Assessing Toxicity In Daphnia Magna: An Oxidative .

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Assessing toxicity in Daphnia magna:an oxidative lipidomic approachThomas Andrew WhiteA thesis submitted to theUniversity of Birmingham for the degree ofDOCTOR OF PHILOSOPHYSchool of BiosciencesUniversity of BirminghamSeptember 2013

University of Birmingham Research Archivee-theses repositoryThis unpublished thesis/dissertation is copyright of the author and/or thirdparties. The intellectual property rights of the author or third parties in respectof this work are as defined by The Copyright Designs and Patents Act 1988 oras modified by any successor legislation.Any use made of information contained in this thesis/dissertation must be inaccordance with that legislation and must be properly acknowledged. Furtherdistribution or reproduction in any format is prohibited without the permissionof the copyright holder.

AbstractLipidomics, an under-utilised and rapidly developing field aims to identify the fullcomplement of hydrophobic constituents in a cell, tissue or organism. Lipidperoxidation, a major consequence of oxidative stress, represents a mechanisticallyilluminating marker for numerous toxicants. Lipidomics offers the ideal technique toacquire a greater level of mechanistic detail compared to currently utilisedmethodologies. Here, I present a study into lipid peroxidation from simple in vitromodels to complex in vivo systems utilising mass spectrometric techniques. Initially,oxidised products from a systematic range of phospholipids were induced andcomprehensively annotated to allow the development of OxyLipidBlast. This is thefirst tool facilitating the identification of oxidised lipids and provides utility to numerousfields. Secondly I present the first annotated lipidome of the keystoneecotoxicological species Daphnia magna and the first annotated lipidome of algalspecies Chlamydomonas reinhardtii published in English. Subsequent oxidation invitro of lipid extracts yielded perturbations, biologically relevant to the following in vivoexposures with well established toxicants and novel silver nanoparticles. Overall thework presented in this thesis enhances both eco and oxidative lipidomics. However,these studies also highlighted the limitations of shotgun lipidomics for ecotoxicologyassessment.ii

This thesis is dedicated to Anthony White.iii

AcknowledgementsI would like to acknowledge the contributions of the following people to this thesis.Professor Mark Viant for all the enthusiastic and encouraging meetings despite theirtardiness. All the people at Cefas including Dr. Brett Lyons for financial support,encouragement and breaks to the seaside. Dr. Ulf Sommer for his instruction on theFT-ICR. Dr. Tobias Kind for his input to the oxidation experiments. Dr. Nadine Taylorfor her culturing, exposure and extraction of C. reinhardtii. Dr. Isabella Rӧmer, Dr.Ruth Merrifield, soon to be Dr. Mila Tejamaya and other members of the Lead groupfor their help with the alchemic science of nanoparticles.In more personal terms my office mates in reverse chronological order for friendshipand academic advice, Ralf, Martin, Rosie, Kate, Eva, Leanne, Alex, Andy, Nadine,Stefania, Anja, Lisa. All members of Mark’s group during my studies, particularly Roband Ralf for always being ready for a beer and considerable support. Greg for lettingme win at babyfoot occasionally. Andy for never showing frustration with mypersistent questions. Kate for being a big orange dot. Alex for all your help withnanoparticles and water fleas. Outside our lab. Chib (I miss your hiccups), Laura,Lauren and Farhat thank-you for making my lab time more bearable.More personally. Dr. Kaat Brulez, I almost certainly would have quit without you towhinge to. Jon (BSc, PGCE), Leigh (A-levels), Tread (GCSEs), Simo (50m swimmingcertificate) and Baz (something boring about building supermarkets?), on our tinanniversary, I love you all but none of you are ever going to be more qualified thanme. Rosie, this thesis wouldn’t exist without you, when it got really tense toward theiv

end, your support and encouragement was pretty much the only thing holding meback from the abyss, thank-you.I’ll never forget how important you all have been to my work and to me.Finally, this thesis marks the end of my formal education and four years of drainingeffort. More broadly, this thesis represents the support of my family both emotionallyand financially throughout my entire life. I will never be able to appropriately expresshow much the encouragement to pursue whatever I wished and the reassurance thatI can do anything I choose if I work hard enough has meant to me.If.v

