Investigating the Role of Oxidative Stress in the Generation of Plausibly Misleading Positive Results for in vitro Genotoxicity Generated by Polyphenolic Antioxidants A THESIS SUBMITTED TO THE UNIVERSITY OF MANCHESTER FOR THE DEGREE OF PHD IN THE FACULTY OF LIFE SCIENCES CHRISTOPHER ADDINSELL 2014 1 Contents 1 Abstract ............................................................................................................................................... 11 2 Introduction ........................................................................................................................................ 14 2.1 Background ..................................................................................................................................... 14 2.2 Genetic Toxicology .......................................................................................................................... 15 2.3 Genotoxicity assessment ................................................................................................................ 21 2.4 Oxidative stress ............................................................................................................................... 31 2.5 Antioxidants .................................................................................................................................... 35 2.6 Assessments of cytotoxicity ............................................................................................................ 36 2.7 Aims and objectives ........................................................................................................................ 37 3 Materials & Methods .......................................................................................................................... 38 3.1 Cell lines and culture ....................................................................................................................... 38 3.2 Chemicals used in these studies ..................................................................................................... 39 3.3 The GADD45a-GFP (‘GreenScreen HC’) genotoxicity assay ............................................................ 40 3.4 Population doubling assessment .................................................................................................... 48 3.5 in silico interrogation of compounds using Derek Nexus™ ............................................................. 53 3.6 Using dichlorofluorescin diacetate to detect intracellular ROS generation ................................... 55 3.7 Detection of early stages of toxicity and mitochondrial membrane depolarisation using JC-1 ..... 59 3.8 Quantifying oxidative DNA damage using antibodies against 8-oxoguanine ................................. 61 3.9 Statistical analysis ........................................................................................................................... 62 4 Results I – Compound Choice .............................................................................................................. 65 4.1 Introduction .................................................................................................................................... 65 4.2 Polyphenolic antioxidants ............................................................................................................... 67 4.3 Monophenolic antioxidants ............................................................................................................ 77 4.4 Non-phenolic antioxidants .............................................................................................................. 80 4.5 Oxidants .......................................................................................................................................... 83 4.6 Genotoxins ...................................................................................................................................... 85 4.7 Non-genotoxic, cytotoxic compounds ............................................................................................ 90 4.8 Miscellaneous compounds ............................................................................................................. 92 4.9 Pro-genotoxins ................................................................................................................................ 95 4.10 Discussion ....................................................................................................................................... 97 5 Results II: Most polyphenolic antioxidants produce positive results in the GADD45a-GFP genotoxicity assay ..........................................................................................................................................100 5.1 Introduction ..................................................................................................................................100 5.2 The autofluorescent and light absorbing properties of propyl gallate, nordihydroguaiaretic acid and epigallocatechin gallate confound spectrophotometric data collection ............................................100 5.3 Results for GADD45a-GFP genotoxicity assay in the absence of S9 .............................................102 5.4 Results for GADD45a-GFP genotoxicity assay with S9 metabolic activation ................................107 5.5 Discussion .....................................................................................................................................110 5.6 Summary .......................................................................................................................................113 6 Results III – Most polyphenolic antioxidants produce alerts of “Plausible” or higher for genotoxic endpoints using in silico tool, Derek Nexus™ .................................................................................................114 6.1 Introduction ..................................................................................................................................114 6.2 Results ...........................................................................................................................................114 6.3 Discussion .....................................................................................................................................134 6.4 Summary .......................................................................................................................................136 7 Results IV – Incubation in the presence of a lowered oxygen concentration does not reduce the genotoxicity of polyphenolic antioxidants in the GADD45a-GFP assay .........................................................137 7.1 Introduction ..................................................................................................................................137 7.2 GADD45a-GFP Assays Incubated with 5% Oxygen........................................................................137 7.3 GADD45a-GFP Assays Incubated with 1% Oxygen........................................................................144 7.4 Discussion .....................................................................................................................................153 7.5 Summary .......................................................................................................................................154 8 Results V – Most polyphenolic antioxidants lead to the generation of intracellular reactive oxygen species in vitro ................................................................................................................................................155 8.1 Introduction ..................................................................................................................................155 8.2 Optimisation of the DCFH-DA assay protocol in TK6 Cells ............................................................155 8.3 Calculation of thresholds for DCF fluorescence and PI permeability ...........................................156 2 8.4 Assessment of 34 compounds in the DCFH-DA assay ................................................................... 159 8.5 Propidium iodide cannot detect a high proportion of cells that have committed to a cytotoxic endpoint within a 4 hour timeframe ......................................................................................................... 164 8.6 Eight phenolic antioxidants reduce the level of ROS in cells treated with oxidant, TBHP ............ 166 8.7 Discussion ..................................................................................................................................... 168 8.8 Summary ....................................................................................................................................... 170 9 Results VI – Most polyphenolic antioxidants are able to depolarise the mitochondrial membrane 171 9.1 Introduction .................................................................................................................................. 171 9.2 Optimising the protocol for TK6 cells ........................................................................................... 171 9.3 Generation of a threshold for JC-1 assessment ............................................................................ 173 9.4 Results .........................................................................................................................................