Metabolism of nonylphenol by human P450-recombinant yeast and assessment of the xeno-hormone potency of different isomers and their chlorinated derivatives [Elektronische Ressource] / vorgelegt von Paola Giulia Cormio

Cormio Paola Giulia

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2008
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

Metabolism of nonylphenol by human P450-recombinant
yeast and assessment of the xeno-hormone potency of
different isomers and their chlorinated derivatives








Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen
University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften
vorgelegt von





Dott.ssa

Paola Giulia Cormio

aus Bari (Italy)




Berichter: Univ.–Prof. Dr.rer.nat. Ingolf Schuphan
Privat-Dozent Dr.rer.nat. Nikolaus Schlaich


Tag der mündlichen Prüfung: 29 August 2008




Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.

Index
1 Introduction ............................................................................................................... 1
1.1 Nonylphenol...............................1
1.2 Aims of this study......................................................................................................................2
2 General Information.................................. 5
2.1 Endocrine system ......................................................................................5
2.1.1 General...............................................5
2.1.2 Hormones and hormone receptors................................................................5
2.1.3 Steroid and thyroid hormone receptors........................................................6
2.1.4 Hormone-Receptor Binding and Interactions with DNA..........................6
2.2 Endocrine disrupter compounds (EDCs) ..............................................................................7
2.2.1 Definition of EDCs............................................................7
2.2.2 Classification of EDCs.....................7
2.2.3 Nonylphenol as endocrine disrupter..............................................................................................8
2.3 Cytochrome P450 monooxigenase and reductase................................9
2.3.1 Cytochrome P450 monooxigenase ................................................................................................. 9
2.3.2 Cytochrome P450 reductase.........11
2.3.3 Human cytochrome P450 2B6......11
2.3.4 Human cytochrome P450 2C19....................................................................................................12
2.4 Metabolism with cell cultures...............................12
2.4.1 Cytochromes P450 and their role in plant metabolism ............................................................12
2.4.2 Cytochrome P450 monooxigenase and reductase in Saccharomyces cerevisiae ...................12
2.4.3 Yeast as heterologous expression system for cytochrome P450..............................................13
2.4.4 Simazine and P450 metabolism....................................................................14
2.4.5 Methoxychlor and P450 metabolism...........................15
2.5 Methods for disinfection of WWTP and DWTP: chlorination........................................15
2.5.1 Chemical disinfection.....................................................................................15
2.5.2 Chlorination in drinking water and wastewater.......16
2.5.3 Chlorination of nonylphenol.........................................................................................................17
2.6 Task of this work.....................................................................................18
2.6.1 Part A: metabolism studies of nonylphenol................................................18
2.6.2 Part B: estrogenic and androgenic potency of branched nonylphenol isomers and their
derivatives .....................................................................................................................19
3 Materials and methods.............................................................21
3.1 Organisms ................................................................................................21
- I - 3.1.1 Bacteria ............................................................................................................................................21
3.1.2 Yeast strains (Saccharomyces cerevisiae)....................21
3.2 Plasmids....................................................................................................................................21
3.2.1 pESC-URA, pESC-TRP................21
3.2.2 pYeDP60..........22
3.2.3 pCW’2b6 and pCW’2c19 ..............................................................................................................22
3.3 Yeast cells culture....................................................................................22
3.3.1 pT2b6 and pT2c19..........................................................22
3.3.2 PY2b6 and PY2c19.........................22
3.4 Culture media and plates.......................................................................................................22
3.4.1 LB and LBA media.........................22
3.4.2 YAPD media....................................................................................................23
3.4.3 YEPD media....................................23
3.4.4 SW(6) media....23
3.4.5 N3 media ..........................................................................................................23
3.4.6 SD and SG media............................24
3.5 Microbiological methods........................................................................................................25
3.5.1 E. coli culture..................................25
3.5.2 3.5.2 Competent cells of E. coli.....................................25
3.5.3 Transformation of competent cells of DH5! E. coli .................................................................26
3.5.4 Preparation of yeast competent cells ...........................................................26
3.5.5 Transformation of whole yeast cells............................27
3.6 Molecular and biochemical methods ...................................................................................27
3.6.1 Isolation of plasmids DNA ............................................27
3.6.2 Polymerase chain reaction (PCR)................................27
3.6.3 Restriction enzyme digestion........................................28
3.6.4 Colony PCR .....................................................................28
3.6.5 3.6.5 Primers for PCR....................................................................................................................29
3.6.6 Isolation and purification of DNA fragments from agarose gel..............30
3.6.7 Ligation reaction.............................................................................................................................30
3.6.8 Agarose gel electrophoresis...........................................30
3.6.9 Sequencing of DNA........................31
3.6.10 Yeast protein extraction ................................................................................31
3.6.11 SDS-PAGE.......................................................................32
3.6.12 Western blot: semi-dry blotting ...................................................................33
3.7 Chemical ...................................................................34
3.7.1 Reference substances......................................................................................34
3.7.2 Radiochemical.................................35
- II - 3.8 Synthetic methods ...................................................................................................................35
3.8.1 Synthesis of 33-nonanol.................35
12 133.8.2 Synthesis of p33-NP, p353-NP, p363-NP C/ C.......................................................................35
3.8.3 Purification of nonylphenol isomers............................................................36
3.8.4 Chlorinated derivatives of nonylphenol isomers.......37
3.9 Metabolism studies..................................................................................................................38
3.9.1 System 1 (over-expressing YR and Cytochrome P450)............................38
3.9.2 System 2 (over-expressing only cytochrome P450) ...................................................................38
3.9.3 Procedures for the metabolism studies .......................................................39
3.9.4 Thin layer chromatography (TLC).............................40
3.9.5 High pressure liquid chromatography (HPLC) ........................................................................40
3.9.6 GC/MS for nonylphenol metabolites...........................................................40
3.10 Yeast estrogenic and androgenic screen (YES and YAS).................................................41
3.10.1 Minimal media (MM) YES/YAS..................................................................41
3.10.2 3.10.2 YES/YAS growing media and test media........................................41
3.10.3 Estrogenic and androgenic assay for possible agonist..............................................................41
3.10.4 Estrogenic and androgenic assay of antagonist.........................................42
4 Results and discu