UNIVERSITY OF CALGARY. Interactions of Inorganic Mercury and Inorganic Cadmium with Biomimetic and Complex

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1 UNIVERSITY OF CALGARY Interactions of Inorganic Mercury and Inorganic Cadmium with Biomimetic and Complex Biological Membranes and their Influence on Membrane Packing and Size by Evan Kerek A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOLOGICAL SCIENCES CALGARY, ALBERTA MAY, 2016 Evan Kerek 2016

2 Abstract Inorganic mercury (Hg 2+ ) and Inorganic Cadmium (Cd 2+ ) are toxic heavy metals linked to the development of cancer, diabetes and neurological dysfunctions. The effect of these metals on the fluidity and phase transition (Tm) of biomimetic and polar extract membranes was investigated using Laurdan Generalized Polarization (GP) and Dynamic Light Scattering (DLS). Hg 2+ and Cd 2+ electrostatically target and induce rigidity in membranes containing cationic and anionic lipids respectively. Hg 2+ also imparts rigidity by acting as a catalyst in the vinyl ether hydrolysis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) plasmalogens. Cd 2+ - induced rigidity of anionic membranes results in a stabilization of the gel phase and a suppression of the Tm of membranes composed of phosphatidic acid (PA), cardiolipin (CL), phosphatidylserine (PS), phosphatidylglycerol (PG) and phosphatidylinositol (PI). Cd 2+ induces more rigidity in rigid anionic membranes compared to more fluid anionic membranes. These results further our understanding of metal-lipid interactions. ii

3 Acknowledgements I would like to thank my supervisor, Dr. Elmar Prenner for taking me on as a summer research student, Honors project student and as a Graduate student. Your advice and guidance when experiments were not working and hands-off style supervision when I started doing well was much appreciated. I am extremely thankful for being given such a complex and unique project which allowed me to showcase what I could accomplish in the lab. I am also very appreciative for being given the rare opportunity to attend two once in a lifetime experiences at the Conferences in Parry Sound and Sassari. In addition to my supervisor I would also like to thank my committee member Dr. Jürgen Gailer for his mentorship during the CanBic and Cadmium Symposium Conferences and Dr. Vanina Zaremberg for her experimental suggestions and expertise on the subject of membrane lipid compositions. I would also like to thank Dr. Sutherland and Dr. Edwards for taking on the respective roles of external examiner and neutral chair. Last but not least I would like to thank my lab colleague Mohamed Hassanin for passing on such an exciting project to me. The initial experiments done by Mohamed were essential in building the groundwork for the experiments I would be able to plan and conduct. I would also like to thank another lab colleague Dr. Patrick Lai for his help with ITC, DLS, Zeta Potential and experiments with the lysosomes and the rest of the Prenner lab for their support. iii

4 Table of Contents Abstract... ii Acknowledgements... iii Table of Contents... iv List of Tables... viii List of Figures... ix List of Abbreviations... xviii Chapter One: Introduction Hg and Cd targets in mammalian systems Hg and Cd speciation in aqueous solution Membrane Lipid Bilayers Phospholipid Nomenclature Membrane Lipids Cholesterol Membrane Lipid Profiles Brain Membrane Lipids Liver Membrane Lipids Heart Membrane Lipids RBC Membrane Lipids E. coli Membrane Lipids Fatty acid compositions of membrane bilayers Lipid Bilayer Asymmetry The effect of Hg and Cd on Membrane Fluidity Hg and Cd target different lipid classes The effect of Hg and Cd on Lipid Bilayer size Hypothesis Detailed Project Goals...31 Chapter Two: Materials and Methods Laurdan Generalized Polarization Introduction to Laurdan Laurdan in Membranes Generalized Polarization Lipid System Preparation Materials Lipid Structures LUV systems Large Unilamellar Vesicle Preparation Lipid Concentration Determination Schiefer and Neuhoff Phospholipid Determination Assay Ames Phosphate Determination Assay Determination of Total Lipid Concentration RBC Ghost Preparation...53 iv

5 2.2.7 RBC Ghost Total Lipid Extraction Laurdan GP Fluorescence Spectroscopy Trials Laurdan General Polarization Procedure Dynamic Light Scattering (DLS) Trials LUV Size Determination Thin Layer Chromatography (TLC) Zeta Potential Measurements...61 Chapter Three: The effect of Cd on the fluidity and size of biomimetic liposomes LUV Size Determination using DLS Interactions of Hg and Cd with Laurdan Interactions of Cd with membranes containing POPC and Cholesterol Laurdan GP with Cd and LUVs containing POPC and Cholesterol DLS with Cd and LUVs containing POPC and Cholesterol Discussion of the interactions of Cd with LUVs containing POPC and...72 Cholesterol 3.4 Interactions of Cd with membranes sulfatide and ganglioside Laurdan GP with Cd and LUVs containing sulfatide and ganglioside DLS with Cd and LUVs containing sulfatide and ganglioside Discussion of the interactions of Cd with LUVs containing sulfatide and...77 ganglioside 3.5 Interactions of Cd with phosphatidic acid (PA) membranes Laurdan GP with Cd and POPA and DMPA LUVs DLS with Cd and POPA and DMPA LUVs Discussion of the interactions of Cd with POPA and DMPA LUVs Interactions of Cd with Cardiolipin (CL) membranes Laurdan GP with Cd and TOCL and TMCL LUVs DLS with Cd and TOCL and TMCL LUVs Discussion of the interactions of Cd with TOCL and TMCL LUVs Interactions of Cd with phosphatidylglycerol (PG) membranes Laurdan GP with Cd and POPG and DMPG LUVs DLS with Cd and POPG and DMPG LUVs Discussions of the interactions of Cd with POPG and DMPG LUVs Interactions of Cd with phosphatidylserine (PS) membranes Laurdan GP with Cd and POPS and DMPS LUVs DLS with Cd and POPS and DMPS LUVs Discussions of the interactions of Cd with POPS and DMPS LUVs Interactions of Cd with membranes containing phosphatidylinositol (PI) Laurdan GP with Cd and LUVs containing PI(3)P and PI(3,5)2P DLS with Cd and LUVs containing PI(3)P and PI(3,5)2P Discussion of the interactions of Cd with LUVs containing PI(3)P and PI(3,5)2P 3.10 A summary of the interactions of Cd with biomimetic LUVs v

6 Chapter Four: The effect of Hg on the fluidity and size of biomimetic liposomes Interactions of Hg with POPC and sphingomyelin membranes Laurdan GP with Hg and POPC and sphingomyelin LUVs DLS with Hg and POPC and sphingomyelin LUVs Discussion of the interactions of Hg with POPC and sphingomyelin LUVs Interactions of Hg with membranes containing cholesterol and DOTAP Laurdan GP with Hg and LUVS containing cholesterol and DOTAP DLS with Hg and LUVs containing cholesterol and DOTAP Discussion of the interactions of Hg with membranes containing cholesterol and DOTAP 4.3 Interactions of Hg with membranes containing ceramide and cerebroside Laurdan GP with Hg and LUVs containing ceramide and cerebroside DLS with Hg and LUVs containing ceramide and cerebroside Discussion of the interactions of Hg with LUVs containing ceramide and..129 cerebroside 4.4 Interactions of Hg with anionic membranes Laurdan GP with Hg and POPG and DMPG LUVs DLS with Hg and POPG and DMPG LUVs Discussion of the interactions of Hg with anionic LUVs Interactions of Hg with PC plasmalogens Laurdan GP with Hg and PC plasmalogen LUVs DLS with Hg and PC plasmalogen LUVs Discussion of the interactions of Hg with plasmalogens A comparison of interactions of Hg with plasmalogen and DOTAP Laurdan GP with Hg and LUVs containing PE plasmalogen and DOTAP DLS with Hg and LUVs containing PE plasmalogen and DOTAP A summary of interactions of Hg with biomimetic membranes The Biological Significance of plasmalogen and Hg Chapter Five: The effects of Hg and Cd on the fluidity and size of polar lipid extracts and RBC ghosts 5.1 Interactions of Hg and Cd with Brain polar extract (porcine) Laurdan GP with Hg, Cd and Brain polar extract LUVs DLS with Hg, Cd and Brain polar extract LUVs Discussion of the interactions of Hg and Cd with Brain polar extract LUVs 5.2 Interactions of Hg and Cd with 60 POPS 40 PC plasmalogen Laurdan GP with Hg, Cd and 60 POPS 40 PC plasmalogen LUVs DLS with Hg, Cd and 60 POPS 40 PC plasmalogen LUVs Discussion of the interactions of Hg and Cd with 60 POPS 40 PC plasmalogen LUVs 5.3 Interactions of Hg and Cd with Liver polar extract (bovine) Laurdan GP with Hg, Cd and Liver polar extract LUVs vi

7 5.3.2 DLS with Hg, Cd and Liver polar extract LUVs Discussion of the interactions of Hg and Cd with Liver polar extract LUVs 5.4 Interactions of Hg and Cd with Heart polar extract (bovine) Laurdan GP with Hg, Cd and Heart polar extract LUVs DLS with Hg, Cd and Heart polar extract LUVs Discussion of the interactions of Hg and Cd with Heart polar extract LUVs 5.5 Interactions of Hg and Cd with E. coli polar extract Laurdan GP with Hg, Cd and E. coli polar extract LUVs DLS with Hg, Cd and E. coli polar extract LUVs Discussion of the interactions of Hg and Cd with E. coli polar extract LUVs 5.6 Interactions of Hg and Cd with RBC ghosts Fluorescence spectra of RBC ghosts Laurdan GP with Hg, Cd and RBC ghosts DLS with Hg, Cd and RBC ghosts Laurdan GP with Hg, Cd and asymmetrical RBC ghosts Discussion of the interactions of Hg and Cd with RBC ghosts Interactions of Hg and Cd with RBC total extract Laurdan GP with Hg, Cd and RBC total extract LUVs DLS with Hg, Cd and RBC total extract LUVs Discussion of the interactions of Hg and Cd with RBC total extract LUVs Chapter Six: Conclusions Future Directions References Appendix vii

8 List of Tables Table 1. The predominant species of Hg and Cd present under physiological conditions of 100 mm NaCl and ph 7.40 which were also used in this thesis....5 Table 2. The common, IUPAC and carboxyl reference names for the fatty acid that are components of the lipids used in this thesis along with the abbreviations by which they will be referred to....8 Table 3. The fatty acid composition of different rat tissues expressed as a percentage of total fatty acids. This table is adapted from Abbot et al (2012)...20 Table 4. The fatty acid composition of E. coli lipids expressed as a percentage of the total fatty acids for each respective lipid. Data is adapted from Avanti Polar Lipids...21 Table 5. A summary of the effect of Hg and Cd on the Tm of anionic PS, PG and PA membranes adapted from Payliss et al. The table summarizes DPH fluorescence anisotropy data collected for MLVs under various conditions as described in the legend below the table. MLVs contain multiple bilayers before extrusion (See Figure 17 in Chapter Two) Table 6. The lipid systems used in laurdan GP and DLS experiments viii

9 List of Figures Figure 1. The possible interaction sites between inorganic Hg and Cd with the components of the bloodstream as well as different targets inside and outside the cell. (Hg/Cd)/Cl2 refers to the neutral chloride species HgCl2 and CdCl2 which may be able to diffuse directly across the lipid bilayer and interact with intracellular targets...4 Figure 2. The speciation of Hg and Cd as a function of chloride concentration and ph in aqueous solution using 1.0 mm Hg/Cd. Curves were determined using thermodynamic parameters from Visual Minteq 3.1 software at 37ºC. The ph curves were determinedat 100 mm NaCl and the chloride curves were determined at ph Figure 3. The general structure of glycerol based phospholipids. The dotted blue box highlights the ester, ether and vinyl ether linkages present in the diacyl, alkyl-acyl and alkenyl-acyl subclasses respectively. Sn1, sn2 and sn3 positions on the glycerol backbone are highlighted in red. Note that for consistency a phosphocholine headgroup is shown for all 3 subclasses...11 Figure P NMR phospholipid levels from human brain tissue. Results are the average of 45 replicates. Abbreviations: PC (Phosphatidylcholine), AAPC (Alkyl-acylphosphatidylcholine), PI (Phosphatidylinositol), SM (Sphingomyelin), PS (Phosphatidylserine), PE (Phosphatidylethanolamine), PEplasm (PE plasmalogen), CL (Cardiolipin), PA (Phosphatidic Acid). This figure is adapted from Pettegrew et al. (2001)...14 Figure 5. Percentage distribution of phospholipid in human liver adapted from Kwiterovich et al. (1970). Results are the average of data from organic extractions performed on 12 livers...15 Figure 6. Percentage distribution of phospholipids in rat heart membranes adapted from Zhao and Dhalla (1991) and Chi and Gupta (1998). NR indicates that the percentage of that lipid was not reported in that respective study...17 Figure 7. Percentage distribution of phospholipids in rabbit RBCs adapted from Nelson (1967) and Sullivan (1996). NR indicates that the percentage of that lipid was not reported in that respective study...18 Figure 8. The proposed mechanism by which HgCl2 interacts with plasmalogens...28 Figure 9. The structure of laurdan ix

10 Figure 10. The fluorescence of laurdan in different polar and nonpolar solvents using a UV lamp...33 Figure 11. A schematic representation of laurdan in a tighter packing/rigid membrane (left) and a less tightly packing/fluid membrane (right)...34 Figure 12. Results from scans of fluorescence intensity vs wavelength of Laurdan in DMPG LUVs hydrated in 100 mm NaCl at ph 7.40 at 13, 23 and 37 ºC. Arrows indicate inensity at 440 and 490 nm...35 Figure 13. Generalized polarization values as a function of temperature for laurdan in large unilamellar vesicles of DMPG. Fluorescence intensity was measured at 440 and 490 nm using an excitation wavelength of 340 nm...36 Figure 14. Generalized polarization values as a function of temperature for laurdan in large unilamellar vesicles of POPC. Fluorescence intensity was measured at 440 and 490 nm using an excitation wavelength of 340 nm...38 Figure 15. The structure of the glycerophospholipids used in this study. Red represents the lipid headgroup, blue represents the phosphate linker, black represents the glycerol backbone and green represents the fatty acyl tails Figure 16. The structures of the sphingolipids, glycerolipids and cholesterol that were used in this study. Excluding cholesterol, red represents the lipid headgroup, black represents the sphingosine backbone, blue represents the phosphate linker and green represents the fatty acyl tail Figure 17. A diagram illustrating the formation of large unilamellar vesicles (LUVs)...47 Figure 18. A schematic depicting the glycerol based anionic lipids studied in Chapter 3 of this thesis Figure 19. A representative DLS trial conducted using 0.3 mm POPC LUVs hydrated in 100 mm NaCl ph 7.40 extruded by using 100 nm diameter pores in a polycarbonate membrane. Each replicate is the average of 10 measurements...66 Figure 20. The fluorescence intensity of 500 nm Prodan in different solvents over a series of wavelengths in the absence and presence of 2 mm Hg and Cd at 25 ºC. Each dataset is the average of 2 replicates which were each the average over 5 scans x

