that the constitutively expressed c-Jun is important in the maintenance of CYP2J2 expression in normoxic cells. The up-regulation of c-Fos in hypoxia stimulates the formation of c-Fos/c-Jun heterodimers, which do not support CYP2J2 transcription, leading to gene down-regulation. Experiments with CYP2J2 promoter deletion constructs revealed that the region between -152 to -50 bp relative to the translation start site was crucial for activation of CYP2J2 by c-Jun. Electrophoretic mobility shift assays (EMSAs) and transfection studies identified two distinct elements within this region that were involved in c-Jun-dependent transactivation: an AP-1-like element at -56 to -63 bp, and an atypical AP-1 element at -105 to -95 bp. c-Jun homodimers interacted specifically with both elements. Separate mutagenesis of either element significantly impaired c-Jun-dependent transactivation of CYP2J2, while mutagenesis of both elements eliminated c-Jun-responsiveness. EMSAs established that c-Jun, but not c-Fos, interacted with both elements in normoxic HepG2 cells. Furthermore, mutagenesis of either c-Jun-response element significantly decreased the basal transcriptional activity of the CYP2J2 promoter in HepG2 cells, while mutagenesis of both elements almost completely suppressed basal promoter activity. These findings indicate a pivotal role for c-Jun in the maintenance of CYP2J2 expression in normoxic cells. Transfection studies indicated that c-Fos suppresses c-Jun-dependent activation of CYP2J2 at both the -56/-63 bp and -105/-95 bp c-Jun-response elements. However, c-Fos-dependent inhibition appears to be mediated by distinct mechanisms at these two regulatory elements. While both elements interacted with c-Jun homodimers, only the -105/-95 bp element was able to interact with c-Fos/c-Jun heterodimers. Thus, the up-regulation of c-Fos in hypoxia, and the shift from c-Jun homodimers to c-Fos/c-Jun heterodimers, directly decreased c-Jun binding and transactivation at the -56/-63 bp element. In contrast, up-regulation of c-Fos in hypoxia altered the composition of proteins bound at the -105/-95 bp element from c-Jun to c-Fos/c-Jun. Inhibition of promoter activity occurs because c-Fos/c-Jun heterodimers can occupy, but not transactivate, the CYP2J2 promoter via the -105/-95 bp element. In summary, this thesis provides novel information on the molecular mechanisms that control the differential expression of the human CYP2J2 gene in normoxia and hypoxia. In particular, this study has established that the AP-1 proteins c-Jun and c-Fos play a crucial role in modulating the transcriptional activation of the CYP2J2 promoter in response to cellular stress. Binding of c-Jun to two distinct c-Jun-response elements within the CYP2J2 proximal promoter induces transcriptional activation of the CYP2J2 gene and is essential for maintenance of CYP2J2 expression in normoxic cells. The up-regulation of c-Fos in hypoxia promotes the formation of c-Fos/c-Jun heterodimers, which inhibit transcriptional activation of the CYP2J2 promoter by c-Jun, thus contributing to decreased CYP2J2 expression in hypoxia. Impaired expression of CYP2J2 may contribute to cellular injury in diseases such as atherosclerosis and stroke, and a greater understanding of the mechanisms responsible for mediating altered CYP2J2 expression may eventually lead to therapeutic strategies that manipulate the expression of this important human gene.

Master of Philosophy / Clozapine is a drug of choice for treatment of refractory schizophrenia, which is primarily metabolized by Cytochrome P450 1A2 (CYP1A2). Norclozapine is its main metabolite. There are reports of wide ranging gastrointestinal side effects associated with clozapine therapy, that result in concomitant administration of proton pump inhibitors to treat acid-related disorders. Omeprazole is an established CYP1A2 inducer, while an in vitro study has shown that rabeprazole is much less potent in this regard. There is no available information about the impact of rabeprazole on CYP1A2 activity in patients. Firstly, this information is essential when prescriptions are changed from omeprazole to rabeprazole to reduce medication costs. Therefore, the aim of this study was to compare the effects of rabeprazole and omeprazole on CYP1A2-mediated clearance (CL/F) of clozapine. Secondly, the effective dosage of clozapine varies widely among patients, making it necessary to individualize drug therapy with clozapine. The reason for dosage variation could be due to the influence of patient-related variables on clozapine plasma concentrations. Therefore, another aim of this study was to investigate