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Investigating the Role of Oxidative Stress in the Generation Of 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 .........................................................................................................................................
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