! INVESTIGATION OF PHYTOPLANKTON AS AN OVERLOOKED MARINE SOURCE OF NATURAL ENDOCRINE DISRUPTING CHEMICALS ! ! ! ! ! ! ! ! ! ! ! WANG XIAOCHONG ! ! ! ! ! ! ! ! NATIONAL UNIVERSITY OF SINGAPORE 2014 ! ! INVESTIGATION OF PHYTOPLANKTON AS AN OVERLOOKED MARINE SOURCE OF NATURAL ENDOCRINE DISRUPTING CHEMICALS ! ! ! ! ! WANG XIAOCHONG (MSc., TECHNISCHE UNIVERSITÄT MÜNCHEN) ! ! ! ! ! A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF OBSTETRICS & GYNAECOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2014 ! DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information that have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ! Wang!Xiaochong! 25!August!2014! ! ! ! ! ! ! ! ! ! ! ! I! ! ACKNOWLEDGEMENTS First and foremost, I would like to express my gratitude to my supervisors, Dr. Gong Yinhan for providing me the opportunity to embark on this valuable research experience and nurturing me over the years. I appreciate the unfailing support from Dr. Gong, and the tremendous amount of patience and guidance he has shown to me during the whole project. Next, I would like to thank all those people, especially to my lab mates who have helped in one way or another during the whole project. I would like to acknowledge the National University of Singapore (NUS) for financial support of this work, and I am grateful for the award of a research scholarship by the NUS. Last, but not least, I am especially grateful to my husband and my family for their encouragement, care, and support. ! II! ! TABLE!OF!CONTENTS! DECLARATION! .!I! ACKNOWLEDGEMENTS! .!II! SUMMARY! .!IX! LIST!OF!TABLES! !XIII! LIST!OF!FIGURES! .!XIV! LIST!OF!ABBREVIATION! .!XVII! CHAPTER!1! INTRODUCTION! 1.1! ENDOCRINEIDISRUPTING!CHEMICALS!(EDCS)! .!1! 1.1.1! Estrogenic!EndocrineIdisrupting!Chemicals! .!6! 1.1.2! Androgenic!EndocrineIdisrupting!Chemicals! .!10! 1.2! SEAWATER!ACTIVITY!OF!POLLUTANTS!AND!PHYTOPLANKTON! !11! 1.2.1! Measurements!of!Bioactivity!of!Seawaters! !11! 1.2.1.1! Individual!Measurement!of!Bioactivity!of!Seawaters!Samples! !13! 1.2.1.2! Total!Assays!of!Bioactivity!of!Seawaters!Samples! .!16! 1.2.2! Unexpected!High!Bioactivity!in!Singapore!Seawaters! .!19! 1.2.3! Phytoplankton! .!20! 1.3! CORRELATION!OF!WATERS!PARAMETERS!WITH!BIOACTIVITY! !23! 1.4! HYPOTHESIS!AND!GENERAL!OBJECTIVES! .!25! CHAPTER!2! INVESTIGATION!OF!THE!CORRELATION!BETWEEN!PHYTOPLANKTON!AND!SEX! HORMONE!RECEPTOR!BIOACTIVITIES!OF!THE!SINGAPORE!SEAWATERS!! 2.1! INTRODUCTION! .!27! 2.2! EXPERIMENTAL! .!28! 2.2.1! Materials!and!Apparatus! !28! 2.2.1.1! Chemical!and!Materials! .!28! 2.2.1.2! Apparatus!used!for!LCIMS!Analysis! .!30! 2.2.1.3! Apparatus!used!for!Bioassay!(Luciferase!activity)! .!31! 2.2.2! Human!CellIbased!Bioassays!(ERα,!ERβ!and!AR)!for!Measurement!of! Estrogen/Androgen!Receptor!Bioactivities!of!Phytoplankton!Extract! !31! 2.2.3! Seawater!Collection!(Sampling)! !33! 2.2.4! Phytoplankton!Culture!Medium!Preparation! !34! ! VIII! 2.2.4.1! Nature!Seawater!Culture!Medium!Preparation! !34! 2.2.4.2! Artificial!Seawater!Culture!Media!Preparation! !36! 2.2.5! Phytoplankton!Culture! !38! 2.2.5.1! Laboratory!Phytoplankton!Culture!Conditions! !38! 2.2.5.2! UpIscaling!of!Phytoplankton!Culture! !39! 2.2.5.3! Stock!Culture!Maintaining! .!39! 2.2.5.4! LargeIscale!Cultivation!of!Phytoplankton! .!41! 2.2.6! Phytoplankton!Growth!and!Bioactivity!Monitoring! .!42! 2.2.7! Harvest!of!the!Phytoplankton!Culture!Samples! .!43! 2.2.7.1! SPE!Extraction!of!Phytoplankton!Cultures! .!43! 2.2.7.2! Phytoplankton!Cell!Extraction! .!45! 2.2.8! LCIMS!Method!for!Phytoplankton!Crude!Media!Extracts! !47! 2.2.8.1! LCIMS!Method!for!Chat!M1!Extracts! .!48! 2.2.8.2! LCIMS!Method!for!PB3!Extracts! !49! 2.2.9! Statistics! .!50! 2.3! Results!and!discussion! !50! 2.3.1! Optimization!of!the!Phytoplankton!Culturing!Conditions! .!50! 2.3.1.1! Optimization!of!Culture!Materials! !50! 2.3.1.2! Cleaning!and!Sterilization!of!Culture!Materials!and!Media! .!51! 2.3.1.3! Phytoplankton!Culturing!Conditions! !53! 2.3.1.4! Water!Sources!for!Phytoplankton!Culture! !55! 2.3.2! Effect!of!Salinity!of!Culture!Medium!on!the!Growth!of!Phytoplankton! !55! 2.3.3! DoesIresponse!Assessment!of!E2!and!DHT! !60! 2.3.4! Optimization!of!Extraction!Methods!for!Bioactive!Compounds!(Secreted!by! Phytoplankton)! .!64! 2.3.5! Phytoplankton!Growth!Monitoring!(PB3)! !69! 2.3.6! Batches!of!Phytoplankton!MassIculture!Under!the!Optimized!Conditions!71! 2.3.7! Phytoplankton!Levels!Demonstrate!a!Positive!Correlation!with!AR!and!ERα! Bioactivities!in!Seawater!Samples! !77! 