11 Figure 21. Generalized Polarization values for Laurdan in 0.3 mm LUVs composed of 0.3 mm POPC (top) and 60 POPC 40 cholesterol (bottom) in the absence and presence of 2 mm Cd as a function of temperature. Results are the average of 3 replicates + standard deviation. Error bars are within the symbol size Figure 22. The average LUV radii measured using DLS in the absence and presence of 2 mm Cd. Results are the average of 3 replicates + standard deviation...71 Figure 23. Generalized Polarization values for Laurdan in LUVs composed of 0.3 mm 80 POPC: 20 sulfatide (Brain) (top) and 90 POPC: 10 ganglioside (Brain) (bottom) in the absence and presence of 2 mm Cd as a function of temperature. Results are the average of 3 replicates + standard deviation Figure 24. The average LUV radii measured using DLS in the presence and absence of 2 mm Cd. Results are the average of 3 replicates + standard deviation...76 Figure 25. The fluorescence intensity of 0.3 mm DMPA LUVs at 50 ºC in the absence and presence of 0.75 mm Cd over a series of wavelengths. Results are the average of 3 replicates...79 Figure 26. Generalized polarization values for Laurdan in LUVs of 0.3 mm POPA (top) and DMPA (bottom) with a series of Cd concentrations as a function of temperature. Results are the average of 3 replicates + standard deviation...80 Figure 27. The change in the phase transition temperatures of LUVs made of 0.3 mm POPA and DMPA over a series of Cd concentrations compared to the control. Results are the average of 3 replicates + standard deviation...81 Figure mm DMPA LUVs hydrated in 100 mm NaCl ph 7.40 incubated with 2 mm Cd and photographed at 0 minutes (left), 1 hour (centre) and 5 hours (right)...82 Figure 29. The average radii of 0.3 mm POPA and DMPA LUVs measured using DLS over a series of Cd concentrations. Results are the average of 3 replicates + standard deviation. * = p < 0.05, ** = p < Figure 30. Generalized polarization values for Laurdan in LUVs made with 0.3 mm TOCL (top) and TMCL (bottom) with a series of Cd concentrations as a function of temperature. Results are the average of 3 replicates + standard deviation...87 Figure 31. The change of the phase transition temperature of LUVs made of 0.3 mm TMCL over a series of Cd concentrations compared to the control. Results are the average of 3 replicates + standard deviation...88 xi

12 Figure 32. The average radii of 0.3 mm TOCL and TMCL LUVs in the presence and absence of 2 mm Cd. Results are the average of 3 replicates + standard deviation. * = p < 0.05, ** = p < Figure 33. Generalized polarization values for Laurdan in LUVs of 0.3 mm POPG (top) and DMPG (bottom) with a series of Cd concentrations as a function of temperature. Results are the average of 3 replicates + standard deviation...92 Figure 34. The change in the phase transition temperatures of LUVs made of 0.3 mm DMPG over a series of Cd concentrations compared to the control. The arrowed line indicates the concentration of lipid. Results are the average of 3 replicates + standard deviation...93 Figure 35. The average radii of 0.3 mm POPG and DMPG LUVs in the absence and presence of 2 mm Cd. Results are the average of 3 replicates + standard deviation...94 Figure 36. Generalized polarization values for Laurdan in LUVs of 0.3 mm POPS (top) and DMPS (bottom) with a series of Cd concentrations as a function of temperature. Results are the average of 3 replicates + standard deviation...98 Figure 37. The ΔTm of LUVs made of 0.3 mm POPS (top) and DMPS (bottom) over a series of Cd concentrations compared to the control. The arrowed line indicates the concentration of lipid. Results are the average of 3 replicates + standard deviation...96 Figure 38. The average radii of 0.3 mm POPS LUVs in the absence and presence of a series of Cd concentrations. Results are the average of 3 replicates + standard deviation. * = p< 0.05, ** = p < 0.01, **** = p < Figure 39. The average radii of 0.3 mm DMPS LUVs in the absence and presence of a series of Cd concentrations. Results are the average of 3 replicates + standard deviation. *** = p < 0.001, **** = p < Figure 40. The changes in the Tm of DMPA, DMPG, DMPS and TMCL LUVs. The dotted line shows the concentration of lipid in all experiments. Results are the average of 3 replicates + standard deviation Figure 41. Generalized polarization values for Laurdan in LUVs made with 0.3 mm 80 POPC: 20 PI(3)P (top) and 80 POPC: 20 PI(3,5)2P (bottom) with a series of Cd concentrations as a function of temperature. Results are the average of 3 replicates + standard deviation xii

13 Figure 42. The average radii of 0.3 mm LUVs of 80 POPC 20 PI(3)P (top) and 80 POPC 20 PI(3,5)2P (bottom) in the absence and presence of a series of Cd concentrations. Results are the average of 3 replicates + standard deviation. * = p < 0.05, ** = p < 0.01, *** = p < Figure 43. The change in General Polarization (GP) of 0.3 mm LUVs incubated with 2 mm Cd compared to the controls as a function of temperature. Results are the average of 3 replicates + standard deviation Figure 44. Generalized polarization values for Laurdan in LUVs of 0.3 mm POPC (top) and Brain SM (bottom) with 2 mm Hg as a function of temperature. Results are the average of 3 replicates + standard deviation Figure 45. The average radii of 0.3 mm POPC and Brain SM LUVs in the absence and presence of 2 mm Hg. Results are the average of 3 replicates + standard deviation Figure 46. Generalized polarization for Laurdan in LUVs of 0.3 mm 60 POPC 40 Cholesterol (top) and 50 POPC 50 DOTAP (bottom) with varying concentrations of Hg as a function of temperature. Results are the average of 3 replicates + standard deviation Figure 47. The average radii of 0.3 mm 60 POPC 40 Cholesterol LUVs in the absence and presence of 2 mm Hg. Results are the average of 3 replicates + standard deviation Figure 48. The average radii of 0.3 mm 50 POPC 50 DOTAP LUVs in the absence and presence of a series of Hg concentrations after 0 and 24 hours. Results are the average of 3 replicates + standard deviation. * = p < 0.05, ** = p < 0.01, *** = p < Figure 49. Generalized polarization values as a function of temperature in LUVs of 0.3 mm 80 POPC 20 ceramide (top) and 80 POPC 20 cerebroside (bottom) in the absence and presence of 2 mm Hg. Results are the average of 3 replicates + standard deviation Figure 50. The average radii of 0.3 mm LUVs of 80 POPC 20 Ceramide and 80 POPC 20 cerebroside in the absence and presence of 2 mm Hg. Results are the average of 3 replicates + standard deviation xiii

14 Figure 51. Generalized polarization for LUVs composed of 0.3 mm POPG (top) and DMPG (bottom) in the absence and presence of 2 mm Hg and 2 mm Cd as a function of temperature. Results are the average of 3 replicates + standard deviation Figure 52. The average radii of 0.3 mm POPG and DMPG LUVs in the absence and presence of 2 mm Hg. Results are the average of 3 replicates + standard deviation Figure 53. Generalized polarization as a function of temperature for 0.3 mm PC plasmalogen LUVs in the absence and presence of 2 mm Hg and 2 mm Cd. Results are the average of 3 replicates + standard deviation Figure 54. Fluorescence intensity at 440 and 490 nm recorded by exciting a 0.3 mm sample of PC plasmalogen LUVs at 340 nm before and after adding Hg to a final concentration of 0.2 mm at 37 ºC. The dotted arrow indicates the time when Hg was added Figure 55. The average radii of 0.3 mm PC plasmalogen LUVs in the absence and presence of 2 mm Hg. Results are the average of 3 replicates + standard deviation **** = p < Figure 56. Generalized polarization for Laurdan in 0.3 mm 88 POPC 12 PE plasmalogen (top) and 88 POPC 12 DOTAP (bottom) LUVs in the absence and presence of 2 mm Hg as a function of temperature. Results are the average of 3 replicates + standard deviation Figure 57. The average difference in the GP of LUVs composed of 0.3 mm 88 POPC and 12 DOTAP and 12 PE plasmalogen in the absence and presence of 2 mm Hg as a function of temperature. Results are the average of 3 replicates + standard deviation Figure 58. The average radii of 0.3 mm LUVs composed of 88 POPC 12 DOTAP and 88 POPC 12 PE plasmalogen in the absence and presence of 2 mm Hg. Results are the average of 3 replicates + standard deviation. * = p < 0.05, ** = p < Figure 59. The phospholipid composition of Brain polar extract from Avanti Polar Lipids Figure 60. Generalized polarization for Laurdan in 0.3 mm Brain polar extract LUVs in the absence and presence of 2 mm Hg, 2 mm Cd and 2 mm Hg + 2 mm Cd as a function of temperature. Results are the average of 3 replicates + standard deviation xiv

15 Figure 61. The average radii of 0.3 mm Brain polar extract LUVs in the absence and presence of 2 mm Hg, 2 mm Cd and a mixture of 2 mm Cd and 2 mm Hg added in different orders. Results are the average of 3 replicates + standard deviation. ** = p < 0.01, *** = p < 0.001, **** = p < st and 2nd refer to the order of addition of the metals Figure 62. Generalized polarization values for Laurdan in 0.3 mm 60 POPS 40 PC plasmalogen LUVs in the presence and absence of 2 mm Hg, 2 mm Cd and 2 mm Hg + 2 mm Cd as a function of temperature. Results are the average of 3 replicates + standard deviation Figure 63. The average radii of 0.3 mm Brain polar extract LUVs in the presence and absence of 2 mm Hg, 2 mm Cd and a mixture of 2 mm Cd and 2 mm Hg added in different order. Results are the average of 3 replicates + standard deviation. *** = p < 0.001, **** = p < The secondary axis in this Figure refers to the size of LUVs measured after exposure to 2 mm Cd Figure 64. The lipid composition of Liver polar extract from Avanti Polar Lipids Figure 65. Generalized polarization for Laurdan in 0.3 mm Liver polar extract LUVs in the absence and presence of 2 mm Hg, 2 mm Cd and 2 mm Hg + 2 mm Cd as a function of temperature. Results are the average of 3 replicates + standard deviation Figure 66. The average radii of 0.3 mm Liver polar extract LUVs in the absence and presence of 2 mm Hg, 2 mm Cd and a mixture of 2 mm Cd and 2 mm Hg. Results are the average of 3 replicates + standard deviation Figure 67. The ΔGP between control and metal treated 0.3 mm Brain and Liver polar extract LUVs as a function of temperature. Results are the average of 3 replicates + standard deviation Figure 68. The phospholipid composition of Heart polar extract from Avanti Polar Lipids Figure 69. A thin layer chromatography plate run with Heart polar extract from Avanti Polar Lipids Figure 70. Generalized polarization values for Laurdan in 0.3 mm Heart polar extract LUVs in the presence and absence of 2 mm Hg, 2 mm Cd and 2 mm Hg + 2 mm Cd as a function of temperature. Results are the average of 3 replicates + standard deviation xv

16 Figure 71. The average radii of 0.3 mm Heart polar extract LUVs in the presence and absence of 2 mm Hg, 2 mm Cd and a mixture of 2 mm Cd and 2 mm Hg added in different orders. Results are the average of 3 replicates + standard deviation ** = p < Figure 72. Zeta potential measurements of 0.3 mm LUVs of different systems. Results are the average of 3 measurements with an applied voltage of 5 V. ** = p < 0.01, **** = p < Figure 73. The phospholipid composition of E. coli polar extract provided by Avanti Polar Lipids Figure 74. Generalized polarization for Laurdan in 0.3 mm E. coli polar extract LUVs in the absence and presence of 2 mm Hg, 2 mm Cd and 2 mm Hg + 2 mm Cd as a function of temperature. Results are the average of 3 replicates + standard deviation Figure 75. The average radii of 0.3 mm E. coli polar extract LUVs in the presence and absence of 2 mm Hg, 2 mm Cd and a mixture of 2 mm Cd and 2 mm Hg. Results are the average of 3 replicates + standard deviation. ** = p < Figure 76. The fluorescence spectra of 0.3 mm RBC ghosts and RBC total extract excited at 280 and 340 nm with 5 nm excitation and emission slits at 25 ºC. Results are the average of 3 scans Figure 77. Generalized polarization for Laurdan in 0.3 mm RBC ghosts with mm Hg, mm Cd and 2 mm Cd + 2 mm Hg as a function of temperature. Results are the average of 3 replicates + standard deviation Figure 78. The average radii of 0.05 mm RBC ghosts in the absence and presence of 2 mm Hg, 2 mm Cd and a mixture of 2 mm Cd and 2 mm Hg. Results are the average of 9 replicates + standard deviation Figure 79. Generalized polarization for Laurdan in 0.3 mm RBC ghosts from different preparations and following the incubation of preparation 2 with 0.6 mm Mg-ATP at 37 ºC for 1 hour as a function of temperature. Results are the average of 3 replicates + standard deviation Figure 80. Generalized polarization for Laurdan in 0.3 mm asymmetrical RBC ghosts with 2 mm Hg, 2 mm Cd and 2 mm Hg/Cd as a function of temperature. Results are the average of 3 replicates + standard deviation xvi

17 Figure 81. Generalized polarization for Laurdan in 0.3 mm RBC ghosts and RBC total extract LUVs as a function of temperature. Results are the average of 3 replicates + standard deviation Figure 82. Generalized polarization as a function of temperature for Laurdan in 0.3 mm RBC total extract LUVs in the absence and presence of 2 mm Hg, 2 mm Cd and a 2 mm Hg/2 mm Cd mixture. Results are the average of 3 replicates + standard deviation Figure 83. The average radii of 0.3 mm RBC total extract LUVs in the absence and presence of 2 mm Hg, 2 mm Cd and a mixture of 2 mm Cd and 2 mm Hg. Results are the average of 3 replicates + standard deviation. **** = p < xvii

18 List of Abbreviations BAM BBPS Cd CL DLS DM DMPA DMPG DMPS DOTAP DPPA DSC GP Hg HPLC Laurdan LUV MLV PA PC Brewster Angle Microscopy Bovine Brain Phosphatidylserine Inorganic Cadmium (Cd 2+ in aqueous solution) Cardiolipin Dynamic Light Scattering Dimyristoyl 1, 2-dimyristoyl-sn-glycero-3-phosphate 1, 2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) 1, 2-dimyristoyl-sn-glycero-3-phospho-L-serine 1,2-dioleoyl-3-trimethylammmonium-propane 1, 2-dipalmitoyl-sn-glycero-3-phosphate Differential Scanning Calorimetry General Polarization Inorganic Mercury (Hg 2+ in aqueous solution) High performance liquid chromatography 6- Dodecanoyl-2-Dimethylaminonapthalene Large Unilamellar Vesicle Multilamellar Vesicle Phosphatidic Acid Phosphatidylcholine xviii

19 PCplasm PEplasm PI PI(3)P PI(3,5)2P PO POPA POPC POPG POPS PS RBC SM TLC Tm TMCL TOCL 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphocholine 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphoethanolamine Phosphatidylinositol 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3'-phosphate) 1, 2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3', 5 -bisphosphate) Palmitoyl-Oleoyl 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine Phosphatidylserine Red Blood Cell (Erythrocyte) Sphingomyelin Thin Layer Chromatography Gel to Liquid Crystalline Phase Transition 3 -bis [1, 2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol 1', 3 -bis [1, 2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol xix

20 1 Chapter One: Introduction Both inorganic mercury (Hg) and inorganic cadmium (Cd) are natural elements that are widely distributed in the crust, ground, sea and air 1. As a consequence of industrial activities there has been an ever increasing redistribution of these metals from the crust and ground into the air and water resulting in an influx of these metals into living systems. It has been estimated that 20,000-30,000 tons of Hg are released into the environment per year with the main source being emissions from coal fired power plants 2. These emissions are expected to rise at a rate of 5% per year making this an increasing global problem 3. It is estimated that in most Western countries 90% of Cd released from the Earth s crust is from anthropogenic sources including rock phosphate fertilizer, fossil-fuel combustion and mining 4. Higher levels of Hg and Cd in living systems have resulted in the increased onset of a number of diseases that are detrimental to human health including damage to the kidney, liver and central nervous system 5,6. Concentrations of Hg below 10 ppm have been linked to many neurological deficits including memory loss, disruption of fine motor function and hearing and vision impairment in which developing infants have been shown to be more sensitive than adults 7. In 1993 Cd was classified as a human carcinogen and has since been shown to induce cancers in multiple animal tissues including the lung, prostate, kidney and liver 8. In addition, Cd has been shown to reduce insulin levels and have a direct cytotoxic effect on the pancreas which increases the risk of developing Diabetes 9. There has been some success in reducing the body burden of Hg through administering 2,3-dimercaptopropane1-sulfonate (DMPS) which forms complexes with some heavy metals including Hg 10. Other studies have also shown that the chelator

21 2 diethylenetriaminepentaacetic acid (DTPA) has been effective in reducing Cd content in the tissues of rats acutely exposed to Cd although this same treatment is much less effective in rats chronically exposed to Cd 11. This particularly concerning as Cd has been shown to have an extremely long biological half-life of years resulting in its accumulation primarily in the kidney and liver 12. Additionally, there are concerns in using these treatment methods as some studies have shown that chelation in cases of acute exposure to Cd may actually exacerbate damage to the kidney tubules 13. As such, there is no widely agreed upon method for removal of Hg and Cd from human tissues which make these metals a continuing problem 14. Accordingly, studies that aim to increase our understanding of the interactions between these metals and their biological targets are of great importance. However, in order to gain a comprehensive understanding of how these metals function in a complex biological environment it is first necessary to study them in a simpler and more controlled environment. It is therefore the initial goal of this thesis to study the interactions of Hg and Cd with well-defined biomimetic membranes before more complicated polar extracts and RBC ghost membranes are investigated. It is important to note that the abbreviations Hg and Cd used in this thesis refer to the inorganic forms of Hg and Cd. Mercury can be taken up in elemental, inorganic and organic forms while Cd is rarely found in its elemental form and is largely present in an inorganic form with carbonates, hydroxides, chlorides and sulphates 15. Although multiple forms of mercury and cadmium are both present in living systems simultaneously, in the context of this thesis the abbreviations Hg and Cd refer to the ionic species formed under the experimental conditions of 100 mm NaCl and ph 7.40.

