{"paper_id":"81fc3a09-6b93-4e22-99b4-2f2fbb0ce4f2","body_text":"Association between endometriosis and\nheavy metals in a group of Sri Lankan \nwomen of reproductive age \nby \nWedikara Arachchige Nalinda Yasanga Silva \nPh.D. \t 2012 \nDOI: 10.31357/fmesphd.2012.00110\n\nAssociation between endometriosis and \nheavy metals in a group of Sri Lankan \nwomen of reproductive age \nby \nWedikara Arachchige Nalinda Yasanga Silva \nThesis submitted to the University of Sri Jayewardenepura for the \naward of the Degree of Doctor of Philosophy in Physiology on \n\"Association between endometriosis and heavy metals in a group \nof Sri Lankan women of reproductive age\", on 12th  December \n2012. \n\nI certify that the candidate has incorporated all corrections, amendments and additions \nrecommended by the examiners. \n- . ........................ \nProfessor Sharaine Fernando \nProfessor in Physiology \nDepartment of Physiology \nUniversity of Sri Jayewardenepura \nDate \t .... \n\nThe work described in this thesis was carried out by me under the supervision of \nProf Sharaine Fernando of University of Sri Jayewardenepura, Prof Hemantha \nSenanayake and Prof Kainani Tennekoon of University of Colombo, Dr. Roshini \nPeiris-Johnof University of Auckland, New Zealand and Prof Rajitha Wickramasinghe \nof University of Kelaniya and a report on this has not been submitted in whole or in part \nto any University or any other institution for another Degree / Diploma. \n................. ~'/ \t L3 \nW.A.N.YSilva \t Date \n\nWe certify that the above statement made by the candidate is true and that this thesis is \nsuitable for submission to the University for the purpose of evaluation. \nIcb \nProfessor Sharaine Fernando \nProfessor in Physiology \nDepartment of Physiology \nUniversity of Sri Jayewardenepura \nDate... \n.............. ........ \nProfessor Hemantha Senanayake \nHead \nDepartment of Obstetrics and (Iynaecology \nFaculty of Medicine \nUniversity of Colombo \n192 Date ......... \nDr. Roshini Peiris-John \nResearch Fellow \nSection of Epidemio logy and \nBiostatistics \nFaculty of Medical and \nHealth Sciences \nUniversity of Auckland \nNew Zealand \nDate ......................... \nJCH 9 \nProfessor Kamani Tennekoon \nProfessor of Molecular Life Sciences \nInstitute of Biochemistry, Molecular Biology \nand Biotechnology \nUniversity of Colombo \nProfessor Rajitha Wickramasinghe \nProfessor of Public Health \nFaculty of Medicine \nUniversity of Kelaniya \nDate ...... \t Date \n\nTABLE OF CONTENTS \nTABLEOF CONTENTS................................................................................................. \nLISTOF TABLES .........................................................................................................ix \nLISTOF FIGURES .......................................................................................................xi \nLIST OF ABBREVIATIONS......................................................................................xiv \nACKNOWLEDGEMENTS........................................................................................xvii \nABSTRACT...................................................................................................................xx \nINTRODUCTION....................................................................................................1 \n1.1 \tObjectives.................................................................................................................. 9 \n1.1.1 \tGeneral objectives \t................................................................................................ 9 \n1.1.2 \tSpecific \tObjectives............................................................................................... 9 \nLITERATURE REVIEW.........................................................................................10 \n2.1 Anatomy and physiology of the female reproductive system........................................11 \n2.1.1 Anatomy of the female reproductive system.........................................................11 \n2.1.2 Physiology of the female reproductive system...................................................... 12 \n2.1.2.1 \t Oestrogen stricture, receptors and functions ..............................................12 \n2.1.2.1.1 \tStructure of oestrogen hormone.............................................................. 12 \n2.1.2.1.2 Bio synthesis \tof oestrogens ....................................................................13 \n2.1.2.1.3 \tOestrogen receptors............................................................................... 14 \n2.1.2.1.4 Biological actions of oestrogens ............................................................. 21 \n2.1.3 \tSummary ........................................................................................................... 23 \n2.2 \tEndometr ios is \t.......................................................................................................... 24 \n2.2.1 \tIntroduction \t....................................................................................................... 24 \n2.2.2 \tIncidence of endometrios is.................................................................................. 24 \n2.2.3 \tImpact of endometrios is...................................................................................... 25 \n\n2.2.3.1 Pain symptoms associated with endometriosis \t . 25 \n2.2.3.2 \tSubfertility .................................................................................................. 27 \n2.2.3.3 \tRisk of malignancy ...................................................................................... 27 \n2.2.4 \tEconomic \tburden \t............................................................................................... 27 \n2.2.5 \tDiagnosis \tof endometriosis ................................................................................. 