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A Comparison of Aquatic Species Responses to Anticancer Drug Exposure

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A Comparison of Aquatic Species Responses to Anticancer Drug Exposure A comparison of aquatic species responses to anticancer drug exposure A thesis submitted for the degree of Doctor of Philosophy by Graham Harris Institute for the Environment Brunel University September 2015 i Table of Contents 1 Introduction .................................................................................................................... 1 1.1 Pharmaceuticals in the environment ...................................................................... 1 1.1.1 Overview .......................................................................................................... 1 1.1.2 Environmental entry routes ............................................................................. 1 1.1.3 Environmental fate........................................................................................... 3 1.2 Environmental toxicology of cytotoxic drugs .......................................................... 3 1.2.1 Overview .......................................................................................................... 3 1.2.2 Ph.D. objective ................................................................................................. 4 1.2.3 Ph.D. aims ......................................................................................................... 5 1.2.4 Hypothesis ........................................................................................................ 5 1.3 Cytotoxic drug selection methodology ................................................................... 5 1.3.1 United Kingdom usage data ............................................................................. 5 1.3.2 Classes of anticancer drug ............................................................................... 7 1.3.3 Chemical characteristics relevant to the aquatic environment ....................... 8 1.4 Mechanisms of Action of the selected cytotoxic drugs. ....................................... 10 1.4.1 5-fluorouracil .................................................................................................. 11 1.4.2 Cyclophosphamide ......................................................................................... 14 1.4.3 Cisplatin & carboplatin ................................................................................... 15 ii 1.4.4 Hydroxycarbamide ......................................................................................... 17 1.4.5 Imatinib .......................................................................................................... 18 1.5 The selected cytotoxic drugs in combination - Drug regimens ............................. 19 1.6 Target Cytotoxics in the environment ................................................................... 20 1.7 An overview of the selected test species .............................................................. 25 1.8 Anticancer ecotoxicology research in the literature at the commencement of this project .............................................................................................................................. 26 1.9 Ph.D. thesis overview ............................................................................................ 28 2 Impact of cytotoxic drugs on algal growth ................................................................... 29 2.1 Introduction ........................................................................................................... 29 2.1.1 Aims ................................................................................................................ 30 2.1.2 Hypothesis ...................................................................................................... 30 2.2 Methods & materials ............................................................................................. 30 2.2.1 Exposures to single cytotoxic drugs ............................................................... 31 2.2.2 Exposure to mixtures of cytotoxic drugs ....................................................... 33 2.3 Results ................................................................................................................... 34 2.3.1 Exposures to single cytotoxic drugs ............................................................... 34 2.3.2 Exposure to mixtures of cytotoxic drugs ....................................................... 36 2.4 Discussion .............................................................................................................. 37 2.4.1 Single drug exposures .................................................................................... 37 2.4.2 Results from the literature ............................................................................. 40 iii 2.4.3 Exposure to simple mixtures of cytotoxic drugs ............................................ 52 3 Impact of cytotoxics on Daphnia mobility .................................................................... 59 3.1 Introduction ........................................................................................................... 59 3.2 Methods & materials ............................................................................................. 60 3.2.1 Single chemical exposures ............................................................................. 61 3.2.2 Mixtures exposure ......................................................................................... 63 3.3 Results ................................................................................................................... 64 3.3.1 Single chemical exposures ............................................................................. 64 3.3.2 Mixtures experiments .................................................................................... 68 3.4 Discussion .............................................................................................................. 68 3.4.1 Single drug experiments ................................................................................ 68 3.4.2 Daphnia immobilisation and the effects of the target cytotoxics in the literature ....................................................................................................................... 72 3.4.3 Mixtures experiments .................................................................................... 78 4 Impact of cytotoxic drugs on zebrafish morphology & blood ...................................... 85 4.1 Introduction ........................................................................................................... 85 4.1.1 Aims ................................................................................................................ 85 4.1.2 Hypothesis ...................................................................................................... 86 4.2 Methods & materials ............................................................................................. 86 4.2.1 Exposure of zebrafish to cytotoxic drugs ....................................................... 86 4.2.2 Chemical analysis of aquarium water ............................................................ 95 iv 4.2.3 Single drug and simple mixture morphological endpoints ............................ 95 4.2.4 Single drug and simple mixture morphology endpoints ................................ 96 4.2.5 Single drug and simple mixture blood endpoints .......................................... 97 4.2.6 Fish blood plasma concentration calculations ............................................. 101 4.2.7 Statistical analysis techniques ...................................................................... 102 4.3 Results ................................................................................................................. 105 4.3.1 Single drug and simple mixture morphological endpoints results .............. 105 4.3.2 Single drug blood endpoint results .............................................................. 112 4.3.3 Measured drug concentrations in aquarium water samples....................... 116 4.3.4 Fish steady state plasma concentration calculations .................................. 119 4.4 Discussion ............................................................................................................ 121 4.4.1 Single drug and simple mixture morphological endpoints .......................... 121 4.4.2 Zebrafish morphology conclusions .............................................................. 140 4.4.3 Blood endpoints ........................................................................................... 141 4.4.4 Zebrafish blood endpoint conclusions ......................................................... 147 5 Impact of cytotoxic drugs on mRNA expression in adult zebrafish ............................ 149 5.1 Introduction ......................................................................................................... 149 5.1.1 Aims .............................................................................................................. 150 5.1.2 DNA damage enzymes RNA expression hypothesis .................................... 150 5.2 Selection of target and housekeeping genes ...................................................... 151 5.3 Quantifying proteins of interest .......................................................................... 155 v 5.4 Methods & materials ........................................................................................... 157 5.4.1 RNA extraction ............................................................................................
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