List of contents1 - Introduction . 11.1 - Aquatic ecotoxicology. 11.1.1 - Model organisms for aquatic toxicology . 21.1.1.1 - Daphnia magna . 31.1.1.2 - Chlamydomonas reinhardtii . 61.1.2 - Aquatic contaminants . 81.1.2.1 - Nano-materials . 81.1.2.2 - Silver nanoparticles (AgNP) . 91.2 - Phospholipids . 111.2.1 - Phospholipid structure and nomenclature . 111.2.2 - Phospholipids in D. magna . 141.2.3 - Phospholipids in C. reinhardtii . 171.3 - Non-enzymatic toxic mediated lipid peroxidation . 171.4 - The emerging role of metabolomics in ecotoxicology . 191.4.1 - Environmental metabolomics . 191.4.2 - Lipidomics . 201.4.2.1 - Lipid extraction from biological tissue . 211.4.2.2 - Pre-analytical, chromatographic separation of lipids . 211.4.2.3 - Analytical platforms for lipidomic analysis . 221.4.2.4 - Statistical analyses in metabolomic studies. 251.4.2.5 - Identification of metabolites . 26vi

1.5 - Aim and objectives . 282 - Materials and methods. 292.1 - Chemicals . 292.2 - Culturing of Daphnia magna . 292.2.1 - OECD modified media preparation for culturing of Daphnia magna. 302.2.2 - Supplement preparation and regimen . 302.2.2.1 - Chlorella vulgaris . 302.2.2.2 - Baker’s yeast . 322.2.2.3 - Marinure . 322.3 - Acute toxicity assessment . 322.3.1 - Exposures . 322.3.2 - EC50 calculation . 332.4 - General workflow to obtain, analyse and process shotgunlipidomic samples . 332.4.1 - Exposures . 342.4.2 - Metabolite extraction . 342.4.3 - Fourier transform ion cyclotron resonance mass spectrometry . 352.4.3.1 - Sample preparation for mass spectrometry . 352.4.3.2 - Acquisition of mass spectra . 362.4.4 - Processing mass spectral data to increase reliability and allowstatistical analysis . 38vii

2.4.5 - Putative metabolite identification . 392.4.6 - Statistical analysis of lipidomic spectra . 402.4.6.1 - Univariate comparison of individual spectral feature intensities . 402.4.6.2 - Multivariate comparison of spectral feature intensities . 403 - In vitro oxidation of lipid standards for creation of OxyLipidBlastdatabase tool . 413.1 - Introduction . 413.2 - Materials and methods . 433.2.1 - Molecular structure of PE lipid standards . 433.2.2 - Oxidation of PE species . 433.2.3 - Analysis of lipid species and oxidised products by FT-ICR and LTQIT mass spectrometry. . 443.2.4 - Peak assignment . 453.2.5 - Creation of OxyLipidBlast conducted by Dr. Tobias Kind (UC Davis). 453.2.6 - Validation of the OxyLipidBlast database . 463.3 - Results and discussion . 473.3.1 - Oxidation time-course . 553.3.2 - Annotation and interpretation of fragmentation patterns . 563.3.2.1 - Unmodified PE (positive ion mode) . 573.3.2.2 - Oxidised PE (positive ion mode) . 583.3.2.3 - Unmodified PE (negative ion mode) . 63viii

3.3.2.4 - Oxidised PE (negative ion mode) . 643.3.3 - Utilisation of OxyLipidBlast to identify oxidised products . 693.3.4 - Validation of the OxyLipidBlast database . 703.4 - Conclusions. 734 - Optimisation of mass spectrometric lipidomics and baselinelipidome characterisation of D. magna and C. reinhardtii . 754.1 - Introduction . 754.2 - Materials and methods . 764.2.1 - Optimising the preparation of samples for lipidomics analysis . 764.2.2 - Optimising the concentration of D. magna and C. reinhardtiiextracts for mass spectrometric analysis in collaboration with Dr. NadineTaylor (U of Birmingham) . 784.2.3 - Definitive identification of selected lipids to form a robust calibrationlist in collaboration with Dr. Nadine Taylor (U of Birmingham). 794.2.4 - Annotation of baseline lipidomes of D. magna and C. reinhardtii . 804.3 - Results and discussion . 814.3.1 - Optimisation of the sample preparation methodology for massspectrometric lipidomic analysis . 814.3.2 - Optimisation of the concentration of D. magna and C. reinhardtiiextracts for mass spectrometric analysis . 834.3.3 - Creation of calibration list from identified lipids. 864.3.4 - Annotation of baseline lipidomes of D. magna and C. reinhardtii . 89ix