the relationship between patient variables, such as age, gender, cigarette smoke, weight and body mass index and clozapine clearance (CL/F). A cross-over study design was used for this study. Twenty patients from Macquarie hospital who were receiving clozapine and rabeprazole (with no other interacting medications) were recruited in this study. Blood samples were taken at 30 min, 1 hr, 2 hr and 12 hr after a dose of clozapine. Rabeprazole was then replaced with omeprazole. After at least 1 month blood samples were again collected at the above corresponding intervals after clozapine. The plasma concentrations of clozapine and norclozapine were determined by high performance liquid chromatography. Abbottbase Pharmacokinetic Systems Software, which utilizes Bayesian forecasting, was used to estimate pharmacokinetic parameters of clozapine. The ratio of plasma norclozapine/clozapine concentrations at trough level was used to reflect CYP1A2 activity. No difference was observed in clozapine clearance (CL/F) and CYP1A2 activity during concurrent therapy with either rabeprazole or omeprazole. According to some studies CYP1A2 induction by omeprazole is dose dependent. Furthermore, since rabeprazole is a weak CYP1A2 inducer in vitro, we conclude that omeprazole and rabeprazole may not induce CYP1A2 activity when used at conventional therapeutic dosage (<40 mg/day). Hence, replacement of omeprazole with rabeprazole at conventional therapeutic dosages (20 or 40 mg daily) offers no advantages in the management of patients with schizophrenia on clozapine and no dose adjustment is required. Consistent with previous studies, clozapine concentrations were found to be significantly lower in cigarette smokers due to CYP1A2 induction. No relationship was found between age, gender, or weight and clozapine clearance (CL/F). However, body mass index showed a significant negative correlation with clozapine clearance (CL/F). Since weight gain and lipid accumulation are common side effects of clozapine they may be associated with a reduction of CYP1A2 activity and clozapine clearance (CL/F). Moreover, high lipoprotein levels may decrease the unbound fraction of clozapine and decrease the availability of clozapine for oxidation by cytochrome P450 enzymes. Therefore, it is concluded that omeprazole and rabeprazole may not induce CYP1A2 activity when used at conventional therapeutic dosage (<40mg/day). Hence, replacement of omeprazole with rabeprazole does not require the dose of clozapine to be adjusted. Moreover, the negative correlation between clozapine clearance (CL/F) and BMI is informative. Further studies are now required to clarify the relationship between BMI, lipoprotein levels and clozapine clearance in patients with schizophrenia.

Includes bibliography / 102 leaves, [19] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1977

The cytochrome P450 (CYP) superfamily of enzymes catalyses the oxidative metabolism of lipophilic xenobiotics such as drugs and environmental chemicals, and also plays an essential role in the biosynthesis and metabolism of endogenous compounds such as cholesterol and bile acids, vitamins, steroids, arachidonic acid and eicosanoids. Cytochrome P450 2J2 (CYP2J2) is a recently identified member of the human CYP protein family that is highly expressed in the heart, vasculature, liver and other tissues. CYP2J2 metabolises arachidonic acid (AA) into epoxyeicosatrienoic acids (EETs), which have a number of potent biological activities including cytoprotective, vasodilatory and anti-inflammatory effects. Given its widespread tissue distribution and the biological actions of EETs, CYP2J2 is likely to play an important role in cellular physiology, and altered expression of CYP2J2 may have pathophysiological consequences. Indeed, recent literature studies have indicated that CYP2J2 protein levels are decreased in vascular endothelial cells exposed to hypoxia and reoxygenation, and that maintenance of CYP2J2 expression enhances cell survival. Thus, CYP2J2 expression may be impaired in diseases or conditions associated with decreased oxygen availability, such as ischaemic heart disease, stroke and atherosclerosis, and this may contribute to their pathogenic consequences. Despite its likely importance in human physiology and pathophysiology, very little is known about the regulation of CYP2J2 gene expression. The aim of this study was to investigate the molecular mechanisms that control expression of the CYP2J2 gene. In particular, this study was designed to identify factors that regulate the expression of the CYP2J2 gene in the liver-derived HepG2 cell line during normoxia and hypoxia. A 2.4 kb fragment of the 5???