2 PARTE EXPERIMENTAL
2.3 Técnicas y Métodos
2.3.4 Parámetros de Calidad de los Extractos Fluidos de Jengibre, Romero y
2.3.4.4 Prueba de Edema Plantar en Rata Inducido por Carragenina
• Characterise and perfect the MCF-7 cell culture system. Determine optimum growth conditions by observing MCF-7 growth characteristics following modification of culture media (e.g. changes in non-essential amino acids (NEAAs)). Determining optimum tryspin methodology to de-adhere cells from culture vessels.
• Investigate the effects of xenoestrogens and combinations of xenoestrogens on MCF-7 cells in culture by exposing cultured MCF-7 cells to varying concentrations of selected xenoestrogens both individually and in combinations and plotting growth curves. • Investigate the estrogenic potency of individual and combinations of xenoestrogens on
both ER isoforms (ERa and ERb) of the CALUXâ assay by exposing ERa and ERb
CALUXâ cells to varying concentrations of selected xenoestrogens both individually and
in combinations and determining EC50 values.
• Use in silico modelling (Schrödinger platform) to investigate the biomolecular
interactions of xenoestrogens with ER ligand binding sites (LBC and AF-2) and explore the interplay between the two binding sites. Using ligand-receptor docking to study the biological chemistry of biomolecular interactions of selected xenoestrogens with ERs, and determine the importance of ligand structure in predicting interactions with LBC and AF-2.
• Assess women’s and pre-pubertal girls’ exposures to xenoestrogens using a dietary and lifestyle questionnaire, and determine the contribution of xenoestrogen exposure to the theoretical total estrogenic load.
• Determine serum markers of xenoestrogen exposure (e.g. BPA, genistein, butylparaben), based on women’s exposures from the dietary and lifestyle questionnaire study, by taking blood samples from a group of women and determining serum levels of a selection of xenoestrogens by LC-MS.
• Bring together data from the cell culture, CALUXâ, in silico and exposure studies to
Chapter 2
Materials and Methods
2.1. Materials
A list of materials used in cell culture and blood analysis are presented in Table 2.1 Table 2.1: Materials used in cell culture and blood analysis
Material Supplier/model Location
Cell culture Chemicals
b-Estradiol (E2) Sigma-Aldrich Auckland, NZ
Estrone Sigma-Aldrich Auckland, NZ
Estriol Sigma-Aldrich Auckland, NZ
17a-Ethinyl estradiol (EE2) Sigma-Aldrich Auckland, NZ
Bisphenol A (BPA) Sigma-Aldrich Auckland, NZ
Genistein LC-Laboratories Massachusetts,
USA
Daidzein LC-Laboratories Massachusetts,
USA
Kaempferol Sigma-Aldrich Auckland, NZ
Curcumin Sigma-Aldrich Auckland, NZ
Tetrahydrocurcumin Sigma-Aldrich Auckland, NZ
Methyl 4-hydroxybenzoate Sigma-Aldrich Auckland, NZ
Butyl 4-hydroxybenzoate Sigma-Aldrich Auckland, NZ
Benzyl 4-hydroxybenzoate Sigma-Aldrich Auckland, NZ
Streptomycin sulphate salt Sigma-Aldrich Auckland, NZ
Penicillin G sodium salt Sigma-Aldrich Auckland, NZ
Commercial phosphate buffered saline (PBS) packet (total weight = 9.55 g
comprising sodium chloride (8 g), potassium phosphate, monobasic (0.2 g), sodium phosphate, dibasic (1.15 g) and potassium chloride (0.2 g))
Sigma-Aldrich Auckland, NZ
Gibco MEM non-essential amino acids
(NEAA) Thermofisher Scientific Christchurch, NZ
Analytical grade ethanol ECP Ltd Auckland, NZ
Sodium bicarbonate ECP Ltd Auckland, NZ
Ethylenediaminetetracacetic acid (EDTA) ECP Ltd Auckland, NZ
Ground dextran coated charcoal Sigma-Aldrich Auckland, NZ
Magnesium chloride hexahydrate ECP Ltd Auckland, NZ
Sucrose ECP Ltd Auckland, NZ
4-(2-Hydroxyethyl)-1-
Trypan Blue Sigma-Aldrich Auckland, NZ
Tris Sigma-Aldrich Amsterdam, NL
Dithiothreitol (DTT) Sigma-Aldrich Amsterdam, NL
1,2-diaminocyclohexane-N,N,N´,N´-
tetraacetic acid (CDTA) Sigma-Aldrich Amsterdam, NL
EDTA Sigma-Aldrich Amsterdam, NL
Glycerol Sigma-Aldrich Amsterdam, NL
TritonâX-100 Sigma-Aldrich Amsterdam, NL
Tricine Sigma-Aldrich Amsterdam, NL
Mg(CO2)4Mg(OH)2.5H2O Sigma-Aldrich Amsterdam, NL
MgSO4.7H2O Sigma-Aldrich Amsterdam, NL
Dextran T500 Sigma-Aldrich Amsterdam, NL
Biological products
RPMI-1640 powder Life Technologies Auckland, NZ
Gibco phenol red-free RPMI-1640 Life Technologies Auckland, NZ
Fetal bovine serum Life Technologies Auckland, NZ
Trypsin powder Sigma-Aldrich Auckland, NZ
Gibco trpLEä express Life Technologies Auckland, NZ