22 3 1.1 Hg and Cd targets in mammalian systems The two main routes by which Hg and Cd enter the bloodstream from the environment are the lungs and the gastrointestinal (GI) tract. These metals enter the blood through inhalation of particulates or gases into the lungs whereas the most important sources for GI tract uptake are contaminated food and water. The interactions between Hg and Cd and various targets present in the blood plasma including human serum albumins 16, platelets 17, and red blood cells (RBCs) 18,19 have been previously characterized. With regards to RBCs, interactions have been elucidated between the lipid membrane and both Hg and Cd 20, intracellular glutathione 18, as well as hemoglobin 21 and integral membrane proteins such as aquaprorin I 22 and hexose transporter 23. After absorption, Cd is rapidly cleared from the bloodstream and is initially sequestered in the liver before being transported more slowly to the kidney where it accumulates over time 12. This is thought to be due to the preferential uptake of free or metallothionein bound Cd (Cd-MT) in the renal proximal tubule by receptor-mediated endocytosis 24. Internalized Cd-MT can then be degraded into free Cd in the cytosol which can generate reactive oxygen species and induce apoptosis 24. Additionally, the presence of the neutrally charged HgCl2 form results in the passage of Hg across the blood-brain and lipid bilayer barriers and where it can then interact with intracellular targets 25. In contrast to the abundant research on metal-protein interactions, much less is known about the interactions between these metals and lipids and the membranes of each tissue. Figure 1 depicts the possible interactions between Hg and Cd and exterior and interior targets of the RBCs.

23 4 Figure 1. The possible interaction sites between inorganic Hg and Cd with the components of the bloodstream as well as different targets inside and outside the cell. (Hg/Cd)/Cl2 refers to the neutral HgCl2 and CdCl2 forms of these metals which may be able to diffuse directly across the lipid bilayer and interact with intracellular targets. 1.2 Hg and Cd speciation in aqueous solution To better understand the interactions between Hg and Cd with membrane lipids it is of critical importance to understand which species of each metal are present in solution under the given experimental conditions (Table 1). The main factors that affect Hg and Cd speciation are the concentration of metal and chloride (Cl - ) and the ph of the solution as shown in Figure 2.

24 5 Table 1. The predominant species of Hg and Cd present under physiological conditions of 100 mm NaCl and ph 7.40 which were used in this thesis. Metal Species of Metal Percentage of Total Species HgCl2 37 Overall Estimated Charge of Metal in Solution Percentage of Total Species Near a Anionic Membrane Percentage of Total Species Near a Cationic Membrane 1 9 HgCl Hg HgCl HgClOH Hg(OH) CdCl Cd Cd CdCl Results in Table 1 were generated using a free equilibrium speciation model program called Visual Minteq This software has been used extensively in the literature to predict the formation of precipitates and allow for the selective removal of metals from solution 27. This is done using available thermodynamic data and equilibrium constants and was used in this thesis to predict the species distribution of Hg and Cd in 100 mm NaCl at ph While the overall estimated charge of metal in solution is based on the speciation of Hg and Cd in the bulk solution, the speciation near the surface of a charged membrane is also provided based on calculations conducted using Gouy Chapman Theory 28.

25 Percentage of total species ph ph [Chloride] (mm) [Chloride] (mm) HgClOH HgCl 2 HgCl 3-2- HgCl 4 Hg(OH) 2 Cd 2+ CdCl 2 CdCl + Cd(OH )+ Cd(OH) 2 Figure 2. The speciation of Hg and Cd as a function of chloride concentration and ph in aqueous solution using 1.0 mm Hg/Cd. Curves were determined using thermodynamic parameters from Visual Minteq 3.1 software at 37ºC. The ph curves were determined at 100 mm NaCl and the chloride curves were determined at ph 7.40.

26 7 The theoretical distribution of Hg and Cd species has been supported by experimental data. For example, inorganic mercury has been shown to exist as multiple unique species that have different chemical shifts that can be detected using 199 Hg NMR 29. These include Hg 2+, HgOH +, Hg(OH)2, HgCl +, HgClOH, HgCl2, HgCl3 - and HgCl4 2-30,29. In the absence of chloride, Hg 2+ and Hg(OH)2 are present as > 95% of the species present in solution at ph 1 and 4 respectively. 0-20% of Hg is present as HgOH + between ph 2-4. However, in the presence of chloride Hg preferentially forms chloride over hydroxide species until above ph 8.5 at which point HgClOH and Hg(OH)2 combined represent over 50% of the total Hg species. Although Cd forms multiple chloride and hydroxide species like Hg, results from Visual Minteq predict that the species distribution is affected differently by ph and chloride concentration. In the absence of chloride, Cd 2+, CdOH +, Cd(OH)2, Cd(OH)3 - and Cd(OH)4-2 are predicted to be most abundant species of Cd present at ph 8, 10, 11, 12 and 14 respectively. In the presence of chloride Cd prefers to form chloride species over hydroxides although Cd does not form CdCl3 - and CdCl4 2- species until much higher chloride concentrations compared to Hg. Thus, at ph 7.4 and 100 mm NaCl the higher affinity of Hg for chlorides results in the formation of species with 1-4 chlorides while Cd forms species with 0-2 chlorides (Table 1). The net effect is that under these conditions the majority of Hg in solution is negatively charged while the majority of Cd is present as positively charged species (Table 1). Also, in the presence of 100 mm Cl -, the speciation of Hg and Cd are predicted to be independent of ph until above 7.0 and 9.0 for Hg and Cd respectively (Figure 2).

27 8 1.3 Membrane Lipid Bilayers Phospholipid Nomenclature When referring to phospholipids a common nomenclature is used to describe the lipids fatty acid chains called the carboxyl reference system. This is done through two numbers represented by: (number of carbons in the fatty acyl chain): (number of double bonds in the acyl chain). When counting the carbons of the fatty acyl chain using this system the carboxyl carbon is counted as number 1. The position of the double bond on the acyl chain can be described using a delta (Δ) symbol followed by the carbon number at which the double bond starts as a superscript (Ex. 16:0 Δ 9 ). Also, in this thesis the fatty acids will be mentioned by their common names rather than their IUPAC names as outlined in Table 2. Table 2. The common, IUPAC and carboxyl reference names for the fatty acid that are components of the lipids used in this thesis along with the abbreviations by which they will be referred to. Common Name IUPAC Name Carboxyl Reference Abbreviation Myristoyl Tetradecanoyl 14:0 M Palmitoyl Hexadecanoyl 16:0 P Stearoyl Octadecanoyl 18:0 S Oleoyl Cis-9-Octadecanoyl 18:1 Δ 9 O In addition to acyl chain abbreviations the headgroup portion of the lipid is also abbreviated with two letters according to the phosphate and the adjacent molecule (Eg. Choline).

28 9 The isolated headgroup with an attached phosphate is referred to as the phospho-headgroup name while the entire phospholipid is referred to as the phosphatidyl-headgroup name. If a glycerol based phosphocholine (PC) headgroup has a sn1 acyl chain that is 16 carbons long with no unsaturation and a sn2 acyl chain that is 18 carbons long with 1 double bond, then it can be abbreviated as 16:0-18:1 PC. Alternatively using the common name abbreviations, the lipid is called POPC. If the same fatty acid is present at both the sn1 and sn2 positions, then the IUPAC prefix Di- (~2x) is used to indicate this. (Ex. DM = Dimyristoyl). In the case of Cardiolipin (CL) which has 4 fatty acyl chains, the letter T in the TOCL/TMCL lipids refers to the Tetra (~4x) prefix Membrane Lipids The 3 main classes of lipids present in biological membranes that were studied in this thesis are glycerol based lipids, sphingosine based lipids and sterols. Both glycerol and sphingosine based lipids are structurally characterized by headgroup, backbone and fatty acyl chain regions. If they also contain a phosphate group as a linker between the headgroup and backbone regions they are termed glycerophospholipids or sphingophospholipids, respectively. Sphingolipids without a phosphate group include ceramides, cerebrosides and gangliosides while the only glycerolipid without a phosphate used in this study was 1, 2-dioleoyl-3- trimethylammonium-propane (DOTAP). Cerebrosides and gangliosides can be also classified as glycosphingolipids composed of a ceramide backbone and a glycosylated headgroup. Cerebrosides are monoglycosylated with either glucose or galactose which are often referred to

29 10 as glucosyl and galactosyl ceramide, respectively. Gangliosides are glycosphingolipids in which the headgroup has multiple sugar groups present as well as characteristic terminal sialic acids. Concerning the lipid composition of biological membranes, the headgroups in these lipid structures can be divided into major and minor classes based on their abundance. The major phospholipid headgroups include phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine 31. Minor headgroups include phosphatidylinositol (PI), phosphatidylglycerol (PG) and phosphatidic acid (PA) 31. The hydrophobic section of the aforementioned lipids consists of fatty acyl tails of varying lengths and degrees of unsaturation. Longer and saturated fatty acyl chains result in a more rigid membrane due to increased van der Waals interactions between neighboring acyl chains. Unsaturation in membrane lipids is most often present as cis double bonds in the acyl chain 32. This results in kinks in the side chain(s) which disrupt the close packing between the fully extended saturated acyl tails. Membranes with unsaturated lipids are more fluid than saturated systems due to reduced van der Waals interactions 32. The 3 carbons in the glycerol backbone of membrane lipids are labelled sn1, sn2 and sn3 as shown in Figure 3. The sn3 carbon contains the headgroup while the sn2 and sn1 carbons contain different fatty acid linkages which can be divided into 3 subclasses 33. The first and by far most common type in mammalian membranes is the 1, 2-diacyl group followed by the 1-alkyl, 2- acyl group. This ether linkage represents only 1-2% of lipids in mammalian membranes. The third group is the 1-alkenyl-2-acyl group more commonly referred to as the plasmalogens. These

30 11 enol-ether lipids make up between 2-40% of the lipids in different mammalian tissue membranes 33. Sn3 Sn2 Sn1 Sn3 Sn2 Sn1 Sn3 Sn2 Sn1 1, 2-Diacyl 1-alkyl, 2-acyl 1-alkenyl, 2-acyl Figure 3. The general structure of glycerol based phospholipids. The dotted blue box highlights the ester, ether and vinyl ether linkages present in the diacyl, alkyl-acyl and alkenyl-acyl subclasses respectively. Sn1, sn2 and sn3 positions on the glycerol backbone are highlighted in red. Note that for consistency a phosphocholine headgroup is shown for all 3 subclasses Cholesterol In addition to the various phospholipids discussed in Section 1.3.2, the presence of sterols in mammalian membranes was considered whereby cholesterol is the most abundant 34. For the purposes of this thesis, the presence of cholesterol will be discussed based on the effect of this

31 12 lipid on membrane fluidity. Indeed, it has been proposed that the ability of cholesterol to modulate the behavior of other lipids in the bilayer is perhaps one of its most important functions 35. The presence of cholesterol in a lipid bilayer is well characterized and has been shown to regulate membrane fluidity depending on the phase of the bilayer 36. In gel phase membranes, cholesterol intercalates between phospholipids and reduces the packing density in the membrane thereby increasing membrane fluidity while the opposite effect is seen in membranes in the more biologically relevant liquid crystalline phase 36. Cholesterol has been shown to increase the order and decrease the motion of phospholipid acyl chains thereby increasing membrane rigidity through an increase in van der Waals interactions 37. In regards to lipid systems with cholesterol in Chapters Three and Four, an increase in membrane rigidity is expected compared to the equivalent sterol free system as it was used in biomimetic membranes formulated with phospholipids present in the liquid crystalline state over the entire temperature range (Eg. POPC). 1.4 Membrane Lipid Profiles Hg and Cd have previously been shown to exert toxic effects on different lipid targets in different species and cell types 1. Thus, for the purpose of this thesis, variations in membrane composition need to be considered. Lipid composition is constantly changing due to many different factors including but not limited to diet, metabolism, age and disease state. Furthermore, both mammalian and bacterial membranes have very complex compositions and may contain over 1000 different unique lipids based on differences in lipid headgroups, backbones and fatty acyl tail lengths and degrees of unsaturation 38.

32 13 Based on the hypothesis described in Section 1.8, it is important to determine the approximate percentages of plasmalogens and anionic lipids in Brain, Heart, Liver, RBC and E. coli membranes as these are the predicted lipid targets for Hg and Cd respectively. In addition, since lipids with identical headgroups and different degrees of acyl chain saturation were compared in this study, the ratio of saturated/unsaturated lipids in each tissue will be briefly highlighted as well. Previous studies have shown that the acyl chain composition of a membrane can be significantly altered based on the unsaturation of dietary fats that are consumed so this ratio is an approximation of a constantly changing property of biological membranes Brain Membrane Lipids Previous studies have used 31 P NMR to analyze the phospholipid composition of this tissue 40. This was done following an organic extraction of human brain using chloroform: methanol (2:1 v/v) 41. These results are shown in Figure 4 as molar percentages. The composition of brain lipids is particularly interesting as lipids make up about 70% of the total mass which is higher than what is reported for most other cell membranes 42. In addition to the phospholipid component of brain lipids, there is also an abundance of cholesterol in brain lipids which is reported as being between 40-50% of total lipids (~1 cholesterol/ 1 phospholipid) 42.

33 Percentage of total moles of lipid PC AAPC PI SM PS PE PEplasm CL PA Figure P NMR phospholipid levels from human brain tissue. Results are the average of 45 replicates. Abbreviations: PC (Phosphatidylcholine), AAPC (Alkyl-acyl-phosphatidylcholine), PI (Phosphatidylinositol), SM (Sphingomyelin), PS (Phosphatidylserine), PE (Phosphatidylethanolamine), PEplasm (PE plasmalogen), CL (Cardiolipin), PA (Phosphatidic Acid). This figure is adapted from Pettegrew et al. (2001). Interestingly, a large portion of PE lipids in brain membranes are plasmalogens. While one study reported that the PE based lipids in the brain myelin of humans was almost entirely plasmalogens 43, this percentage is reduced when the lipids from the plasma membranes of nerve cells are taken into account 40. Based on results from total brain lipids (myelin + plasma membranes) in Figure 4, plasmalogens are shown as approximately 60% of PE lipids in brain tissue. This is agreement with other data which suggested that plasmalogens accounted for 50-75% of PE lipids in grey and white matter 43.

34 Percentage of total moles of lipid 15 Concerning negatively charged lipids, multiple studies have shown that PS is present as about 14-15% of brain lipids in both humans and mice making this the most abundant anionic lipid in brain tissue 40,44,45. Minor percentages (< 5%) of other anionic lipids including PI, PA and CL were also observed bringing the total percentage of negative lipids in the brain to ~22% (Figure 4). Based on these percentages and the hypothesis of this thesis, approximately 42% of brain lipids are targets for these metals (22% for Cd + 20% for Hg) Liver Membrane Lipids The composition of human liver plasma membranes has previously been studied using gas chromatography as shown in Figure PC Lyso PC PE PI PS SM CL Unknown Figure 5. Percentage distribution of phospholipid in human liver adapted from Kwiterovich et al. (1970). Results are the average of data from organic extractions performed on 12 livers. PC and PE lipids are the largest components of liver membranes which is similar to what was found in brain membranes while neither PC or PE plasmalogens were reported in this study.