2.3.8! Identification!of!Phytoplankton!Species!Related!to!Estrogenic!and! Androgenic!Bioactivity! !84! 2.3.9! 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(around the mainland of Singapore) and a large amount of phytoplankton cultures were successfully cultivated in our research laboratory under controlled conditions The phytoplankton cells and culture media were extracted and screened for estrogenic and androgenic activities via human cell-based bioassays The extracts of phytoplankton cultures were purified and fractionated by a series of chromatographic... C.L., 2008; Tanabe S., 2002; Andrea C.G., 2008), e.g placental and ovarian function An endocrine- disrupting chemical is defined by the World Health Organization (2002) as an exogenous substance or mixture that alters function(s) of the endocrine system and consequently produces adverse health effects in an intact organism, or its progeny, or (sub) populations” Pollutants including pesticides, natural products,... suggest that phytoplankton can secrete some estrogenic/androgenic endocrine disrupting chemicals (EDCs) into marine environment However, the chemical and biological properties of the secreted chemicals are still unknown In order to investigate the properties of the secreted compounds, five species of phytoplankton, Gymnodinium catenatum, Prorocentrum minimum, Alexandrium leei, Chattonella marina, and Fibrocapsa... filtration! system! in! Tropical Marine Science Institute (TMSI) Figure 2.2 Schematic of stock phytoplankton cells maintaining Figure 2.3 EGS incubator for maintaining stocks of phytoplankton Figure 2.4 EGS incubator used for mass culture of phytoplankton Figure 2.5 Procedure of Solid Phase Extraction (SPE) Figure 2.6 Procedure of Phytoplankton cell extract Figure 2.7 Comparison of PB3 growth in two different... batch of seawater samples from the same sample sites (AR bioactivity) Figure 2.17 Bioactivity of different phytoplankton culture extracts Figure 2.18 Growth curves and bioactivity profiling of Singapore phytoplankton isolates C marina (Chat M1) Figure 2.19 Growth curves and bioactivity profiling of Singapore phytoplankton isolates P minimum (PB3) Figure 2.20 Growth curves and bioactivity profiling of. .. phytoplankton are one of natural marine sources of endocrinedisrupting chemicals (EDCs) The harmful nature of EDCs may be largely due to their bioaccumulation in the aquatic food chain As such, these findings indicated that EDCs from phytoplankton sources needed to be thoroughly investigated as they may have significant impact on the food chain, especially our food sources from the sea ! X! ! LIST !OF! PUBLICATIONS!ARISING!FROM!THIS!THESIS!... High estrogenic and androgenic activities for seawater samples were previously reported in the confined clusters close to the mainland of Singapore Further investigations revealed a hitherto unsuspected link between estrogenic/androgenic activity and phytoplankton Phytoplankton is a type of microscopic organism in the marine environment, which forms the foundation of the food chain in the marine ecological... effects of Epimedium extracts on breast cancer and uterine growth in nude mice, Planta Medi., 80 (2014), pp 22-28 3 Tan H.M., Wang X.C.* (joint first author), Soh S.F., Tan S., Zhao J Yong E L., Lee H.K., and Gong Y.H., Preparation and application of mixed octadecysilyl- and (3-(C-methylcalix[4]resorcinarene)-2- hydroxypropoxy)-propylsilyl-appended silica particles as stationary phase for high-performance... germination and stress signalling (Ruiz-Larrea M.B et al., 1997; Wei H et al., 2003; Manthey J.A et al., 2002) Phytoestrogens not only can be detected in certain plant or plantderived products, but are also found in water as a result of plant decomposition They are represented by hundreds of different types of molecules and can be divided into three main classes based on their ! 7! chromone backbones and hydroxylation . ! INVESTIGATION OF PHYTOPLANKTON AS AN OVERLOOKED MARINE SOURCE OF NATURAL ENDOCRINE DISRUPTING CHEMICALS ! ! ! ! ! ! ! ! ! ! ! WANG XIAOCHONG ! ! ! ! ! ! ! ! NATIONAL UNIVERSITY OF. OF SINGAPORE 2014 ! ! INVESTIGATION OF PHYTOPLANKTON AS AN OVERLOOKED MARINE SOURCE OF NATURAL ENDOCRINE DISRUPTING CHEMICALS ! ! ! ! ! WANG XIAOCHONG (MSc., TECHNISCHE UNIVERSITÄT. Yong E.L., Phytoplankton blooms: an overlooked marine source of natural endocrine disrupting chemicals, Ecotox. Environ. Safe., 107 (2014), pp. 126-132. 2. Indran I.R., Zhang S., Zhang Z.,