Studies on the Effects of Paraben Mixtures on MCF-7 Breast Cancer Cells in Culture A thesis submitted in partial fulfilment of the requirements for the Degree of Masters of Science in Biochemistry In the Department of Chemistry At the University of Canterbury New Zealand By Kristie Webber University of Canterbury 2013 Table of Contents Acknowledgements .............................................................................................................................. i Abstract ............................................................................................................................................... ii Abbreviations ..................................................................................................................................... iii List of figures and tables .................................................................................................................... iv 1 Introduction ......................................................................................................................................... 1 1.1 Hormones and the endocrine system ........................................................................................... 2 1.2 Estrogens ....................................................................................................................................... 5 1.2.1 What are estrogens? .............................................................................................................. 5 1.2.2 17β-Estradiol .......................................................................................................................... 7 1.2.3 Estriol ..................................................................................................................................... 8 1.2.4 Estrone ................................................................................................................................... 9 1.2.5 Transport of estrogen in the body ....................................................................................... 10 1.3 The estrogen receptor ................................................................................................................ 11 1.4 Xenoestrogens ............................................................................................................................ 14 1.5 Experimentally testing xenoestrogens ........................................................................................ 16 1.6 MCF-7 cell line ............................................................................................................................. 16 1.7 Parabens ..................................................................................................................................... 18 1.7.1 What are parabens? ............................................................................................................. 18 1.7.2 Uses of parabens .................................................................................................................. 18 1.7.3 Human exposure to parabens – are they safe? ................................................................... 20 1.8 Xenoestrogen combinations ....................................................................................................... 21 1.9 Aims and objectives .................................................................................................................... 22 2 Materials and methods ...................................................................................................................... 23 2.1 Chemicals .................................................................................................................................... 24 2.2 Sterilisation ................................................................................................................................. 25 2.2.1 Autoclaving .......................................................................................................................... 25 2.2.2 Filtration ............................................................................................................................... 25 2.2.3 70% ethanol (v/v) aerosol .................................................................................................... 25 2.2.4 Cell procedures .................................................................................................................... 25 2.3 Preparation of antibiotic solution ............................................................................................... 25 2.4 Inactivation of fetal bovine serum .............................................................................................. 26 2.5 Charcoal stripping of fetal bovine serum .................................................................................... 26 2.6 Preparation of culture medium .................................................................................................. 27 2.7 Preparation of Minimum Essential Medium (phenol red free) .................................................. 27 2.8 Preparation of Dulbecco’s Phosphate Buffered Saline ............................................................... 27 2.9 Cell Culture .................................................................................................................................. 28 2.9.1 MCF-7 Cells........................................................................................................................... 28 2.9.2 Maintaining MCF-7 cell cultures .......................................................................................... 28 2.9.3 Cryopreservation of MCF-7 cells .......................................................................................... 28 2.9.4 Passaging MCF-7 cell cultures .............................................................................................. 29 2.9.5 Counting cells ....................................................................................................................... 29 2.9.5.1 Manually counting cells ................................................................................................ 29 2.9.5.2 Countess® Automated Cell Counter ............................................................................. 30 2.9.6 Seeding a well plate ............................................................................................................. 30 2.9.7 Method development – determining a method for effective dispersal of MCF-7 cells using TrypLE™ Express ............................................................................................................................ 31 2.9.8 Removing cells from plate wells for counting ...................................................................... 32 2.9.9 MCF-7 growth curve ............................................................................................................ 32 2.9.9.1 MCF-7 growth curve with estrogen present (10 pM) ................................................... 32 2.9.9.2 MCF-7 control growth curve ......................................................................................... 33 2.9.9.3 MCF-7 growth curve in PRFMEM .................................................................................. 33 2.9.9.4 MCF-7 growth curve in MEM ........................................................................................ 33 2.9.9.5 MCF-7 growth curve with estrogen present (18.3 pM) ................................................ 34 2.9.9.6 MCF-7 growth curve with estrogen present (3.67 pM) ................................................ 34 2.9.9.7 MCF-7 growth curve with estrogen present (0.367 pM) .............................................. 34 2.9.10 MCF-7 cell exposure experiments...................................................................................... 34 2.9.10.1 Exposing MCF-7 cells to 17β-estradiol ........................................................................ 35 2.9.10.2 Exposing MCF-7 cells to methylparaben and butylparaben ....................................... 35 2.9.10.3 Exposing MCF-7 cells to methylparaben and butylparaben combinations ................ 35 2.10 Statistical analysis using Analysis of Variance ........................................................................... 36 2.11 UV spectroscopy of methylparaben and butylparaben cocktails ............................................. 36 3. Results ............................................................................................................................................... 37 3.1 Method development – use of stripped FBS .............................................................................. 38 3.2 Method development – determining a method for effective dispersal of MCF-7 cells using TrypLE™ Express ................................................................................................................................ 39 3.3 Method development – estrogen exposure and the use of Countess® Automated Cell Counter .......................................................................................................................................................... 40 3.4 Method development – growth curves in the presence of 17β-estradiol to reduce variability using the Countess® Automated Cell Counter .................................................................................. 42 3.5 MCF-7 growth curve in the presence of 10 pM 17β-Estradiol