4.4 - Conclusions. 1105 - Oxidation of the D. magna and C. reinhardtii lipidomes by in vitroand in vivo stressors . 1125.1 - Introduction . 1125.2 - Materials and methods . 1145.2.1 - In vitro oxidation of lipid extracts of D. magna and C. reinhardtii . 1145.2.1.1 - Oxidation of D. magna and C. reinhardtii lipid extracts byexposure to air . 1145.2.1.2 - Oxidation of D. magna lipid extracts by hydrogen peroxide andcopper chloride . 1155.2.1.3 - Putative annotation of potential oxidation . 1155.2.2 - Assessing the acute toxicity of AgNO3, and H2O2 to D. magna andC. reinhardtii . 1165.2.3 - In vivo exposure of D. magna and C. reinhardtii to oxidativestressors . 1175.3 - Results and discussion . 1185.3.1 - In vitro oxidation of D. magna and C. reinhardtii lipid extracts . 1185.3.2 - Acute toxicity of AgNO3 and H2O2 to D. magna and C. reinhardtii. 1315.3.3 - Lipidomic responses of D. magna and C. reinhardtii to in vivooxidative stressors . 1345.4 - Conclusions. 144x

6 - Lipidomic analysis of silver nanoparticle toxicity in D. magnafollowing extensive exposure optimisation . 1466.1 - Introduction . 1466.2 - Materials and methods . 1486.2.1 - Acid washing of equipment. 1486.2.2 - Silver nanoparticle preparation conducted by Dr. Isabella Rӧmerand Mila Tejamaya (U of Birmingham) . 1486.2.2.1 - Silver nanoparticle characterisation . 1496.2.3 - Media dilution acute stress assessment . 1496.2.4 - Media dilution chronic stress assessment . 1506.2.5 - Acute toxicity conducted in collaboration with Alex Gavin (U ofBirmingham) . 1506.2.6 - Nanoparticle dissolution conducted in collaboration with Alex Gavin(U of Birmingham) . 1516.2.6.1 - Ultracentrifugation separation of ionic and nano silver . 1516.2.6.2 - Ultrafiltration separation of ionic and nano silver . 1526.2.6.3 - Inductively coupled plasma mass spectrometry analysis of silverconducted by Dr Steve Baker (U of Birmingham) . 1526.2.6.4 - Calculation of nano and ionic silver concentrations from ICP-MSresults . 1526.2.7 - Lipidomic analysis of D. magna exposed to PVP capped silvernanoparticles conducted in collaboration with Alex Gavin . 1546.3 - Results and discussion . 155xi

6.3.1 - Silver nanoparticle characterisation. 1556.3.2 - Acute effects of diluting culture media on D. magna . 1576.3.3 - Chronic effects of diluting culture media on D. magna . 1576.3.4 - Acute toxicity of silver nanoparticles capped by citrate and PVP toD. magna . 1586.3.5 - Dissolution of silver nanoparticles over the time-course of an acuteexposure . 1616.3.6 - Lipidomic perturbations caused by silver nanoparticles to D. magna. 1646.4 - Conclusions. 1737 - General conclusions and future work. 1758 - References . 180A - Appendix . 197AI - Tables showing mass spectra from oxidised PE species . 197AI-I - Tables showing mass spectra of oxidised PE(14:0,0:0) in negativeand positive ion mode. 197AI-II - Tables showing mass spectra of oxidised PE(18:1,0:0) in negativeand positive ion mode. 198AI-III - Tables showing mass spectra of oxidised PE(14:0,14:0) in negativeand positive ion mode. 199AI-IV - Tables showing mass spectra of oxidised PE(P-18,18:1) innegative and positive ion mode . 200xii

AI-V - Tables showing mass spectra of oxidised PE(18:1,18:1) in negativeand positive ion mode. 201AI-VI - Tables showing mass spectra of oxidised PE(16:0,20:4) in negativeand positive ion mode. 203AII - Manually annotated fragmentation patterns from oxidised PEproducts . 205AII-I - Fragmentation patterns from PE(14:0,0:0) and oxidised productsthereof in negative ion mode . 205AII-II - Fragmentation patterns from PE(14:0,0:0) and oxidised productsthereof in positive ion mode . 206AII-III - Fragmentation patterns from PE(18:1,0:0) and oxidised productsthereof in negative ion mode . 206AII-IV - Fragmentation patterns from PE(18:1,0:0) and oxidised productsthereof in positive ion mode . 208AII-V - Fragmentation patterns from PE(14:0,14:0) and oxidised productsthereof in negative ion mode . 210AII-VI - Fragmentation patterns from PE(14:0,14:0) and oxidised productsthereof in positive ion mode . 211AII-VII - Fragment

ecotoxicological species Daphnia magna and the first annotated lipidome of algal . these studies also highlighted the limitations of shotgun lipidomics for ecotoxicology . 5.2.2 - Assessing the acute toxicity of AgNO 3, and H 2O2 to D. magna and

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