35 16 This is in agreement with another manuscript which stated that PE plasmalogen accounts for less than 5% of PE lipids in the liver 33. Results in Figure 5 show that negatively charged lipids represent 18.3% of the total phospholipids, which is comparable to the 22% determined in brain membranes. Cholesterol has been found to be another major component in rat liver membranes with a cholesterol/phospholipid molar ratio between which is lower than what is observed in brain membranes 47. Overall, while plasmalogen lipids are much lower in the liver compared to the brain, the amount of anionic lipids is comparable to what is observed in brain membranes Heart Membrane Lipids Figure 6 illustrates the lipids classes in rat heart plasma membranes that were quantified in two different studies using High Performance Liquid Chromatography (HPLC) 48 and 31 P NMR respectively 49. One of the major distinctions between heart lipids and many other membranes is the presence of high concentrations of choline plasmalogen in addition to ethanolamine plasmalogen 50. These lipids represent 40% and 32% of the total glycerophospholipids in bovine and human heart tissues respectively and approximately 60-70% of choline and 30-40% of ethanolamine glycerophospholipids in both human and bovine heart tissues 51. This is considerably higher than the plasmalogen content of brain membranes (~20%) as well as the liver which contains few plasmalogens (< 5%). Results from these studies show that between about 11-15% of lipids are negatively charged which is lower than in both liver and brain membranes. Understanding the interactions of metals with lipids in the cardiovascular

36 Percentage of total moles of lipid 17 system is of importance as Hg has been previously linked to the onset of hypertension and cardiovascular disease 52. Data from the study using HPLC showed that the cholesterol/phospholipid molar ratio was 0.25 suggesting a lower amount of cholesterol compared to both liver and brain membranes. The percentage of cholesterol was not reported in the 31 P NMR work HPLC Study 31P NMR Study NR NR NR PC PE PEplasm PI SM LPC CL PS Figure 6. Percentage distribution of phospholipids in rat heart membranes adapted from Zhao and Dhalla (1991) for HPCL results and Chi and Gupta (1998) for 31 NMR. NR indicates that the percentage of that lipid was not reported in that respective study RBC Membrane Lipids As RBCs from rabbits were used in this thesis, the lipid composition of these membranes are the focus of this section. The lipid composition of rabbit RBCs has been extensively studied

37 Percentage of total moles of lipid 18 in the literature by different groups 53,54,55. Results from studies conducted in 1967 and 1996 are shown in Figure 7. Like in previously discussed mammalian membranes, PC and PE are the most abundant lipids in the RBC membrane whereby about 67% of PE and 10% of the PC are plasmalogens respectively 56. Concerning anionic lipids, the study conducted by Nelson in 1967 reported smaller percentages of PA and PI, whereas more recent data reported ~13% PS as the only anionic lipid in the membrane. Thus, approximately 13-15% of the lipids in rabbit RBCs are anionic which is about the same as in heart membranes and lower than brain and liver membranes Nelson 1967 Sullivan et al NR PA PE PS PI PC SM NR Figure 7. Percentage distribution of phospholipids in rabbit RBCs adapted from Nelson (1967) and Sullivan (1996). NR indicates that the percentage of that lipid was not reported in that respective study.

38 19 While Sullivan et al. did not report the percentage of cholesterol, the earlier work reported a cholesterol/phospholipid ratio of 1:2. This is in contrast to results from other studies that have reported a molar cholesterol/phospholipid ratio in both rabbit and human RBCs as being about 1:1 57,58. Nonetheless, it is clear that the cholesterol/phospholipid ratio is higher than what is observed in Heart and Liver membranes and about equal to what is observed in Brain membranes E. coli Membrane Lipids Unlike the membranes discussed so far, E. coli membranes have been found to be unusually simple with a profile generally reported to be 75% PE, 20% PG and 5% CL 59. This is a very unique composition as these membranes contain a large concentration of PG which is not present in appreciable amounts in mammalian membranes. Other important distinctions between bacterial and mammalian membranes include a lack of cholesterol as well as PC which are both major components in Brain, Heart, Liver and RBC membranes. Moreover, plasmalogens and sphingolipids are also not found in E. coli membranes Fatty acid compositions of membrane bilayers As the effect of Hg and Cd on lipids with an identical headgroup and varying degrees of acyl chain unsaturation was studied in Chapters Three and Four of this thesis, the fatty acid composition of lipids present in polar extracts and RBC ghosts in Chapter Five must also be considered. An excellent study on the fatty acid composition of different rat membranes according to the amount of unsaturated fat in the diet is available and will be referred to for this section 60. The three categories of fatty acids include saturated fatty acids (SFAs),

39 20 monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs). SFAs are lipids without any double bonds in the acyl tails while MUFAs contain 1 double bond whereas PUFAs contain more than 1 double bond. The abundance of SFAs, MUFAs and PUFAs in the Brains, Livers, Hearts and RBCs of rats is summarized in Table 3. As higher percentages of SFAs and lower percentages of MUFAs and PUFAs result in a more rigid membrane, based on the data in Table 3 it would be expected that the rigidity of these membranes is in the following order: (most rigid to least rigid) Brain > RBCs > Liver > Heart. In addition to the high percentage of SFAs in Brain and RBC membranes, the high cholesterol/phospholipid ratio also increases the rigidity of these membranes compared to the liver and heart which contain less cholesterol by comparison. Results in Chapter Five showed that there was not a clear trend in rigidity between polar lipid extract membranes at all temperatures. However, at 37 ºC the order was RBC > Brain > Heart ~ Liver. Table 3. The fatty acid composition of different rat tissues expressed as a percentage of total fatty acids. This table is adapted from Abbot et al (2012). Rat Membrane SFAs (%) MUFAs (%) PUFAs (%) Brain 50% 25% 25% Liver 45% 5% 50% Heart 30% 15% 55% RBCs 50% 10% 40%

40 21 Concerning E. coli, a study conducted using Liquid Chromatography- Mass Spectrometry (LC-MS) showed that the most common fatty acid species in PE lipids were 16:0 and 18:1 fatty acids 61. This is in agreement with data from Avanti Polar Lipids, which has characterized the fatty acid distributions of PE, PG and CL in E. coli as shown in Table 4. Overall, a higher percentage of saturated fatty acids particularly in PG and CL lipids as well as the absence of PUFAs in E. coli membranes results in a very rigid membrane compared to most mammalian membranes. While the lack of cholesterol is expected to increase membrane fluidity, the hydrogen bonding from the high percentage of PE lipids would work to increase membrane rigidity. Table 4. The fatty acid composition of E. coli lipids expressed as a percentage of the total fatty acids for each respective lipid. Data is adapted from Avanti Polar Lipids. E. coli lipid SFAs (%) MUFAs (%) PUFAs (%) PE 56.6% 43.4% 0% PG 79.7% 20.3% 0% CL 74% 26% 0% 1.5 Lipid Bilayer Asymmetry A common characteristic of all eukaryotic membranes is the unequal distribution of certain lipids in either the extracellular (outer) or intracellular leaflet (inner) of the membrane bilayer which is referred to as lipid asymmetry 62. This phenomenon is primarily due to ATPdriven proteins (flippases and floppases) that assist in creating and maintaining this asymmetry

41 22 and less so because of the lipids ability to spontaneously flip their headgroups through the hydrophobic core of the membrane. Not surprisingly, the significant expenditure of energy required to maintain this asymmetry is important for proper function and is significant for blood coagulation, membrane fusion, phagocyte recognition and the induction of apoptosis 63. While lipid asymmetry is observed in Golgi, endosomal and plasma membranes of Eukaryotic cells, the interactions of Hg and Cd were not studied with organelle membranes in this thesis. Thus, the focus of this study is on the asymmetry present in the plasma membranes of RBCs where PC, SM and the glycosphingolipids are found mostly in the extracellular leaflet while PS, PI, PA and PE are enriched in the inner leaflet while cholesterol is distributed evenly between leaflets 63. This asymmetry is particularly important in RBCs as the externalization of PS is part of a normal apoptosis induction pathway used to degrade aging RBCs for recycling by the macrophages in the bloodstream 64. This is relevant for metal-lipid interactions as studies have shown that the addition of Hg and Cd to RBCs can disrupt phospholipid asymmetry leading to cell death in part due to premature externalization of PS lipids. For example, one study showed that incubation with 1 µm Hg 2+ at 37 ºC for 24 hours resulted in 10x more exposure of PS in the external lipid leaflet compared to the controls 65. However, this result may have been influenced by the use of 32 mm HEPES buffer that was found in this thesis to interact with Hg and form a precipitate within hours. Another study showed the Hg and Cd induced activation of sphingomyelinase whereby the resulting ceramides made the phospholipid scramblases more prone to activation by Ca 2+. Their subsequent activation lead to increased exposure of PS in the extracellular leaflet. Scramblases are membrane proteins that move lipids bidirectionally down

42 23 their concentration gradients, which works against flippases, and floppases that maintain asymmetry 62. A method has been developed to make liposomes with asymmetrical leaflets although there are some disadvantages 66. Briefly, this is done by making a monolayer containing the exterior leaflet lipids on an aqueous phase. Two organic phases of different densities that contain the lipids of the interior leaflet are then added on top of the aqueous phase. Centrifugation is then done to transfer the lipids from the organic phase to the aqueous phase where the liposomes are collected. Unfortunately this method does not allow the researcher to control the size of the liposome population and the asymmetry is only stable for ~24 hours 66. Therefore, for the purposes of this thesis, experiments with LUVs were conducted with symmetrical leaflets. Experiments with RBC ghosts with intact leaflet asymmetry were performed to determine how this asymmetry affects Hg and Cd induced fluidity changes. 1.6 The effect of Hg and Cd on membrane fluidity Membrane fluidity refers to how tightly the lipids in the membrane pack together. If Hg and/or Cd bind and interact with lipids, it is conceivable that this interaction may disrupt the packing of the lipids and alter membrane fluidity. However, the propensity of these metals to interact with the lipid bilayer could also be dependent on the membrane fluidity prior to the addition of the metals. Thus, if metal induced fluidity changes are observed, the question arises if this effect is more pronounced for membranes in the more rigid gel phase or the less rigid liquid crystalline phase or at the Tm between these phases.

43 24 Previous studies have examined the effect of Hg and Cd on membrane fluidity and their phase transition 67,19,68. One of the most common methods to study membrane fluidity employs fluorophores that respond differently to changes in lipid packing and hydration. For example, diphenylhexatriene (DPH) exhibits strong fluorescence when it incorporates into lipid membranes allowing the measurement of membrane fluidity in the acyl chain region. Another fluorophore called laurdan is sensitive to membrane fluidity in the interface and headgroup regions of the lipid bilayer and will be discussed in further detail in Chapter Two. Studies with DPH showed that 0.5 mm Hg induced a 7 and 25% increase in the rigidity of RBC and Dimyristoyl phosphatidylcholine (DMPC) membranes respectively 68. This was done at 37ºC using phosphate buffered saline for the RBC studies and unbuffered water for DMPC trials. The same experiment using Laurdan showed a 20% and 104% increase in membrane rigidity in RBC and DMPC systems, respectively. The use of multiple probes that monitor fluidity at different locations of the bilayer showed that Hg induced more rigidity at the headgroup/interface region compared to the acyl chain region of the bilayer. In addition, it was speculated that the large difference between results from DMPC and the RBCs was due to the presence of membrane proteins and the membrane cytoskeleton of the RBCs although it should be noted that the different buffer conditions may have played a role as well. Differences in the ph and chloride concentration affect both the speciation of the metals as well as the environment around the liposomes (See Figure 2). Experiments with DMPC membranes containing Laurdan showed that mm Cd increased membrane rigidity at 18ºC while the opposite effect was seen at 37ºC 19. As the Tm of

44 25 DMPC occurs at 24ºC the nature of the phase of the membrane encountered by Cd affects the metal lipid interaction. Experiments with RBCs at 37ºC showed that Cd between mm metal increased membrane fluidity in a concentration dependent manner 19. The observed opposing effects of Hg and Cd on RBC membranes suggests that these metals target different lipid classes based on their varying speciation in aqueous solution. The large variance in the experimental data for Hg and Cd with different anionic lipids has been previously discussed 67. The effect of Hg and Cd on the Tm of PS, PG and PA is summarized in Table 5. Particularly interesting are the results for Cd and DPPA which show that depending on the concentration of salt and the ph, the Tm can be abolished, increased or remain unaffected by Cd 69. This was also true of Hg which increased the Tm of DMPA at ph 5.8 in 74 mm acetate but did not affect the Tm at ph 7.8 in 50 mm phosphate 70. The implication that metal lipid interactions strongly depend on the experimental conditions has made the stringent control of ph, ionic strength necessary in addition to avoiding the use of any buffers to eliminate a potential influence due to their interactions with Hg and/or Cd.

45 26 Table 5. A summary of the effect of Hg and Cd on the Tm of anionic PS, PG and PA membranes adapted from Payliss et al. The table summarizes DPH fluorescence anisotropy data collected for MLVs under various conditions as described in the legend below the table. MLVs contain multiple bilayers before extrusion (See Figure 17 in Chapter Two) Hg and Cd target different membrane lipid classes Given that the speciation of Hg and Cd under the experimental conditions used in this thesis are very different (See Table 1), it is reasonable to expect that these metals may target different lipids. This is especially true if it is assumed that metal-lipid interactions are driven by electrostatic attraction. In this case, Hg would be expected to target cationic membranes while Cd would be expected to target anionic membranes. There is some support for this in the literature as it has been previously shown that Hg does not target the negatively charged phosphate group found in many lipids 70. X-ray diffraction has been used to show that 10 µm Hg (in water) is capable of inducing disorder in the lipid bilayer of DMPC vesicles 68. Furthermore,

46 27 neutral HgCl2 is able to deeply penetrate into the hydrophobic core of PS containing liposomes at ph Also, the diffusion rate of Hg across membranes composed of PC and cholesterol was shown to be highly dependent on the chloride concentration which is the main factor that determines how much HgCl2 is present 25. At ph 7.0 and [Cl - ] ranging from mm it was found that only the HgCl2 form was able to pass through the bilayer at a significant rate 25. Consequently, the negatively charged species of Hg that are formed should target cationic lipids. Since biomembranes do not contain cationic lipids there have not been any studies examining a potential interaction between Hg and these lipids. However, synthetic cationic lipids have become increasingly popular in recent years for various in vitro and in vivo applications such as transfection experiments 71. One of the most common cationic lipids is DOTAP which is structurally similar to 18:1-18:1-PC but the removal of the phosphate linker results in a net charge of +1 originating from the choline headgroup. To study the possibility that Hg targets cationic membranes, LUVs containing DOTAP were formulated to assess the membrane binding affinity of negatively charged chloride species. In addition to possible electrostatic interactions, Hg has also been shown to specifically target the enol-ether bond in PC and PE plasmalogens 72. This interactions leads to the irreversible cleavage of this bond resulting in the formation of an aldehyde and an alcohol via the proposed mechanism below (Figure 8) 72,73 :

47 Figure 8. Proposed mechanism by which HgCl2 interacts with plasmalogens. This reaction occurs very quickly as the release of the aldehyde degradation product has previously been detected in PC and PE plasmalogens from heart lipids instantaneously at 25 and 50 ºC 72. However, other studies have not detected this aldehyde at lower temperatures of 0 ºC which suggests that the binding of Hg could be the fast step while the cleavage of the vinyl ether bond is a slower and temperature dependent step 74. NMR studies with 113 Cd and 31 P ranked the affinity of Cd for lipids in the order of PS >> PA > PG >> PC~PE 69. The fact that only the anionic PS, PA and PG where targeted implied that the interaction of Cd with these lipids are largely driven by electrostatic forces with the negative carboxyl and phosphate groups being the probable targets. The effect of Hg on the plasmalogens is specific for Hg and thus Cd does not target the vinyl ether bond of the plasmalogens.

