Influence of quercetin and kaempferol on benzo[a]pyrene-mediated effects via AhR- and Nrf2-pathways in human and rat intestinal celll lines [Elektronische Ressource] / vorgelegt von Jeanette Niestroy

Niestroy

Sujets

• Biologie

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2010
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Influence of quercetin and kaempferol on
benzo[a]pyrene-mediated effects via AhR- and Nrf2-
pathways in human and rat intestinal cell lines







Inaugural-Dissertation



zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf


vorgelegt von

Jeanette Luise Niestroy

aus Heydebreck-Cosel





Düsseldorf, November 2009



The present dissertation was performed according to the Graduate College
“Food constituents as triggers of nuclear receptor-mediated intestinal signalling”,
Heinrich-Heine-Universität Düsseldorf,
at the Leibniz Research Centre for Working Environment and Human Factors.






















Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf


Referent: PD Dr. Peter H. Roos
Koreferent: Prof. Dr. Peter Proksch

Tag der mündlichen Prüfung: 10. Dezember 2009



I
Table of contents........................................................................................................................
Abbreviations..........................................................................................................................IV

1. Introduction ......................................................................................................................... 1
1.1. Gastrointestinal tract ...................................................................................................... 1
1.2. Polycyclic aromatic hydrocarbons (PAH) are environmental contamination................ 2
1.3. Chemo-prevention by secondary plant components ...................................................... 3
1.4. Aryl hydrocarcon receptor (AhR)-pathway ................................................................... 6
1.5. NF-E2-related factor 2 (Nrf2-)-pathway........................................................................ 9
1.6. Aims of the study ......................................................................................................... 11

2. Experimental....................................................................................................................... 13
2.1. Chemicals...................................................................................................................... 13
Chemicals for molecular biology ..................................................................................... 14
Buffers and solutions........................................................................................................ 14
Antibodies, enzymes and other proteins .......................................................................... 14
Kits ................................................................................................................................... 14
Consumables .................................................................................................................... 14
Cell lines........................................................................................................................... 15
Cell culture medium......................................................................................................... 15
Solution for cell culture.................................................................................................... 15
TaqMan® Real time PCR ................................................................................................ 15 e PCR solutions................................................................................. 16 e PCR probes..................................................................................... 16
Housekeeping target genes............................................................................................... 16
Human target genes.......................................................................................................... 16
Rat target genes ................................................................................................................ 16
Instruments....................................................................................................................... 16
Software ........................................................................................................................... 17
Service provider ............................................................................................................... 17
2.2. Methods......................................................................................................................... 18
Cell culture 18
Neutral-red cytotoxicity assay.......................................................................................... 19
Exposure of cells to the testing chemicals ....................................................................... 19 II
Determination of the RNA-concentration and purity....................................................... 21
Reverse transcription........................................................................................................ 21
TaqMan® Real Time PCR............................................................................................... 23
Protein isolation................................................................................................................ 23
Total cell-lysate protein isolation..................................................................................... 25
Determination of the total protein concentration from the cell-lysate ............................. 26
Preparation of samples ..................................................................................................... 27
Standards .......................................................................................................................... 27
Molecular weight-marker................................................................................................. 27
Preparation of gels............................................................................................................ 27
Gel electrophoresis........................................................................................................... 28
Immuno detection............................................................................................................. 29
EROD activity.................................................................................................................. 30
Microarray analysis.......................................................................................................... 30

3. Results ................................................................................................................................. 31
3.1. Effects of B[a]P and flavonoids on Caco-2 cells .......................................................... 31
3.1.1. Cytotoxicity of B[a]P and flavonoids on the CaCo-2 cell line .............................. 31
3.1.2. Effects of B[a]P, Q and K on gene expression in CaCo-2 cells: kinetics .............. 34
3.1.3. Effects of B[a]P, Q and K on gene expression in CaCo-2 cells: dose response .... 35
3.1.4. Influence of quercetin and kaempferol on the B[a]P-induced gene expression..... 37
3.1.5. Influence of quercetin and kaempferol on the B[a]P-induced protein expression. 40
3.1.6. Effect of B[a]P, quercetin and kaempferol on the CYP1-dependent EROD activity
in CaCo-2 cells................................................................................................................. 43
3.1.7. Modulation of the B[a]P-induced CYP1A1 gene and protein expression by
quercetin and kaempferol related to the EROD activity in CaCo-2 cells ........................ 44
3.2. Effects of B[a]P and flavonoids on IEC-6 cells ............................................................ 45
3.2.1. Cytotoxicity of B[a]P and flavonoids on the IEC-6 cell line ................................. 45
3.2.2. Time-dependent gene expression pattern in IEC-6 cells........................................ 47
3.2.3. Assessing an effective concentration of B[a]P, quercetin and kaempferol for the
studies with IEC-6 cells.................................................................................................... 49
3.2.4. Influence of quercetin and kaempferol on the B[a]P-induced gene expression..... 51
3.2.5. Influence of quercetin and kaempferol on the B[a]P-induced protein expression. 54 III
3.2.6. Effect of B[a]P, quercetin and kaempferol on the enzymatic EROD activity in
IEC-6 cells........................................................................................................................ 56
3.2.7. Gene expression profiling of B[a]P and flavonoid treated IEC-6 cells by
microarrays....................................................................................................................... 57
3.3. Effects of B[a]P and flavonoids on HuTu-80 cells ........................