The bioeffects of a high frequency electromagnetic field in the microwave range Submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy By The Hong Phong Nguyen Faculty of Science, Engineering and Technology Swinburne University of Technology 2018 ABSTRACT The World Health Organization (WHO) has stated that there is no evidence available to confirm the existence of any health consequences arising from human exposure to low level EMFs. Some gaps in knowledge exist regarding the biological effects that may be triggered by exposure to high levels of EMFs and hence these possible effects require further research. Notwithstanding the sterilization/inactivation effects that come about from EMF exposure, a number of specific effects have been observed that cannot be explained by the increase in bulk temperature that occurs through EMF exposure alone. The exact mechanism/s of those effects is/are not fully understood and therefore have been the subject of debate. In this thesis, the effects of high frequency (18 GHz) EMF exposure have been studied using typical representatives of prokaryotic and eukaryotic taxa, including two Gram-negative bacteria (Branhamella catarrhalis ATCC 23246 and Escherichia coli ATCC 15034), six Gram-positive bacteria (Kocuria rosea CIP 71.15T, Planococcus maritimus KMM 3738, Staphylococcus aureus CIP 65.8T, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 14990T, and Streptomyces griseus ATCC 23915), a eukaryotic unicellular organism (yeast Saccharomyces cerevisiae ATCC 287) and red blood cells obtained from a New Zealand rabbit. Three parameters of the EMF exposure (power, duration, and exposure number) were varied to determine their effect on the biological system being studied. Advanced microscopy techniques were employed, comprising Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Confocal Scanning Laser Microscopy (CSLM) together with fluorescent probes, in order to allow a thorough examination of the cell membrane morphology and permeability following EMF exposure/s to be studied. It was determined, for the first time, that regardless of the differences in cell wall/ membrane structure, exposure to 18 GHz EMF induced cell permeabilization, as confirmed via the ability of the cells to uptake silica nanospheres (23 nm and 46 nm in diameter), in all the cell types studied, as confirmed directly by transmission electron microscopy (TEM) and indirectly by confocal laser scanning microscopy (CLSM). A dosimetry analysis revealed that the EMF exposure required to induce cell permeation such that the membrane was able to uptake 23 nm and 46 nm nanospheres was between three and six EMF doses with a specific absorption rate (SAR) of 5 kW/kg and 3 kW/kg per exposure, respectively, depending on the cell types being studied. This specific EMF bioeffects could not be duplicated using conventional heating methods under similar temperature conditions. The cells remained viable (85%) and permeable for at least nine minutes after EMF exposures. It is suggested that the taxonomic affiliation and cell wall/membrane structures (e.g., the presence of peptidoglycan layer, mannoprotein/β-glucan layer, phosphatidyl-glycerol and/or pentadecanoic fatty acid) may affect the extent of permeabilization to allow the uptake of 46 nm nanopsheres. A new mechanism of cell permeability using 18 GHz EMF is postulated; EMF exposure disturbs the stability of the charged lipid bilayers within the cell membrane, thus affecting its fluidity. This mechanical stimulation, in turn, alters the tension of the membrane, causing it to deform, which results in an enhanced opportunity for transport through the membrane, which occurs via a quasi- exocytosis/endocytosis process. ACKNOWLEDGMENTS First and foremost, I would like to express my deepest and most sincere gratitude to my mentors and supervisors: Professor Elena Ivanova for her infinite support and encouragement in conducting research as well as writing for publications; Professor Russell J. Crawford for his supervision and guidance throughout my candidature. Without their support, none of this would have been possible and for which I am extremely grateful. Secondly, I would like to thank Professor Rodney J. Croft from University of Wollongong, Dr. Vladimir A. Baulin from Universitat Rovira I Virgili, Professor Andrew W. Wood and Dr. Robert L. McIntosh from Swinburne University of Technology for their guidance and useful advice in this research. I would like to sincerely thank my parents, siblings, relatives and friends for their perpetual love and unconditioned support during my PhD. Finally, I would also like to extend my gratitude to Dr. Vi Khanh Truong, Dr. Yury Shamis, Dr. Hayden K. Webb, Dr. Jafar Hasan, Dr. Thi Song Ha Nguyen, Dr. Thi