48 The effect of Hg and Cd on Lipid Bilayer Size In addition to understanding the effect of Hg and Cd on membrane fluidity and lipid phase transitions, changes in LUV size were also studied. The rationale was that metal-induced changes of LUV size are possible consequences of alterations in membrane fluidity. The propensity of some metals such as Ca 2+ and Zn 2+ to firstly induce liposome aggregation followed by fusion of membranes containing anionic lipids has been previously studied 75. Given that Cd 2+ has a similar ionic radius to Ca 2+ and a similar electronegativity with Zn 2+ it is reasonable to expect that some Cd species may behave similarly to these metals 76. In contrast Hg has never been shown to be fusogenic or induce aggregation of liposomes containing zwitterionic or anionic lipids 75. Thus the main questions concerning Hg were how it would affect the size of cationic liposomes and how Hg catalyzed breakdown of plasmalogens would affect liposome size. Metal-induced changes in liposome size are thought to result from changes in intervesicle interactions (trans-interactions) and changes in the properties of lipids in one vesicle (cisinteractions) 77. Consider a population of liposomes containing negatively charged lipids such as PS. These liposomes are stable structures that would not spontaneously aggregate or fuse largely due to the electrostatic repulsion between the negatively charged headgroups. Therefore, vesicle aggregation is more likely when electrostatic repulsion is decreased, as observed in the presence of multivalent metals. The binding of Ca 2+ and the resulting neutralization of the negative surface charges has been shown to lead to interactions of PS lipids in neighboring liposomes representing trans-interactions 77. In lipid mixtures containing anionic lipids, this aggregation

49 30 may be enhanced by the propensity of multivalent metals to induce PS enriched microdomains representing cis-interactions. It has previously been proposed that the formation of these microdomains could be one of the first steps leading to membrane fusion 77. Such a drastic reorganization of membrane lipids is likely to also result in a change in membrane fluidity which may be detected by Laurdan GP which is discussed in detail in Chapter 2. In addition to the electrostatic barrier that prevents liposome-liposome interactions, another obstacle is the shell of polar water molecules surrounding the surface of each membrane which prevents the interaction of hydrophobic portions of neighboring liposomes. It has been shown that metals that neutralize surface charges promote liposome aggregation resulting in a large increase in membrane size. However, in contrast to membrane aggregation, fusion also results in the mixing of the inner aqueous compartments of liposomes. Previous work has shown that this only occurs if the metal can both neutralize the lipid charge as well as dehydrate the polar headgroup region of the bilayer 77. Screening different divalent metals has shown that Cd induced aggregation and fusion in liposomes containing 20-50% PS in DPPC/BBPS mixtures at concentrations as low as 0.5 mm while Ca 2+ did not induce fusion until concentrations above 15 mm 77. This was done under conditions of 10 mm HEPES, 100 mm NaCl at ph 7.4 which is similar to the experimental conditions of this thesis. It is still unclear how Cd changes the size of anionic liposomes with different headgroups and fatty acyl chain properties. While data are available for the interactions of Cd with liposomes containing PS, much less is known for membranes containing PI, PA, PG and CL.

50 Hypothesis Thermodynamic data suggests that under conditions of 100 mm NaCl and ph 7.40 available Hg and Cd predominantly form positively and negatively charged species respectively. Given this, it is predicted that Cd will electrostatically target negatively charged membranes containing PA, CL, PG, PS and PI while Hg will electrostatically target cationic membranes formulated from DOTAP. Additionally, Hg induced cleavage of the vinyl ether bond of PC and PE plasmalogens is predicted to impact the fluidity and size of LUVs containing these lipids. Previous data has shown that interactions between Cd and anionic membranes results in an increase in rigidity and membrane phase transition (Tm) 69. When Cd is incubated with anionic membranes with identical headgroups but different acyl chains, tighter packing between saturated lipids is predicted to facilitate headgroup bridging better than in unsaturated lipids. In addition, a stronger interaction is predicted between Cd and PA membranes compared to CL membranes due to the higher surface charge density of PA compared to CL. 1.9 Detailed Project Goals The goals of this thesis are as follows: 1. To assess Hg and/or Cd induced fluidity changes in biomimetic membranes, polar extract LUVs and RBC ghosts under nearly physiological chloride and ph conditions (100 mm NaCl, ph 7.40) by using Laurdan General Polarization (GP). a. To investigate how Cd affects the fluidity and membrane phase transition (Tm) of different anionic membranes with a focus on comparing lipids with identical

51 32 headgroups but different acyl chain properties as well as lipids with different headgroups but identical acyl chain properties. b. To determine if negatively charged species of Hg formed under nearly physiological conditions target cationic membranes using DLS and Laurdan GP. 2. To study how Hg and/or Cd induced fluidity changes in systems studied by using Laurdan GP affect LUV and RBC size by using Dynamic Light Scattering (DLS). 3. To compare the individual effects of Hg and Cd on membranes with the impact of metal mixtures.

52 33 Chapter Two: Materials and Methods 2.1 Laurdan Generalized Polarization (GP) The effect of Hg and Cd on the fluidity of membranes formulated from biomimetic lipid mixtures, polar lipid extracts and RBC ghosts was examined using the fluorescent dye 6- Dodecanoyl-2-Dimethylaminonaphthalene (laurdan) Introduction to laurdan Laurdan was synthesized in 1979 by Gregorio Weber as a fluorescent probe that responded to the polarity of its environment 78. The structure of laurdan shown below is composed of a hydrophobic lauroyl tail, a carbonyl group, a naphthalene moiety and a dimethylamino headgroup (Figure 9). Figure 9. The structure of laurdan. The fluorescent naphthalene group experiences a dipole moment due to the partial charge separation due to the electron poor carbonyl moiety and the electron rich dimethyl-amino group 79. This dipole moment is increased upon excitation and can cause reorientation of surrounding solvent molecules 79. Exposure of the excited state of a fluorophore to a solvent results in a red shift or a blue shift of the emitted photon depending on the polarity of the solvent. When the excited state is stabilized a red shift is observed which corresponds to a higher

53 34 wavelength and a lower energy between the excited and ground states. A blue shift is observed when the solvent increases the energy of the excited state thereby increasing the energy of the emitted photon. In the case of laurdan, a blue-shift is seen in apolar/hydrophobic solvents while a red-shift is observed in polar/hydrophilic solvents which can be observed visually (Figure 10) Figure 10. The fluorescence of laurdan in different polar and nonpolar solvents using a UV lamp Laurdan in Membranes The hydrophobicity of laurdan limits its application in highly polar solvents. While laurdan is insoluble in aqueous solutions, its structure facilitates an efficient incorporation into hydrophobic environments such as lipid bilayers. Upon uptake into phospholipid bilayers its acyl chain orients towards the hydrophobic interior of the membrane while the fluorescent

54 35 naphthalene moiety faces the aqueous environment 80. This localization is due to laurdan s long and hydrophobic lauroyl chain which anchors the fluorophore deep within the membrane with the fluorescent naphthalene moiety of laurdan positioned at the level of the glycerol backbone which has relatively few water molecules around it 81. The use of laurdan as a neutral polarity sensor in biomembranes has been extensively discussed in the literature 80,82. The dye has been widely used to measure membrane fluidity based on the fact that its fluorescence is independent of both the lipid headgroup as well as ph values between A schematic below depicts how laurdan responds to changes in membrane fluidity (Figure 11). Figure 11. A schematic representation of laurdan in a tighter packing/rigid membrane (left) and a less tightly packing/fluid membrane (right). Membrane fluidity measurements are based on differences in the accessibility of water molecules to the dye within the bilayer; the subsequent changes in polarity are recorded by

55 Intensity (arbitrary units) 36 laurdan. Some factors that affect membrane fluidity include changes in ph, ionic strength and temperature. For the purpose of this thesis ph and ionic strength were always kept constant and membrane fluidity was monitored as a function of temperature General Polarization As described above, the fluorescence emission spectra of laurdan is different in rigid and fluid membranes. An example of this is shown below in DMPG LUVs at temperatures above, below and equal to the Tm of the system (Figure 12). The intensity at 440 nm corresponds to the more ordered gel phase while the intensity at 490 nm corresponds to the more fluid liquid crystalline phase nm 490 nm degrees 23 degrees 37 degrees Wavelength (nm) Figure 12. Results from scans of fluorescence intensity vs wavelength of Laurdan in DMPG LUVs hydrated in 100 mm NaCl at ph 7.40 at 13, 23 and 37 ºC using an excitation wavelength of 340 nm. Arrows indicate intensity at 440 and 490 nm.

56 General Polarization (GP) 37 The fluidity of a membrane containing laurdan can be quantified with the generalized polarization function using the respective emission maxima as shown below (Eq 1) 80 : GP = I 440 nm I 490 nm I 440 nm +I 490 nm (1) Gel Phase T m Liquid Crystalline Phase Temperature ( C) Figure 13. Generalized polarization values as a function of temperature for laurdan in large unilamellar vesicles of DMPG. Fluorescence intensity was measured at 440 and 490 nm using an excitation wavelength of 340 nm. From Equation 1, GP values range from a maximum of +1 to a minimum of -1 although most membranes have GP values between 0.6 and Laurdan detects fluidity changes as a function of temperature for membranes in the gel and liquid crystalline phases with the largest change occurring during the membrane phase transition (Tm) between these two phases. The Tm of a lipid system is observed as a sharp and sudden decrease in the GP over a short temperature

57 38 range. Data in Figure 13 shows the GP of DMPG as a function of temperature which was chosen as an example because the Tm for this lipid is conveniently near room temperature. GP vs temperature graphs for lipid systems that have a Tm above 0 ºC result in a sigmoidal curve with unique sections as illustrated in Figure 13. Temperatures lower than 18 ºC do not produce higher GP values as the membrane is in its most rigid state which gives the gel phase portion of the GP curve a nearly flat slope. In this state the lipids pack tightly together which results in fewer water molecules in the proximity of laurdan. Between temperatures of ºC the GP value is relatively unchanged as this system is still in the rigid gel phase. As the gel phase begins to melt at ºC the GP starts to decrease slowly until the Tm is reached between ºC at which point the GP sharply decreases. Temperatures above the Tm result in a continuous increase in the number of solvent molecules that permeate into the bilayer and affect the spectroscopic response of laurdan. Interestingly, changes in membrane fluidity can be monitored in systems that are already melted above 0 ºC as asymptotic trends reflect gradual changes in a progressively more fluid membrane with increasing temperature. A representative GP curve for such systems is shown in Figure 14 for POPC which has a Tm of -4 ºC. Measurements below 0 ºC are not feasible because of the formation of ice crystals and condensation of water vapor onto the outside cuvette walls. Both of these factors scatter light and prevent accurate fluorescence measurements. The GP vs. temperature data can then be fit to the sigmoidal Boltzmann function (Eq 2) using Origin Pro 8.0 software 83 to determine the Tm of the sample which is equal to X0.

58 General Polarization (GP) 39 Y = A 2 + A 1 A 2 (x x0 ) 1+e dx (2) Temperature (ºC) Figure 14. Generalized polarization values as a function of temperature for laurdan in large unilamellar vesicles of POPC. Fluorescence intensity was measured at 440 and 490 nm using an excitation wavelength of 340 nm. Tm s determined using laurdan in this thesis were compared to Tm s determined by using other techniques under similar experimental conditions to ensure that laurdan did not impact the properties of the membrane. The method of choice was Differential Scanning Calorimetry (DSC) as this technique determines the Tm of a sample without the addition of any fluorophore. 2.2 Lipid System Preparation Materials N-palmitoyl-D-erythro-sphingosine (Ceramide), Cholesterol, 1, 2-dimyristoyl-sn-glycero-3- phosphate (DMPA), 1, 2-dimyristoyl-sn-phosphoglycerol (DMPG), 1, 2-dimyristoyl-sn-glycero- 3-phospho-L-serine (DMPS), 1, 2-dioleoyl-3-trimethylammmonium-propane (DOTAP), D-

59 40 glucosyl-β-1-,1 -N-palmitoyl-D-erythro-sphingosine (Glucosyl ceramide), 1-(1Z-octadecenyl)-2- oleoyl-sn-glycero-3-phosphocholine (PCplasm), 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3- phosphoethanolamine (PEplasm), 1, 2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3'- phosphate) (PI(3)P), 1, 2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3', 5 -bisphosphate) (PI(3,5)P2), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), Sphingomyelin (Brain), Sulfatide (Brain), 1, 3-bis [1, 2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol (TMCL), Total Ganglioside Extract (Brain) and 1, 3-bis [1, 2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (TOCL) were obtained from Avanti Polar Lipids (Alabaster, AL) (Figures 15 and 16). Brain, Heart, Liver and E. coli polar extracts were purchased from Avanti Polar Lipids (Alabaster, AL). HgCl2, CdCl2 and 6-dodecanoyl-2-dimethyl-aminonaphthalene (laurdan) were purchased from Sigma-Aldrich (Oakville, ON). Spectrosil Far UV Quartz cuvettes with a path length of 5 mm were purchased from Starna Cells, Inc (Atascadero, CA). All reagents and lipid solutions were made using doubly distilled water (ddh2o) filtered by a Millipore Synergy 185 water purification system (Billerica, MA). All chemicals were used without further purification. It has to be emphasized that all buffers examined prior to this work or tested during this study complexed Hg and/or Cd which made them unusable for experiments 20. Cd forms complexes with most anions including phosphate and bicarbonate buffers while Hg forms complexes with Tris, HEPES, MOPS and PIPES buffers 20. To avoid interference from metalbuffer interactions particularly in trials conducted with metal mixtures containing both Hg and

60 41 Cd, the experiments in this thesis were conducted in unbuffered 100 mm NaCl adjusted to ph Lipid Structures POPC TMCL PCplasm TOCL PEplasm DMPA DMPS POPA POPS DMPG PI(3)P POPG PI(3,5)2P Figure 15. The structure of the glycerophospholipids used in this study. Red represents the lipid headgroup, blue represents the phosphate linker, black represents the glycerol backbone and green represents the fatty acyl tails.

61 42 Cholesterol Ganglioside Ceramide Glucosyl Ceramide DOTAP Cerebroside Sulfate Sphingo myelin Figure 16. The structures of the sphingolipids, glycerolipids and cholesterol that were used in this study. Excluding cholesterol, red represents the lipid headgroup, black represents the sphingosine backbone, blue represents the phosphate linker and green represents the fatty acyl tail.