-flanking region of the CYP2J2 gene (corresponding to nucleotides -2341 to +98, relative to the translation start site) was isolated from a human genomic library. Automated searching of the upstream regulatory region of CYP2J2 identified several putative binding sites for the transcription factor activator protein-1 (AP-1). Because AP-1 activity is altered in hypoxia, the possibility that AP-1 may participate in the regulation of CYP2J2 expression in hypoxia was explored. Cell culture studies examined the relationship between the expression of CYP2J2, and the AP-1 genes c-fos and c-jun, in HepG2 cells cultured in normoxia and hypoxia. Down-regulation of CYP2J2 mRNA and protein in hypoxic HepG2 cells was associated with the pronounced up-regulation of c-Fos protein from an undetectable level in normoxic cells; c-Jun protein levels were readily detectable in normoxia, and were also increased in hypoxia. Transient transfection studies revealed distinct effects of Fos and Jun proteins on CYP2J2 promoter activity. While the CYP2J2 promoter was strongly activated by c-Jun, c-Fos was inactive, and also abolished gene transactivation elicited by c-Jun. These results suggest that the constitutively expressed c-Jun is important in the maintenance of CYP2J2 expression in normoxic cells. The up-regulation of c-Fos in hypoxia stimulates the formation of c-Fos/c-Jun heterodimers, which do not support CYP2J2 transcription, leading to gene down-regulation. Experiments with CYP2J2 promoter deletion constructs revealed that the region between -152 to -50 bp relative to the translation start site was crucial for activation of CYP2J2 by c-Jun. Electrophoretic mobility shift assays (EMSAs) and transfection studies identified two distinct elements within this region that were involved in c-Jun-dependent transactivation: an AP-1-like element at -56 to -63 bp, and an atypical AP-1 element at -105 to -95 bp. c-Jun homodimers interacted specifically with both elements. Separate mutagenesis of either element significantly impaired c-Jun-dependent transactivation of CYP2J2, while mutagenesis of both elements eliminated c-Jun-responsiveness. EMSAs established that c-Jun, but not c-Fos, interacted with both elements in normoxic HepG2 cells. Furthermore, mutagenesis of either c-Jun-response element significantly decreased the basal transcriptional activity of the CYP2J2 promoter in HepG2 cells, while mutagenesis of both elements almost completely suppressed basal promoter activity. These findings indicate a pivotal role for c-Jun in the maintenance of CYP2J2 expression in normoxic cells. Transfection studies indicated that c-Fos suppresses c-Jun-dependent activation of CYP2J2 at both the -56/-63 bp and -105/-95 bp c-Jun-response elements. However, c-Fos-dependent inhibition appears to be mediated by distinct mechanisms at these two regulatory elements. While both elements interacted with c-Jun homodimers, only the -105/-95 bp element was able to interact with c-Fos/c-Jun heterodimers. Thus, the up-regulation of c-Fos in hypoxia, and the shift from c-Jun homodimers to c-Fos/c-Jun heterodimers, directly decreased c-Jun binding and transactivation at the -56/-63 bp element. In contrast, up-regulation of c-Fos in hypoxia altered the composition of proteins bound at the -105/-95 bp element from c-Jun to c-Fos/c-Jun. Inhibition of promoter activity occurs because c-Fos/c-Jun heterodimers can occupy, but not transactivate, the CYP2J2 promoter via the -105/-95 bp element. In summary, this thesis provides novel information on the molecular mechanisms that control the differential expression of the human CYP2J2 gene in normoxia and hypoxia. In particular, this study has established that the AP-1 proteins c-Jun and c-Fos play a crucial role in modulating the transcriptional activation of the CYP2J2 promoter in response to cellular stress. Binding of c-Jun to two distinct c-Jun-response elements within the CYP2J2 proximal promoter induces transcriptional activation of the CYP2J2 gene and is essential for maintenance of CYP2J2 expression in normoxic cells. The up-regulation of c-Fos in hypoxia promotes the formation of c-Fos/c-Jun heterodimers, which inhibit transcriptional activation of the CYP2J2 promoter by c-Jun, thus contributing to decreased CYP2J2 expression in hypoxia. Impaired expression of CYP2J2 may contribute to cellular injury in diseases such as atherosclerosis and stroke, and a greater understanding of the mechanisms responsible for mediating altered CYP2J2 expression may eventually lead to therapeutic strategies that manipulate the expression of this important human gene.