Hong Vy Pham for their immense academic and emotional support throughout my research. Dr. James Wang from Faculty of Science, Engineering and Technology, Swinburne University of Technology assisted me in the use of Scanning Electron Microscope. Dr. Igor Sbarski from Faculty of Science, Engineering and Technology, Swinburne University of Technology provided me technical support in the use of Peltier plate. Mr. Phil Francis, Dr. Matthew Field, and Dr. Chaitali Dekiwadia from the RMIT Microscopy and Microanalysis Facility, RMIT University assisted me in the use of Transmission Electron Microscope and sample preparations. Associate Professor Brian Phillips from Faculty of Health, Arts and Design, Swinburne University of Technology provided me valuable statistics advice and support in the use of SPSS software. DECLARATION I, The Hong Phong Nguyen, declare that this thesis is my original work and contains no material which has been submitted for the award of any other degree or diploma from another university. To the best of my knowledge, I certify that this thesis contains no material previously published or written by another person except where due reference has been made. Wherever contributions of others were involved, every effort has been made to acknowledge contribution of the respective workers or authors. The Hong Phong Nguyen LIST OF PUBLICATIONS Peer-reviewed articles Nguyen, T. H. P., Pham, T. H. V., Baulin, V., Croft, R. J., Crawford, R. J. & Ivanova, E. P. (2017) The effect of a high frequency electromagnetic field in the microwave range on red blood cells. Sci Rep 7, 10798. Nguyen, T. H. P., Pham, T. H. V., Nguyen, S. H., Baulin, V., Croft, R. J., Phillips, B., Crawford, R. J. & Ivanova, E. P. (2016) The bioeffects resulting from prokaryotic cells and yeast being exposed to an 18 GHz electromagnetic field. PLoS ONE 11, e0158135. Nguyen, T. H. P., Shamis, Y., Croft, R. J., Wood, A., McIntosh, R. L., Crawford, R. J. & Ivanova, E. P. (2015) 18 GHz electromagnetic field induces permeability of Gram-positive cocci. Sci Rep 5, 10980. Pogodin, S., Hasan, J., Baulin, V. A., Webb, H. K., Truong, V. K., Nguyen, T. H. P., Boshkovikj, V., Fluke, C. J., Watson, G. S., Watson, J. A., Crawford, R. J. & Ivanova, E. P. (2013) Biophysical model of bacterial cell interactions with nanopatterned cicada wing surfaces. Biophys J 104, 835-840. Conference poster presentations with published abstracts Nguyen, T. H. P., Perera, P. G. T., Pham, T. H. V., Croft, R., Crawford, R. J. & Ivanova, E. P. Super High Frequency (18 GHz) electromagnetic field induced membrane permeability on erythrocytes and PC-12 neuronal cells. Science and wireless 2016, 2016 Melbourne, Australia. Nguyen, T. H. P., Shamis, Y., Croft, R., Crawford, R. J. & Ivanova, E. P. Specific electromagnetic effects of microwave radiation on bacteria. 34th Annual Meeting of the Bioelectromagnetics Society, 2012 Brisbane, Australia. TABLE OF CONTENTS ABSTRACT ............................................................................................................ 2 ACKNOWLEDGMENTS ....................................................................................... 4 DECLARATION ..................................................................................................... 6 LIST OF PUBLICATIONS ..................................................................................... 7 LIST OF TABLES ................................................................................................ 12 LIST OF FIGURES ............................................................................................... 13 Chapter 1. Introduction ........................................................................................... 22 1.1. Overview ..................................................................................................... 23 1.2. The aims ...................................................................................................... 25 Chapter 2. Literature review ................................................................................... 27 2.1. Overview ..................................................................................................... 28 2.2. The nature of electromagnetic fields (EMFs) ............................................. 28 2.2.1. Electromagnetic spectrum ................................................................... 28 2.2.2. Electromagnetic fields ........................................................................ 29 2.2.3. Electromagnetic phenomena ............................................................... 33 2.2.4. Dosimetry ............................................................................................ 34 2.2.5. Operational