62 Lipid Systems Table 6 provides a summary of the lipid systems investigated in this study and provides the molar percentages of the lipids as well as a brief rationale about why the system was selected. Table 6. The Lipid systems used in laurdan GP and DLS experiments. System # Composition Molar Percentages Reason for Study 1 POPC 100 Used as a negative control for the remaining systems 2 Brain sphingomyelin 100 Test for interactions between Hg, Cd and the sphingosine backbone 3 PCplasm 100 To compare the effect of Hg on PCplasm to POPC 4 POPC : Cholesterol 60 : 40 To test for an interaction between Hg, Cd and Cholesterol 5 POPC : PEplasm 50 : 50, 88:12 To compare the effect of Hg on PEplasm compared to PCplasm 6 POPC : Ceramide (16:0) 7 POPC : Glucosyl Ceramide 8 POPC : Sulfatide 80 : : 20 To test for an interaction between Hg, Cd and Ceramide 80 : 20 To test for an interaction between Hg, Cd and Glucosyl Cerebroside To test for an interaction between Hg, Cd and Sulfatide 9 POPC : Ganglioside 90 : 10 To test for an interaction between Hg, Cd and Ganglioside 10 POPA 100 To test for an interaction between Hg, Cd and PA

63 44 System # Composition Molar Percentages Reason for Study To see if Cd targets the 11 DMPA 100 saturated form of PA more or less than the partially unsaturated form To see how the increased 12 TOCL 100 distance between negative charges in CL affects Cd binding compared to PA To see if Cd targets the 13 TMCL 100 saturated CL more or less than the unsaturated form To see how the presence of 14 POPG 100 the glycerol headgroup affects Cd binding compared to PA To see if Cd targets the 15 DMPG 100 saturated PG more or less than the unsaturated form To see how Cd affects 16 POPS 100 anionic membranes in which the negative charge is located further from the hydrophobic core of the bilayer To see if Cd targets the 17 DMPS 100 saturated PS more or less than the unsaturated form To see how Cd affects 18 POPC : PI(3)P 80 : 20 membranes that have a lipid with a net charge of -2 To see how Cd affects 19 POPC : PI(3,5)2P 80 : 20 membranes that have a lipid with a net charge of -3

64 45 System # Composition Molar Percentages Reason for Study To test if Hg 20 POPC : DOTAP 50 : 50, 88 : 12 electrostatically targets cationic membranes To compare the effect of Hg on DOTAP to its effect on plasmalogens Polar Extracts To understand how Hg and Brain Cd affect membranes with 21 Heart complex lipid percentages, Liver - headgroup mixtures and E. Coli fatty acyl distributions Red blood cell To determine how Hg and 22 POPS : PC plasmalogen 60 : 40 Cd affect their unique targets when present simultaneously To gain an overall 23 RBC ghosts - understanding for how Hg, Cd affect RBC membrane fluidity Large Unilamellar Vesicle Preparation A schematic showing how LUVs were prepared for this study is shown in Figure 17. Three ml glass vials used for sample preparations were cleaned with 4 solvents acetone, methanol, hexane and chloroform and left to dry overnight before use. Dry lipid powders used in the preparation of LUVs were weighed using a Sartorius Microbalance MC 5 (Göttingen, Germany). A 500 µm stock solution of laurdan was prepared by dissolving the required amount of dry powder in 10 ml of chloroform. LUVs were prepared by dissolving lyophilized lipid

65 46 powder in a 1:1 (v/v) chloroform/methanol solution. Enough lipid was weighed out so that the final concentration of lipid in 1.8 ml of 100 mm NaCl would be about 1.5 mm. Based on the amount of lipid transferred to the glass vial, the volume of laurdan needed to achieve the desired 1 mole laurdan: 550 moles lipid was calculated and added to the vial. Following sonication and vortexing to ensure complete dissolution of the lipid powder and an even distribution of the laurdan the solvent was removed by argon followed by applying-vacuum overnight to ensure complete evaporation of traces of organic solvent. This resulted in the formation of a laurdan/lipid film at the bottom of the glass vial. Lipid mixtures were made by combining aliquots of the individual lipid solutions to achieve the desired molar ratios. Lipid films were then hydrated in 100 mm NaCl adjusted to ph 7.4 followed by heating the samples above the Tm and sonication until a homogenous suspension of multilamellar vesicles (MLVs) was formed. These samples were degassed for 4 mins using a ThermoVac unit to minimize the acidification of the solution from dissolved CO2. Samples were sonicated and exposed to freeze thaw cycles until visual inspection showed that the lipid film was completely removed from the glass surface. LUVs were prepared by using an extruder apparatus (Avanti Mini Extruder, Alabaster, AL) and by passing the MLVs at least 20 times through 100 nm Nucleopore polycarbonate membranes. For each preparation, the solution was kept 5-10 C above the Tm of the lipid during extrusion. The initial concentration of lipid was between mm based on how much lipid was weighed out. A higher lipid concentration was used so that if the sample was diluted during extrusion that it would still be well above the final lipid concentration of 0.3 mm that was used for experiments. It was estimated that 1.8 ml of 1.5 mm lipid would be

66 47 enough sample to perform both GP and DLS experiments for the controls and trials with Hg, Cd and Hg/Cd mixtures in triplicate. To further account for any ph drift that could occur between the times the solution was adjusted and when fluorescent and DLS measurements were completed, separate batches of LUVs were made in 20 mm HEPES and 100 mm NaCl at ph 7.40 as ph control experiments with Cd which does not form a complex with HEPES 84. In contrast, Hg forms a HEPES-Hg precipitate. It was found that Hg had buffering capacity in 100 mm NaCl due to the formation of different hydroxylated species under the selected chloride and ph conditions. As such, the ph was buffered in trials conducted with Hg and Hg/Cd mixtures due to the buffering capacity of Hg while separate experiments with HEPES for metal-free controls and Cd ensured buffering capacity for these experiments as well. The results from controls and experiments with Cd in the presence and absence of HEPES were compared and no statistical differences were found between the data sets based on Student s T-test. This ruled out the possibility that a ph drift was responsible for metal induced fluidity and size changes on the LUVs and RBC ghosts.

67 48 Dissolve in organic solvent Add Laurdan Dry Rehydrate Heat Sonicate Vortex Weigh lipid(s) into clean glass vial Vortex Sonicate Evaporate off organic solvent using Argon Gas Add buffer to lipid film Large Unilamellar Vesicles 100 nm Extrude Solution Multilamellar Vesicles Figure 17. A diagram illustrating the formation of large unilamellar vesicles (LUVs).

68 Lipid Concentration Determination Because the results were highly sensitive to the lipid/metal ratio in solution, it was essential to determine the lipid concentration before fluorescence and DLS measurements were performed. The use of an analytical balance to weigh the lipid powders and the fact that the dried lipid film was rehydrated in a defined aqueous volume provided a good approximation of the lipid concentration. Nevertheless, some losses have to be expected during the extrusion process. This could result from lipids sticking to the glass vials or the membrane filters as well as minor leaks from the extruder and evaporation of water from lipid systems that were extruded above room temperature for an extended period of time Schiefer and Neuhoff Phospholipid Determination Assay Lipid determination of biomimetic membranes was performed by using a sensitive fluorescence assay. This method utilizes the fluorophore Rhodamine 6G which experiences an increase in fluorescence at 563 nm specifically when bound to phospholipids 85. Two 25 µl aliquots were taken from MLVs before extrusion where concentration was known by assuming 100% recovery of lipids and transferred to microcentrifuge tubes. After extrusion, two 25 µl aliquots of the LUV suspension were also deposited into 2 more microcentrifuge tubes. The aqueous solution of all 4 tubes was removed by drying overnight in a Thermolyne Oven/Incubator followed by their resuspension in 100 µl of cold chloroform the next day. The tubes were then sonicated and vortexed until the lipid pellets were dissolved in chloroform. The tubes were then centrifuged at 5000 rpm for 2 mins using a Thermo IEC Micromax centrifuge (Thermo Scientific, Ottawa, ON) to pellet the insoluble NaCl from the initial aliquots.

69 50 The lipid determination was then conducted as follows. 20 µl of a 10 mg/ml stock solution of Rhodamine 6G dissolved in chloroform, 2 ml of cyclohexane and 1 µl of glacial acetic acid were added to a Shanghai glass cuvette. Fluorescence measurements were recorded using a Varian Cary Eclipse Spectrofluorometer (Aglient Technologies, Santa Clara, CA) using excitation and emission wavelengths of 535 and 563 nm respectively. 5 nm bandpass filters were used for both excitation and emission wavelengths. Standard curves were then created using the initial fluorescence measurement followed by measurements after the injection of 2 µl of the lipid/chloroform solution. Seven injections were done before the data were fit using a linear regression. This protocol was applied to pre and post extrusion samples and the concentration of lipid after extrusion was calculated as follows: If the average slope was 50 before extrusion in a 1 mm lipid sample and 45 after extrusion the concentration after extrusion is: Post Extrusion Concentration = (1 mm Pre Extrusion) *(45/50) Post Extrusion Concentration = 0.9 mm One of the disadvantages of the Neuhoff assay is that the observed fluorescence changes of Rhodamine 6G vary with the properties of the phospholipid being injected. This is not an issue when accurate pre-extrusion standard curves can be created as was the case for the biomimetic experiments of this thesis. However, in the polar lipid extract and RBC ghost experiments no initial standard concentration was available as a comparison for the unknown slope. In these cases, lipid concentrations were determined by using the Ames phosphate determination assay.

70 Ames Phosphate Determination Assay Unlike the Neuhoff assay which measures fluorescence changes upon the binding of Rhodamine 6G to phospholipids, the Ames assay measures the concentration of inorganic phosphate 86 and was conducted as follows. Twelve 20 ml glass vials were 4 solvent washed as was done for glass vials used to make MLVs. Six tubes were used for phosphate determination of the extruded sample of LUVs, 3 tubes were used for the negative control (100 mm NaCl) and 3 tubes were used for the positive control (10 mm sodium phosphate buffer). 5 µl of the lipid, negative and positive controls were transferred to specified tubes. 30 µl of 10% (w/v) Mg(NO3)2 dissolved in 95% ethanol was then added to each tube followed by a short vortexing to mix the contents. The tubes were then exposed to high temperature yielding ash by applying a Bunsen burner flame until all the ethanol was evaporated. This was evident by a color change in the sample from dark brown to white. 300 µl of 0.5 M HCl was then added to each tube followed by vortexing until the white precipitate was redissolved. The tubes were capped with aluminum foil and placed in boiling water for 15 minutes. Mg(NO3)2 + HCl result in the cleavage of the inorganic phosphate from the phospholipids in the sample. While the samples were boiling a mixture of 1 part 10% (w/v) ascorbic acid and 6 parts 0.42% (w/v) ammonium molybdate was prepared in 1 M H2SO4. After the samples finished boiling and were allowed to cool, 700 µl of the ascorbic acid/ammonium molybdate mixture was added to each tube. Tubes were vortexed to ensure complete mixing followed by a 1-hour incubation at 37ºC in a Fisher Scientific Isotemp 500 series incubator. The solution from each tube was transferred into quartz cuvettes to measure absorbance at 820 nm

71 52 using a Shimadzu UV-1700 UV-VIS Spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Acidified ammonium molybdate reacts with inorganic phosphate to produce ammonium phosphomolybdate which has an absorption peak at 820 nm 86. The absorbance at 820 nm was averaged between the 6 sample tubes followed by the subtraction of the average absorbance of the 3 negative controls. The concentration of phosphate was then determined using standard curves generated using sodium phosphate standards Determination of Total Lipid Concentration It should be noted that the Neuhoff and Ames assays are only able to detect the concentration of the phospholipids in the sample. Lipids without a phosphate in their structure including cholesterol, ceramide, cerebroside, ganglioside, etc. cannot be detected by either of these assays. For experiments using the Neuhoff assay it was assumed that the concentration of the phospholipids and non-phospholipids changed to the same extent during extrusion. This meant that if the concentration of LUVs composed of 50 POPC: 50 Cholesterol decreased by 10% after extrusion that both the POPC and Cholesterol were both diluted by 10%. For experiments using the Ames assay only the phospholipid concentration was determined using the A820 and the concentration of non-phospholipids was estimated from the lipid composition analysis provided by the supplier Avanti Polar lipids. An example for this calculation is shown below. The Heart polar extract profile provided by Avanti determined that 42.3% of the weight is composed of phospholipids and the other 57.3% is composed of neutral lipids. Before the total

72 53 lipid concentration was calculated the contribution from Cardiolipin had to be accounted for as well. Because 1 Cardiolipin lipid has 2 phosphates in its structure its contribution was divided by 2 to match the other phospholipids. If the concentration determined by the Ames assay after correcting for the Cardiolipin was e.g. 1 mm phospholipid then the total lipid concentration was calculated as: Total Lipid Concentration = 1 mmphospholipid + (1 mmphospholipid*0.573) Total Lipid Concentration = mm Lipid For the RBC ghosts it was determined from the literature that cholesterol makes up 50% of the total membrane lipids 87 so the concentration of phospholipid determined using the Ames assay was doubled to give the total lipid concentration for these experiments. It should be noted that these calculations are only approximations of the total lipid content RBC Ghost Preparation Rabbit RBC ghosts were isolated using a previously optimized method 88. The obtained blood was evenly divided into 4 tubes containing the anticoagulant sodium heparin. Tubes were then centrifuged at 3,000 rpm at 4ºC for 10 mins using a Sorvall Legend X1R Centrifuge (Thermo Scientific, Ottawa, ON) to pellet the red blood cells. The supernatant fraction was decanted followed by 4 washes of the cells with 3 ml of Krebs-Ringer Buffer (Sigma, Oakville, ON) to which 1.26 g/l of sodium bicarbonate was also added and adjusted to ph The pellet was gently resuspended each time using the lowest speed of vortexing possible to remove the cells from the walls off the vial. After the tubes were centrifuged at 1,600 rpm at 4ºC for 10

73 54 minutes the supernatant layer containing the platelets and leukocytes was decanted and discarded. This washing step was repeated a total of four times. Three ml of washed RBCs were then transferred into 40 ml centrifuge tubes to which 27 ml of 5 mm phosphate buffer (ph 7.40) was added to lyse the RBCs and cause the intracellular hemoglobin to leak from the cells. This is due to large osmotic difference that is created between the low salt environment outside the RBCs and the high salt environment inside the RBCs. Each tube was gently mixed by pipetting and centrifuged at 20,000x g at 4ºC for 40 mins using a Sorvall RC SC Plus Centrifuge (Thermo Scientific, Ottawa, ON). The supernatant layer was removed and the absorbance spectra was collected for a 1 ml portion of each wash between nm to monitor the hemoglobin content using a Shimadzu UV 1700 Spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Washes were repeated 6 times until the pelleted RBCs had a white color and the absorbance spectra of the supernatant fraction showed no characteristic peaks from hemoglobin at 425, 550 and 575 nm. As phosphate buffer would form a precipitate in subsequent Cd experiments, the pellet formed after the last wash was resuspended in 30 ml of 100 mm NaCl (ph 7.40) followed by centrifugation at 20,000x g for 40 minutes. This was repeated 3 times to remove the phosphate buffer from the RBC ghosts. The pellet from the last wash was resuspended in 6 ml of 100 mm NaCl (ph 7.4) which was evenly divided into one 4 ml and one 20 ml glass vial. The smaller sample was used for experiments with the intact RBCs while the larger fraction was used for a total lipid extraction. 5 µl aliquots were taken from the vial with intact RBCs for phospholipid determination using the Ames assay while the rest of the ghosts was incubated for 40 minutes at 37ºC to allow for reannealing of the

74 55 membrane. Once the total concentration of lipid was known from the Ames assay, the volume of laurdan needed to reach a final laurdan: lipid molar ratio of 1: 550 was calculated based on a 500 µm laurdan stock dissolved in dimethyl sulfoxide (DMSO). Laurdan is added in DMSO to existing membranes at Laurdan: lipid molar ratios between 1:300 and 1:1000 to keep the concentration of organic solvent as low as possible 79,89,90. The RBC ghost suspensions were wrapped in tin foil and left at room temperature for 2 hours to allow for complete incorporation of laurdan into the membranes. For trials in which the asymmetry of the membrane was restored, a sample of the RBC ghosts was aliquoted into a separate 4 ml glass vial to which Mg-ATP was added to a final concentration of 0.6 mm and left for an hour at 37 ºC as was done in a previous study 91. The presence of phosphate molecules from both ATP and hydrolyzed ATP is a potential problem as a Cd-phosphate precipitate would form during fluorescence trials and obscure measurements. This is a result of both an increase in solution turbidity due to the formation of this precipitate as well as a reduction in the concentration of free Cd which are both problematic events. To correct for this, the sample in which asymmetry was restored was centrifuged at 20,000 g for 30 mins and the supernatant was carefully pipetted off followed by a resuspension of the pellet in 100 mm NaCl adjusted to ph This was done two times followed by fluorescence measurements conducted according to section RBC ghost total lipid extraction In order to assess how the presence of the cytoskeleton, membrane asymmetry and membrane proteins of the RBC affect Hg and/or Cd induced fluidity changes, an organic extraction was performed to isolate the complex lipid fraction of the RBC ghosts from the