The cytochrome P450 (CYP) superfamily of enzymes catalyses the oxidative metabolism of lipophilic xenobiotics such as drugs and environmental chemicals, and also plays an essential role in the biosynthesis and metabolism of endogenous compounds such as cholesterol and bile acids, vitamins, steroids, arachidonic acid and eicosanoids. Cytochrome P450 2J2 (CYP2J2) is a recently identified member of the human CYP protein family that is highly expressed in the heart, vasculature, liver and other tissues. CYP2J2 metabolises arachidonic acid (AA) into epoxyeicosatrienoic acids (EETs), which have a number of potent biological activities including cytoprotective, vasodilatory and anti-inflammatory effects. Given its widespread tissue distribution and the biological actions of EETs, CYP2J2 is likely to play an important role in cellular physiology, and altered expression of CYP2J2 may have pathophysiological consequences. Indeed, recent literature studies have indicated that CYP2J2 protein levels are decreased in vascular endothelial cells exposed to hypoxia and reoxygenation, and that maintenance of CYP2J2 expression enhances cell survival. Thus, CYP2J2 expression may be impaired in diseases or conditions associated with decreased oxygen availability, such as ischaemic heart disease, stroke and atherosclerosis, and this may contribute to their pathogenic consequences. Despite its likely importance in human physiology and pathophysiology, very little is known about the regulation of CYP2J2 gene expression. The aim of this study was to investigate the molecular mechanisms that control expression of the CYP2J2 gene. In particular, this study was designed to identify factors that regulate the expression of the CYP2J2 gene in the liver-derived HepG2 cell line during normoxia and hypoxia. A 2.4 kb fragment of the 5???-flanking region of the CYP2J2 gene (corresponding to nucleotides -2341 to +98, relative to the translation start site) was isolated from a human genomic library. Automated searching of the upstream regulatory region of CYP2J2 identified several putative binding sites for the transcription factor activator protein-1 (AP-1). Because AP-1 activity is altered in hypoxia, the possibility that AP-1 may participate in the regulation of CYP2J2 expression in hypoxia was explored. Cell culture studies examined the relationship between the expression of CYP2J2, and the AP-1 genes c-fos and c-jun, in HepG2 cells cultured in normoxia and hypoxia. Down-regulation of CYP2J2 mRNA and protein in hypoxic HepG2 cells was associated with the pronounced up-regulation of c-Fos protein from an undetectable level in normoxic cells; c-Jun protein levels were readily detectable in normoxia, and were also increased in hypoxia. Transient transfection studies revealed distinct effects of Fos and Jun proteins on CYP2J2 promoter activity. While the CYP2J2 promoter was strongly activated by c-Jun, c-Fos was inactive, and also abolished gene transactivation elicited by c-Jun. These results suggest that the constitutively expressed c-Jun is important in the maintenance of CYP2J2 expression in normoxic cells. The up-regulation of c-Fos in hypoxia stimulates the formation of c-Fos/c-Jun heterodimers, which do not support CYP2J2 transcription, leading to gene down-regulation. Experiments with CYP2J2 promoter deletion constructs revealed that the region between -152 to -50 bp relative to the translation start site was crucial for activation of CYP2J2 by c-Jun. Electrophoretic mobility shift assays (EMSAs) and transfection studies identified two distinct elements within this region that were involved in c-Jun-dependent transactivation: an AP-1-like element at -56 to -63 bp, and an atypical AP-1 element at -105 to -95 bp. c-Jun homodimers interacted specifically with both elements. Separate mutagenesis of either element significantly impaired c-Jun-dependent transactivation of CYP2J2, while mutagenesis of both elements eliminated c-Jun-responsiveness. EMSAs established that c-Jun, but not c-Fos, interacted with both elements in normoxic HepG2 cells. Furthermore, mutagenesis of either c-Jun-response element significantly decreased the basal transcriptional activity of the CYP2J2 promoter in HepG2 cells, while mutagenesis of both elements almost completely suppressed basal promoter activity. These findings indicate a pivotal role for c-Jun in the maintenance of CYP2J2 expression in normoxic cells. Transfection studies indicated that c-Fos suppresses c-Jun-dependent activation of CYP2J2 at both the -56/-63 bp and -105/-95 bp c-Jun-response elements. However, c-Fos-dependent inhibition appears to be mediated by distinct mechanisms at these two regulatory elements. While both elements interacted with c-Jun homodimers, only the -105/-95 bp element was able to interact with c-Fos/c-Jun heterodimers. Thus, the up-regulation of c-Fos in hypoxia, and the shift from c-Jun homodimers to c-Fos/c-Jun heterodimers, directly decreased c-Jun binding and transactivation at the -56/-63 bp element. In contrast, up-regulation of c-Fos in hypoxia altered the composition of proteins bound at the -105/-95 bp element from c-Jun to c-Fos/c-Jun. Inhibition of promoter activity occurs because c-Fos/c-Jun heterodimers can occupy, but not transactivate, the CYP2J2 promoter via the -105/-95 bp element. In summary, this thesis provides novel information on the molecular mechanisms that control the differential expression of the human CYP2J2 gene in normoxia and hypoxia. In particular, this study has established that the AP-1 proteins c-Jun and c-Fos play a crucial role in modulating the transcriptional activation of the CYP2J2 promoter in response to cellular stress. Binding of c-Jun to two distinct c-Jun-response elements within the CYP2J2 proximal promoter induces transcriptional activation of the CYP2J2 gene and is essential for maintenance of CYP2J2 expression in normoxic cells. The up-regulation of c-Fos in hypoxia promotes the formation of c-Fos/c-Jun heterodimers, which inhibit transcriptional activation of the CYP2J2 promoter by c-Jun, thus contributing to decreased CYP2J2 expression in hypoxia. Impaired expression of CYP2J2 may contribute to cellular injury in diseases such as atherosclerosis and stroke, and a greater understanding of the mechanisms responsible for mediating altered CYP2J2 expression may eventually lead to therapeutic strategies that manipulate the expression of this important human gene.