MCF-7 human breast cancer cells American Type Culture
Collection (ATCC) Manassas, USA
ERa-CALUXâ cells BioDetection Systems Amsterdam, NL
ERb-CALUXâ cells BioDetection Systems Amsterdam, NL
D-Luciferin Sigma-Aldrich Amsterdam, NL
ATP Sigma-Aldrich Amsterdam, NL
Dulbecco’s Modified Eagle Medium:
Nutrient Mixture F-12 (DMEM/F12) ThermoFisher Scientific Amsterdam, NL
Gibco Sodium pyruvate (1 M) Life Technologies Auckland, NZ
Equipment
T-75 sterile cell culture flasks In Vitro Technologies Auckland, NZ
T-25 sterile cell culture flasks Sigma-Aldrich Auckland, NZ
Glass vials (4 mL, 7 mL and 20 mL)
24 Well plate Sigma-Aldrich Auckland, NZ
White 384 well plate Sigma-Aldrich Amsterdam, NL
50 mL Centrifuge tubes Automatic pipette and tips
Sterile filter (Steritop-GP, 0.22 µm,
polyethersulfone, 500 mL 45 mm ThermoFisher Scientific Melbourne, AUS Sterile syringe filter (17 mm, 0.2 µm PTFE
filter) ThermoFisher Scientific Christchurch, NZ
Autoclave
Laminar flow cabinet Cytoguard CG2000
series, model CGA-180 Sydney, AUS
Inverted microscope CKX41, Olympus Melbourne, AUS
Microscope camera ProSciTech Kirwan, AUS
Toup camera software ProSciTech Kirwan, AUS
Veriplast plastic counting chambers Thermo Fisher
Scientific Melbourne, AUS
Centrifuge MultiFuge 1 S-R Hanau, Germany
Glassware Hot-plate stirrer
Microwell plates (24, 384 wells) ThermoFisher
Scientific Auckland, NZ
Schott bottles (100 mL, 500 mL, 1000 mL)
Cytomat Incubator ThermoFisher
Scientific Amsterdam, NL
Hamilton Robot STARlet Bonaduz
Switzerland
EL406 Washer-Dispenser BioTek Amsterdam, NL
Luminometer Tecan Mannedorf,
Switzerland Blood analysis
Chemicals
Analytical grade acetonitrile ECP Ltd Auckland, NZ
Analytical grade Methyl tert-butyl ether Sigma-Aldrich Auckland, NZ
Analytical grade chloroform ECP Ltd Auckland, NZ
Sodium sulphate ECP Ltd Auckland, NZ
Equipment
Sterile needles Becton Dickinson Auckland, NZ
Vacutainer Becton Dickinson Auckland, NZ
50 mL Centrifuge tubes
Centrifuge MultiFuge 1 S-R Hanau, DE
High performance liquid chromatography
(HPLC) vials Micro-Analytix Auckland, NZ
Whatman filter paper ThermoFisher
Scientific Auckland, NZ
Glassware
Speed Vac ThermoFisher
Scientific
Auckland, NZ Glass vials
LC-MS
C18 reverse phase HPLC column Phenomex North Shore, NZ
2.1.1 MilliQ Water
All water used in MCF-7 and Blood analysis experiments was purified using the Milli-Q®
system (Merck Millipore, Auckland, New Zealand). Resistivity (25⁰C) = 18.2 MΩ.cm.
2.1.2. Demineralised Water
All water used in CALUXâ assay experiments was purified using a HOH RO 51 Reverse
Osmosis Plant from HOH Water Technology (Greve, Denmark). Water quality = 40 µS/cm (0.25 MΩ.cm).
2.1.2. Computational Modelling
2.1.2.1. Receptor Models
Computational docking relies on models of the receptor protein structure. These models are developed from published x-ray crystal structures. For this work, the x-ray crystal structures of the ER LBD (Table 2.2) were obtained from the RSCB Protein Data Bank (PDB;
http://www.rcsb.org). The models developed from the crystal structure are identified by the PDB code.
Table 2.2: X-ray crystal structures of the human ER ligand binding domains obtained from the PDB.
PDB
code Receptor Ligand Resolution (Å)
Chain Coregulatory
protein peptide Reference
1ERE ERa E2 3.10 A no (Brzozowski,
et al., 1997)
3OLS ERb E2 2.20 A yes (Mocklinghoff,
et al., 2010)
2.1.2.2. Ligand Structures
Computational docking requires a model for each ligand which is developed from their 3D structures. The 3D structures for all ligands were constructed using the software tools (see Section 2.2.10.1.) from the Schrodinger Suite. Models of 16 xenoestrogens (estrone, E2, estriol, progesterone, testosterone, genistein, daidzein, equol, kaempferol, curcumin,
tetrahydrocurcumin, EE2, BPA, methylparaben, butylparaben and benzylparaben (see Figs. 1.1 and 1.19) were required for the study.
All ligands were energy minimised using the Schrödinger software to achieve an optimised conformation of the ligand that had the lowest internal energy. This calculation searched the entire conformation space of the ligand, including ring conformations (e.g., 6-member ring boat/chair conformations) and rotational degrees of freedom. For the ring conformation search, only ligands with minimised energy within 50 kJ/mol of the lowest energy
conformation were retained. For most ligands only one conformation was found that fitted this parameter.