75 56 previously mentioned factors. The organic extraction was conducted as follows: 3.0 ml of a 2:1 (v/v) ratio of a chloroform/methanol mixture 41 was added to 3.0 ml of RBC ghosts in a 20 ml glass vial, which was then thoroughly mixed via sonication and vortexing. The samples were then ultrasonicated for 30 seconds to break the RBC ghosts using a Microson XL-2000 series Ultrasonicator (Newtown, CT, USA). In order to separate the emulsion into organic and aqueous phases, the tube was centrifuged at 2,500x g for 4 mins using a Sorvall Legend X1R Centrifuge (Thermo Scientific, Ottawa, ON). This divided the emulsion into the less dense aqueous layer floating on top, the denser organic layer and a thin, white layer containing proteins associated with the RBC ghosts in between these layers. The organic layer was then removed using a pasteur pipette and stored in a 20 ml glass vial. The extraction process was repeated 4 more times by pooling the organic layer in the same glass vial after each wash. The organic layer was then washed 4 times with an equal volume of deionized water to remove residual salt from the lipid extract. The tubes were centrifuged at 1000xg for 4 mins and the aqueous phase was removed with a Pasteur pipette and discarded. To remove residual water after the last wash sodium sulfate was added as a drying agent until none of the aqueous emulsion layer was visible. The samples were then centrifuged at 1,000g for 2 mins to pellet the sodium sulfate to the bottom of the tube followed by the transfer of the organic solvent to a clean 4 ml glass vial. The organic solvent was then dried using argon resulting in a dry lipid film. LUVs from the RBC total extract lipid film were prepared as described in section (LUV preparation). The terminology total extract in this thesis refers to the extraction of both the polar and nonpolar lipids in the RBC ghost membranes but this does not imply that 100% of the lipids in the RBC ghosts were

76 57 recovered by the extraction procedure. Following the Ames phosphate assay, the volume of laurdan needed to yield a 1 laurdan: 550 lipid molar ratio was calculated and added from a 500 µm laurdan stock dissolved in DMSO. After the necessary 2-hour incubation to ensure complete laurdan incorporation into the RBC total extract LUVs, fluorescence measurements were conducted (as described in section 2.3.1). 2.3 Laurdan GP Fluorescence Spectroscopy Trials Laurdan General Polarization Procedure Laurdan containing LUVs were studied using a Varian Cary Eclipse Spectrofluorometer (Aglient Technologies, Santa Clara, CA). After the concentration was determined, the lipids were diluted to a final concentration of 0.3 mm and a final volume of 500 µl in Far UV Quartz Cuvettes with a 5 mm path length. The temperature of the sample was controlled within ºC using a circulating water bath. Each sample was allowed to equilibrate for 10 minutes before measurements were taken. For systems where a Tm determination was feasible (Tm > 10 ºC) the sample was incubated at a temperature at least 10 ºC below the Tm of the system. The temperature was then increased in 5 ºC increments until about 5 ºC before the Tm at which point measurements were taken every 1-2 ºC depending on how sharp the Tm was. Sharper Tm s required collection of data points every 1 ºC near the Tm. More data point s 2-4 ºC before and after the Tm yielded a better fit to the sigmoidal function in Eq 2. Initial experiments showed that an optimal time to let the sample incubate before taking a measurement was 1 min/δºc. This meant that for a temperature change of 5 ºC, the sample was incubated for 5 mins before taking a

77 58 measurement. This was determined by repeatedly measuring the GP of a sample after changing the temperature and monitoring how long it took the value to stabilize. For metal containing trials, Hg and/or Cd were added to the sample at the start of the initial incubation. Fluorescence measurements were recorded using an excitation wavelength of 340 nm and emission intensities at 440 nm and 490 nm using 5 nm bandwidth slits for both excitation and emission slits. A range of ºC was selected for systems in which the Tm was below 0 ºC to sample temperatures that were below, near and above the physiological body temperature of 37 ºC. After recording fluorescence intensities at 440 and 490 nm, the GP values were calculated using Eq 1 and plotted versus temperature. When metal induced changes in the GP compared to controls were observed the experiment was repeated with half the concentration of metal to determine minimal effective metal concentrations. This process was repeated until GP values after metal addition were no longer statistically different from controls based on Student s T-Test. Scattering contributions in the data were corrected by subtracting readings from LUVs without laurdan. In order to ensure high quality data and to avoid contributions from the inner filter effect, the absorbance of all samples at 340, 440 and 490 nm was measured by using a Shimadzu UV 1700 Spectrophotometer (Shimadzu Corporation, Kyoto, Japan). The inner filter effect was due to the observation that some LUV preparations became more turbid in the presence of Cd making the excitation light inconsistent throughout the sample. The result was that a smaller percentage of excitation light reached the center of the cuvette where the emission light is collected resulting in increased error for the intensities at 440 and 490 nm 92. In

78 59 cases where the absorbance exceeded 0.05 the inner filter effect was corrected for using Eq This was only necessary for DMPA and DMPS preparations above 0.1 mm Cd. The GP was then calculated using these corrected fluorescence intensities. 2.4 Dynamic Light Scattering Trials LUV Size Determination F Corrected = F Observed x 10 Abs exc+absem 2 (3) The LUV size and size distributions by DLS were done for two reasons. The first was to check the size and uniformity of the LUVs formed by extrusion process as a relatively monodisperse population with a diameter below the 100 nm polycarbonate pore cut-off was expected. The second reason was to assess the effect of Hg and/or Cd on the size of the LUVs. DLS is able to measure the size of particles based on their Brownian or random motion in a fluid since large particles move slower than smaller ones. LUV size distributions were analyzed using the Zetasizer Nano ZSP (Malvern Instruments, Malvern, UK). A 500 µl volume of 0.3 mm lipid suspension was degassed for 4 mins to remove air bubbles which would scatter light and interfere with DLS measurements. This solution was then transferred to Far UV Quartz cuvettes with a 5 mm path length. Each sample was measured in triplicate at 25 C to determine the mean peak radius of the LUV population. All preparations resulted in a narrow size distribution with a polydispersity index (PDI) that ranged from Data in the presence and absence of Hg and Cd were compared to the controls and analyzed to assess metal induced changes. It was confirmed that presence of laurdan in the membranes did not affect metal induced changes by using LUVs with and without laurdan.

79 Thin Layer Chromatography (TLC) TLC was conducted on polar extract membranes with lipid standards in order to further characterize the components of these complex mixtures to help explain the observed effects of Hg and Cd. TLC was used in this thesis as a chromatography technique which separates compounds based on their polarity by spotting them onto a thin polar silica glass plate. A solvent mixture is then drawn up the plate by capillary action to a certain height. Based on the structural differences of the lipids in these polar extract mixtures, a separation is achieved with more polar lipids migrating lower on the plate while less polar lipids migrate higher on the plate. TLC experiments were conducted as follows: 5 cm wide and 10 cm long Silica Gel 60 F254 precoated plates were used with a 250 µm thickness from EMD Millipore (Darmstadt, Germany). Before the spotting of lipid samples onto the plate, the two solvents used to separate the lipid mixtures were run to bring any contaminants on the plate surface to the very top of the plate. These solvent mixtures were used from the literature and work well for the separation of different lipid classes 94. Solvent mixture 1 is composed of CH2Cl2/ethyl acetate/acetone (80:16:4, v/v/v) while solvent mixture 2 is composed of CHCl3/ethyl acetate/ acetone/ isopropanol/ ethanol/ methanol/ water/ acetic acid (30:6:6:6:16:28:6:2, by volume). After leaving the plates to dry, lipid samples were spotted 1.5 cm from the bottom of the plate and run with solvent 1 followed by solvent 2 to 1 cm from the top of the plate to avoid contact with the contaminants. The separated lipids were then visualized according to a procedure from the literature in which the plates are sprayed with 1% (w/v) sulfuric acid, allowed to dry and then heated to 200 ºC until the plate started to char 95.

80 Zeta Potential Measurements Not all of the polar lipid extracts that were purchased from Avanti Polar Lipids had completely defined lipid profiles. As the interactions of Hg and Cd are hypothesized to be driven primarily by electrostatics, zeta potential is a useful technique in estimating the surface potential of a membrane particularly in complex mixtures in which a certain percentage is unknown. The zeta potential of a membrane is correlated to how negative or positive it is compared to the bulk solution meaning that membranes containing cationic lipids have a positive zeta potential while membranes containing anionic lipids have a negative zeta potential. Zeta potential measurements were conducted using zetasizer zeta potential folded capillary cells (DTS1060, Worcestershire, UK). 0.3 mm lipid was dissolved in 20 mm HEPES 10 mm NaCl adjusted to ph 7.40 and degassed for 4 mins to ensure no bubbles were introduced while loading the cell. 10 mm NaCl was used because the instrument was unable to measure data under 100 mm NaCl conditions. 1 ml of sample was then carefully added to the cells and were run at 5 V using a Zetasizer Nano ZSP (Malvern Instruments, Malvern, UK). 10 measurements were conducted for each trial and each sample was measured 3 times. Experiments were conducted with zwitterionic POPC LUVs as a negative control.

81 62 Chapter Three: The effect of Cd on the fluidity and size of biomimetic liposomes Laurdan has been widely used for studying the fluidity and phase transition of different lipid membranes as well as the impact of different metals on these properties. The effects of Thallium (I), Thallium (III) and Thallium (III) oxide on the fluidity of liposomes composed of 80 Brain PC: 20 Brain PS (Porcine) were studied using Laurdan GP 96. It was found that 25 µm Tl + and Tl(OH) 3 increased the GP by compared to the controls while 25 µm Tl 3+ increased the GP by 0.1 which are both indicative of an increase in membrane rigidity 96. Another study showed the effects of µm Al 3+, Sc 3+, Ga 3+, Be 2+, Y 3+ and La 3+ on liposomes composed of 60 DMPC: 40 DMPS with 20 mm Tris, 140 mm NaCl, ph 7.40 at 37ºC 97. All metals increased membrane rigidity in the order of La 3+ > Y 3+ > Sc 3+ > Al 3+ > Ga 3+ > Be 2+. These studies have generally concluded that the negatively charged PS lipid is a common target for most multivalent metals with trivalent metals inducing more rigidity at much lower concentrations than divalent metals. However, it has to be noted that not all metal-lipid interactions are limited to membranes with a net charge as 1.0 and 10 mm ZnCl 2 has been shown to increase the GP in zwitterionic DMPC membranes from to and respectively 98. The effects of Hg and Cd on DMPC and RBC membranes were previously discussed in Chapter 1 highlighting the general disordering effect induced by these metals in the absence of salt. In order to better understand how Hg and Cd affect the membrane fluidity of complex polar lipid extracts and RBC ghosts in the presence of 100 mm NaCl, simple biomimetic systems were analyzed first. Studies conducted using Isothermal Titration Calorimetry (ITC) before this study

82 63 showed very weak binding of Hg and no binding of Cd to POPC 20. As such, Hg/Cd-induced fluidity changes were not expected making this lipid a good negative control for other systems including the binary mixtures containing POPC in this study. The lack of binding between POPC and Cd lead to the hypothesis that Cd exclusively targeted anionic membranes in an electrostatically driven process. To further explore this, LUVs were formulated with different zwitterionic, cationic and neutral lipids. Glycerol based lipids included POPC and DOTAP. Sphingosine based lipids included ceramide, sphingomyelin and glucosyl ceramide. Neutral lipids included cholesterol. Because previous data showed no interaction between Cd and POPC, the phosphocholine headgroup was ruled out as a target through which Cd could induce changes in membrane fluidity and LUV size. Thus, the incubation of Cd with sphingomyelin and ceramide LUVs tested if Cd targeted any part of the sphingosine backbone. Next, LUVs containing cerebrosides, sulfatides and gangliosides were used to see if Cd induces membrane fluidity changes by targeting the sugar headgroup(s) of these lipids. Note that ceramide, cerebroside, sulfatide and ganglioside alone are not stable as LUVs due to the large headgroups of the glycosphingolipids and the hydrophobicity of ceramides. Previous studies have shown that if ceramide is present as more than 30 mol % of the total lipids then the LUVs will aggregate over time 99. LUVs were formulated with 80% POPC and 20% cerebroside/sulfatide/ceramide to avoid aggregation in addition to being able to make comparisons to previous data from our lab conducted by using

83 64 this ratio of PC to PE and PS lipids. Previous studies with liposomes utilized 7-15% ganglioside as this lipids very large headgroup needs more space to be stable 100. Negatively charged glycerol based lipids used in this thesis were amenable to single lipid LUV formulation with the exceptions of PI(3)P and PI(3,5)2P which have large headgroups with multiple negative charges. For this reason, LUVs were made with 80% POPC and 20% PI for consistency with the above mentioned glycolipids and to maintain stability in the bilayer. Pure anionic membranes were then studied in the order: POPA, DMPA, TOCL, TMCL, POPG, DMPG, POPS and DMPS. This order was selected based on the proximity of the negative charge to the aqueous phase, the horizontal distance between negative charges and the absence and presence of groups near these negative charges. A cartoon depicting this is shown in Figure 18. Each of these lipids was studied with partially unsaturated and fully saturated acyl chains to determine how the effects of Cd on anionic membranes change for a given headgroup when the acyl chain composition is altered. PA lipids were studied first as they are structurally the simplest anionic lipid in which the phosphate linker is also the lipid headgroup. CL was studied next as its headgroup structure is similar to PA but with a glycerol group horizontally connecting two PA lipids. The large distance between charged phosphate groups in CL membranes changes the surface charge density of the membrane compared to PA. PG lipids were then studied to see how the addition of the large, polar glycerol headgroup would affect Cd induced fluidity changes compared to PA membranes in which the negative charge is more accessible to Cd. While the group that bears the negative charge is the same in PA, CL and PG membranes, PS and PI have

84 65 multiple negative charges some of which are on groups further away from the hydrophobic core of the bilayer. The lipids were used to assess the impact of net charge on Cd induced changes. Headgroup Region Interface Region Acyl Tail Region PA CL PG PS PI(3)P PI(3,5)2P Net Charge = -1 Net Charge = -2 Net Charge = -3 Acyl Tails Glycerol Phosphate Serine Carboxyl Group Serine Carbon Skeleton and Amine Inositol Figure 18. A schematic depicting the glycerol based anionic lipids studied in Chapter 3 of this Thesis.

85 LUV size determination using DLS DLS was used to characterize the size of LUVs made by extrusion as described in Chapter Two. As polycarbonate filters with 100 nm diameter pores were used for the extrusion process, LUVs with an average radius of near 50 nm were expected (+ 5 nm). In cases where large deviations from this expected radius were observed immediately after extrusion, the result was always larger radii (>10 nm). This can be explained in part by vesicle lysis tension which is defined by the pressure at which vesicles are ruptured 101. As MLVs are much larger than the pores in the polycarbonate membrane, pressure is applied during extrusion to rupture these large structures and force them through the smaller pores where they are reformed as LUVs after a number of passes 101. However, it was observed that in some preparations that contained many lipids that impart rigidity to the bilayer the observed radii were nm. One example is brain polar extract (bovine) which formed LUVs with an average radius of 61.2 nm whereby the presence of cholesterol and PE may have increased the vesicle lysis tension resulting in an increased resistance to rupture during extrusion. This could be responsible for a significantly larger radius than expected. Regardless of the average radius after extrusion, the result was a narrow size distribution of vesicles (Figure 19). The mean vesicle radius that is reported in this thesis is the Zavg radius which is the most abundant size in the distribution curve. In addition to the size distribution, the polydispersity index (PDI) is also reported which is a measure of heterogeneity with a value between 0-1. PDI in DLS is calculated from the distribution peak using Eq 4.

86 67 PDI = ( Width Mean )2 (4) Based on this distribution, a perfectly uniform population of liposomes would have a PDI of 0.0 while PDI s between are said to be monodisperse. The PDI of a liposome population is useful in determining if the extrusion process was successful in producing a monodisperse population of liposomes. Secondly, metal induced aggregation of liposomes is known to cause a large increase in vesicle size. The PDI was used to analyze if the aggregates produced in some systems were of a variable size or not following exposure to Hg and/or Cd. Unless otherwise stated, liposomes produced by extrusion before metal incubation had a PDI below 0.1. Figure 19. A representative DLS trial conducted using 0.3 mm POPC LUVs hydrated in 100 mm NaCl ph 7.40 extruded by using 100 nm diameter pores in a polycarbonate membrane. Each replicate is the average of 10 measurements.

87 Interactions of Hg and Cd with Laurdan In order to avoid misinterpreting changes in the GP during lipid trials as metal-induced changes in the fluorescence of Laurdan, control experiments were conducted with the water soluble analogue Prodan. The only structural difference between these compounds is in the length of their acyl tails which are 12 and 3 carbons long for Laurdan and Prodan respectively. The shorter acyl tail in Prodan reduces the overall hydrophobicity of the molecule compared to Laurdan. This makes Prodan more suitable for control experiments with free dye in an aqueous solvent in the presence and absence of Hg and Cd. As this acyl tail is an electronically neutral hydrocarbon, it is not a target for Hg or Cd while the dimethylamino headgroup of this class of dye molecules is a potential target. Thus, the results observed from Prodan were representative of what is likely to occur with Laurdan. As Prodan in 100 mm NaCl is completely surrounded by water molecules, no fluorescence intensity was observed at 440 nm in this solvent. In order to determine if Hg and/or Cd affect the fluorescence intensity of this class of dye molecules at 440 nm, experiments were conducted in 9:1 (v/v) Isopropyl alcohol: 100 mm NaCl ph 7.40 which is a more hydrophobic solvent than water for which intensity was able to be recorded at 440 nm. It was necessary for 10% of the volume to be water in order to dissolve 100 mm NaCl as this salt is insoluble in pure isopropanol. 500 nm Prodan was chosen as this is close to the concentration of Laurdan in 500 µl of 0.3 mm lipid that is present in the lipid trials in this thesis. Results for these experiments are shown in Figure 20.

88 Intensity (Arbitrary units) nm Prodan in 100 mm NaCl 2 mm Cd 100 mm NaCl 2 mm Hg 100 mm NaCl 500 nm Prodan in Isopropanol 2 mm Hg Isopropanol 2 mm Cd Isopropanol Wavelength (nm) Figure 20. The fluorescence intensity of 500 nm Prodan in different solvents over a series of wavelengths in the absence and presence of 2 mm Hg and Cd at 25 ºC. Each dataset is the average of 2 replicates which were each the average over 5 scans. Results in Figure 20 showed that neither 2 mm Cd or Hg affected the intensity of 500 nm Prodan at 490 nm in either solvent that was tested. While Hg seemed to induce minor (< 10%) quenching in Prodan in 100 mm NaCl between nm, this was not observed in Isopropanol and is likely a result of random error. However, if minor quenching is present in this range, it does not affect the results of this thesis as the GP was always calculated using intensities at 440 and 490 nm. Experiments in Isopropanol showed no effects of Hg or Cd on the

89 70 fluorescence of Prodan at 440 nm. These results show that changes in the GP of Laurdan are not an artifact of the interactions of these metals with the dye. 3.3 Interactions of Cd with membranes containing POPC and Cholesterol As Laurdan GP results for Cd and different zwitterionic and neutral lipids confirmed that Cd only induced fluidity changes via electrostatic interactions with anionic lipids, results will be shown for POPC and cholesterol as lipids representing zwitterionic and neutral lipids respectively. Negative results for POPC and cholesterol with Cd were representative of what was observed for other membranes containing Brain sphingomyelin, ceramide, cerebroside and the cationic lipid called DOTAP. These experiments ruled out the negative phosphate in zwitterionic membranes as a target through which Cd could induce rigidity. Additional targets for Cd that were ruled out include the glycerol and sphingosine backbones, carbohydrate headgroups from cerebrosides and the cationic quaternary amine from DOTAP Laurdan GP with Cd and LUVs containing POPC and Cholesterol As both POPC and sphingomyelin share a phosphocholine headgroup, differences in Cd induced fluidity may be ascribed to the different glycerol and sphingosine backbones.

90 General Polarization (GP) General Polarization (GP) POPC POPC + 2 mm Cd POPC 40 Cholesterol 60 POPC 40 Cholesterol + 2 mm Cd Temperature (ºC) Figure 21. Generalized Polarization values for Laurdan in 0.3 mm LUVs composed of 0.3 mm POPC (top) and 60 POPC 40 cholesterol (bottom) in the absence and presence of 2 mm Cd as a function of temperature. Results are the average of 3 replicates + standard deviation. Error bars are within the symbol size. Results from POPC showed decreases in the GP of the membrane, which became progressively smaller as a function of temperature (Figure 21). This asymptotic pattern is indicative of a membrane in the liquid crystalline phase. The addition of cholesterol to POPC

91 Mean LUV radius (nm) 72 resulted in a much more rigid membrane at all temperatures compared to pure POPC. For example, at 25 ºC the GP of 60 POPC 40 Cholesterol was 0.35 compared to for pure POPC. The addition of 2 mm Cd to both these systems resulted in no changes in the GP at any temperature compared to the control DLS with Cd and LUVs containing POPC and Cholesterol DLS results for POPC and 60 POPC 40 cholesterol LUVs are shown in Figure Control 2 mm Cd 45 POPC 60 POPC 40 Cholesterol Figure 22. The average LUV radii measured using DLS in the absence and presence of 2 mm Cd. Results are the average of 3 replicates + standard deviation Discussion of the interactions of Cd with LUVs containing POPC and Cholesterol Cd did not induce any fluidity changes at any temperature in POPC and 60 POPC 40

92 73 cholesterol LUVs hydrated in 100 mm NaCl at ph As, cholesterol is an abundant component in many mammalian membranes discussed in Chapter 5 it was included in this study to test if this sterol is a potential target by which Cd could affect fluidity and induce size changes in polar extracts and/or RBC ghosts (Chapter Five). Because cholesterol is uncharged and mainly hydrophobic except for a lone polar hydroxyl headgroup, the lack of metal induced changes supports the hypothesis that Cd interactions are driven by electrostatic forces. However, Cd has been previously shown to interact with zwitterionic membranes including PC in the absence of chloride 19. Under these conditions and at ph 7.40 Cd is predicted to be present as Cd 2+ ions, which may bind the negatively charged phosphate in POPC membranes. However, the presence of 100 mm NaCl results in a saturation of these phosphates with sodium ions in addition to the formation of Cd species with charges of +1 and 0. Under the conditions of this study only about 20% of Cd is present in the Cd 2+ form (Table 2). The inability of Cd to affect both the fluidity and size of POPC LUVs may indicate that the CdCl + form is unable to effectively bind the phosphate in these lipids. This may be in part due to the binding of sodium ions that neutralize much of this negative charge and/or due to the larger size of the Cd ion that is coordinated to chlorine being unable to accommodate an optimal orientation to bind this phosphate. The bulky size of the positively charged choline headgroup is also a factor that likely prevents access of Cd to the negative phosphate group through both steric hindrance as well as electrostatic repulsion. DLS data supported results from Laurdan GP since no size changes were found for either POPC or 60 POPC 40 cholesterol LUVs. As was previously discussed in Section 1.7 (pages 28-30), in order for metal-induced aggregation to occur in a liposome population, the metal must

93 74 bind and neutralize excess surface charges. This was unable to occur likely due to POPC and cholesterol being zwitterionic and neutral respectively which makes the binding of Cd unfavorable. 3.4 Interactions of Cd with membranes sulfatide and ganglioside The negative result that was found between Cd and LUVs containing sphingomyelin, ceramide and cerebroside ruled out the choline and galactose headgroups, phosphate linker and sphingosine backbone as targets through which Cd could induce rigidity and size changes in LUVs. Negatively charged sphingolipids including cerebroside sulfate (sulfatide) and ganglioside were tested next to challenge the hypothesis that Cd targets anionic lipids. Due to the previously talked about zwitterionic and neutral lipids, any changes in LUV size and membrane fluidity can be ascribed to an interaction between Cd and these negative charges Laurdan GP with Cd and LUVs containing sulfatide and ganglioside The results from Laurdan GP trials with LUVs containing POPC in binary mixtures with 20% cerebroside and 10% ganglioside are shown in Figure 23.

94 General Polarization (GP) General Polarization (GP) POPC 20 Sulfatide 80 POPC 20 Sulfatide + 2 mm Cd POPC 10 Ganglioside 90 POPC 10 Ganglioside + 2 mm Cd Temperature (ºC) Figure 23. Generalized Polarization values for Laurdan in LUVs composed of 0.3 mm 80 POPC: 20 sulfatide (Brain) (top) and 90 POPC: 10 ganglioside (Brain) (bottom) in the absence and presence of 2 mm Cd as a function of temperature. Results are the average of 3 replicates + standard deviation.

95 76 A higher GP in POPC/sphingolipid mixtures compared to pure POPC was observed in all lipid sphingo based systems and at all temperatures with an exception being the mixture containing 10% ganglioside which was more fluid than pure POPC between ºC. This general increase in GP values can be explained by the previously discussed rigidity imparted by saturated acyl chains in addition to the hydrogen bonding capability of the sphingosine backbone. Exposure of LUVs containing 10% Brain ganglioside to 2 mm Cd resulted in an increase in membrane rigidity compared to the control at temperatures higher than 25 ºC while no changes were seen in LUVs containing 20% Brain sulfatide exposed to the same concentration of Cd at any temperature (Figure 23). The degree to which Cd induced rigidity in GM containing LUVs compared to the control seemed to increase as a function of a temperature and began to plateau at 50 ºC DLS with Cd and LUVs containing sulfatide and ganglioside DLS results for POPC in binary mixtures with 20% sulfatide or 10% ganglioside are shown in Figure 24.

96 General Polarization (GP) Control 2 mm Cd POPC 20 Sulfatide 90 POPC 10 Ganglioside Figure 24. The average LUV radii measured using DLS in the absence and presence of 2 mm Cd. Results are the average of 3 replicates + standard deviation. The results for these systems showed no statistically significant size changes compared to the control following exposure to 2 mm Cd based on Student s T-Test Discussion of the interactions of Cd with LUVs containing sulfatide and ganglioside While no interaction was expected between Cd and the uncharged ceramide and cerebroside lipids, some effect was expected for the negatively charged sulfatide and ganglioside lipids. Surprisingly, 2 mm Cd did not induce any fluidity changes in LUVs containing 20% sulfatide while a very moderate increase in rigidity was seen with 10% ganglioside although only

97 78 at temperatures above 25 ºC. The unexpected results for sulfatide may be explained by the weak Cd-sulfate ion pair that forms in solution. Cadmium sulfate is highly soluble in water (~76.4 g/100 ml at 25 ºC) which favors dissociation into free Cd and sulfate ions 102. This weak association could explain why Cd does not strongly bind the negatively charged sulfate group in the sulfatide lipid. Concerning the results from LUVs containing ganglioside, it is possible that Cd did not induce rigidity between temperatures ºC because the ganglioside lipids were already packing somewhat tighter in this temperature range because the very large ganglioside headgroups provide a significant steric hindrance. The increased membrane fluidity at higher temperatures provide more favorable conditions for Cd to exert its rigidifying effect on this lipid. To my knowledge there are presently no studies available reporting on the interactions of Cd with ceramide, cerebroside, sulfatide or ganglioside lipids. Some studies have shown that Cd may indirectly affect the percentage of ceramide in the lipid bilayer by upregulating sphingomyelinase 103. This enzyme activity results in the hydrolysis of sphingomyelin to produce phosphocholine and ceramide while a direct interaction with ceramide is less likely based on the results of this thesis. 3.5 Interactions of Cd with phosphatidic acid (PA) membranes The GP values collected by screening neutral and negatively charged sphingolipids suggest that Cd exclusively targeted negatively charged lipids with the exception of the sulfatide. However, it is currently unknown how Cd discriminates between different negatively charged

98 79 lipids. The local environment of the charge, membrane rigidity as well as the proximity of the charge to the aqueous phase were considered in the following experiments Laurdan GP with Cd and POPA and DMPA LUVs GP results for LUVs composed of POPA and DMPA are shown in Figure 26. In contrast to earlier systems, POPA and DMPA have Tm s at temperatures of 24.0 and 50.1 ºC respectively, which result in sigmoidal GP curves exhibiting inflection points at the Tm. These transitions determined with Laurdan are in agreement with previous data determined by using the fluorescent probe 1, 6-Diphenyl-1, 3, 5-hexatriene (DPH) for POPA and by Differential Scanning Calorimetry (DSC) for DMPA 104,105. Cd concentrations from µm added to POPA and µm added to DMPA increased the GP at all temperatures compared to the control indicating more rigid membranes. This effect was less pronounced for both PA s in the gel phase between temperatures C and C for POPA and DMPA respectively (Figure 26). Raw data showing the change in fluorescence intensity over a series of wavelengths for DMPA LUVs in the presence and absence of Cd is shown in Figure 25. Cd-induced rigidity of PA membranes began within 2-3 ºC of the Tm resulting in both an increase as well as a broadening of the T m in both membranes (Figure 26). A more detailed analysis in Figure 27 illustrates that changes of the Tm s of POPA and DMPA strongly depended on the Cd concentration. The ΔT m of DMPA followed a logarithmic relationship with the concentration of Cd while the largest changes occurred below the concentration of lipid (0.3 mm) between µm Cd. The ΔTm s in DMPA were larger than in POPA at all Cd

99 Intensity (Arbitary units) 80 concentrations (Figure 27). The Tm of POPA seemed to respond differently to a series of Cd concentrations where a biphasic double logarithmic pattern was observed (Figure 27). At 125 µm Cd the ΔTm of POPA seemed to plateau only to begin increasing again at 250 µm Cd and plateau once more at 2.0 mm Cd. Interestingly, this increase after the initial plateau was found to be close to the concentration of lipid in the study mm DMPA 0.3 mm DMPA mm Cd Wavelength (nm) Figure 25. The fluorescence intensity of 0.3 mm DMPA LUVs at 50 ºC in the absence and presence of 0.75 mm Cd over a series of wavelengths. Results are the average of 3 replicates. Results in Figure 25 showed that the increase in the GP was due to an increase in intensity at 440 and a decrease in intensity at 490 compared to the control which is consistent with an increase in membrane rigidity.

100 General Polarization (GP) General Polarization (GP) Temperature ( C) Temperature ( C) POPA Control mm Cd mm Cd mm Cd mm Cd mm Cd DMPA Control mm Cd mm Cd mm Cd mm Cd mm Cd Figure 26. Generalized polarization values for Laurdan in LUVs of 0.3 mm POPA (top) and DMPA (bottom) with a series of Cd concentrations as a function of temperature. Results are the average of 3 replicates + standard deviation.

101 ΔT m (ºC) Concentration of lipid POPA DMPA Cd (µm) Figure 27. The change in the phase transition temperatures of LUVs made of 0.3 mm POPA and DMPA over a series of Cd concentrations compared to the control. Results are the average of 3 replicates + standard deviation. The upper concentration of Cd for which DLS could be conducted was 2.0 and 0.25 mm Cd for POPA and DMPA, respectively. While DLS sizes were highly inconsistent between mm Cd due to the formation of large aggregates for the latter, Laurdan GP was still conducted as these aggregates did not begin to sediment in this range. Above 0.75 mm Cd, aggregation became so extensive that millimeter sized aggregates began to form and sediment in the cuvette which prevented fluorescence measurements. Pictures of these aggregates are shown in Figure 28. Once formed, it was found that these aggregates are very stable and do not break up in the presence mm EDTA.

102 83 Figure mm DMPA LUVs hydrated in 100 mm NaCl ph 7.40 incubated with 2 mm Cd and photographed at 0 minutes (left), 1 hour (centre) and 5 hours (right) DLS with Cd and POPA and DMPA LUVs Results from DLS experiments with POPA and DMPA LUVs incubated with a series of Cd concentrations are shown in Figure 29. The data showed a Cd-induced increase in the average LUV radius in a manner that was dependent on the concentration of Cd followed by a sharp increase in size leading to the large aggregates in Figure 28. As DLS measures the size of particles in solution these aggregates were difficult to characterize as they sedimented in the cuvette. The limited results that could be obtained showed that these aggregates are highly variable in size with a PDI > 0.4 and a large size distribution that increases over a period of hours until one large millimeter sized aggregate is formed (Figure 28, right).