28 \n2.2.6 Treatment options for endometrios is .................................................................... 31 \n2.2.7 \tHistory \t.............................................................................................................. 32 \n2.2.8 Theories on the aetiology of endometriosis ........................................................... 33 \n2.2.8.1 \tSampson's theory of retrograde transplantation .............................................. 33 \n2.2.8.2 \tMetaplastic \ttheory \t....................................................................................... 34 \n2.2.8.3 \tIn situ development theory ........................................................................... 34 \n2.2.9 Current concepts on the aetiopathology of endometriosis ...................................... 34 \n2.2.10 \tEndogenous aetiological factors........................................................................ 37 \n2.2.10.1 Eutopic endometrium in women with endometriosis..................................... 37 \n2.2.10.1.1 \tAdhesion \t............................................................................................ 37 \n2.2.10.1.2 \tInvasion.............................................................................................. 38 \n2.2.10.1.3 \tProliferation ........................................................................................ 39 \n2.2 \t.l0.1.4Apoptosis ........................................................................................... 39 \n2.2.10.1.5 \tAngiogenesis ....................................................................................... 40 \n2.2.10.1.6 Protein \tprofile \t..................................................................................... 41 \n2.2.10.1.7 Gene \texpression .................................................................................. 41 \n2.2.10.2 Ectopic \tendometrium \t................................................................................. 42 \n2.2.10.3 Inflammation and immune response ............................................................ 43 \n2.2.10.4 Hormonal factors ....................................................................................... 45 \n2.2.10.4.1 Biosynthesis of oestrogens in endometriosis .......................................... 45 \n2.2.10.4.2 Oestrogen receptors in endometriosis .................................................... 47 \n\n2.2.10.4.3 Consequences of oestrogen activity in endometriosis \t .47 \n2.2.10.5 \tGenetics \t.................................................................................................... 48 \n2.2.11 \tExogenous aetiological factors .......................................................................... 50 \n2.2.11.1 \tDemographic characteristics ....................................................................... 50 \n2.2.11.1.1Age \t.................................................................................................... 50 \n2.2.11.1.2 \tEthnic ity \t............................................................................................. 51 \n2.2.11.1.3 \tLevel of education ............................................................................... 52 \n2.2.11.1.4 Occupation.......................................................................................... 52 \n2.2.11.1.5 \tSocioeconomic \tstatus........................................................................... 52 \n2.2.11.2 Biological characteristics............................................................................ 53 \n2.2.11.2.1 \tBody \thabitus ....................................................................................... 53 \n2.2.11.2.2 Menstrual and reproductive characteristics ............................................ 54 \n2.2.11.3 \tDietary characteristics ................................................................................ 55 \n2.2.11.4 Environmental factors................................................................................ 56 \n2.2.12 \tSummary ......................................................................................................... 59 \n2.3 \tHeavy melals ............................................................................................................ 60 \n2.3.1 \tDefinition \t.......................................................................................................... 60 \n2.3.2. \tSources of heavy metals..................................................................................... 60 \n2.3.3 \tSri \tLankan \tsituation............................................................................................ 62 \n2.3.4 Routes of entry into the human body.................................................................... 63 \n2.3.5 \tEffects on the \thuman.......................................................................................... 66 \n2.3.5.1 \tToxic \teffects................................................................................................ 66 \n2.3.5.2 Metalloestrogens .......................................................................................... 69 \n2.3.5.2.1 \tIn \tvitro evidence.................................................................................... 69 \n2.3.5.2.2 \tIn vivo evidence.................................................................................... 75 \n2.3.5.2.3 Metalloestrogens and endometriosis ....................................................... 77 \n\n2.3.6 Methods of evaluating the heavy metal status in humans.......................................80 \n2.3.6.1 \tAssessment of exposure ............................................................................... 80 \n2.3.6.1.1 \tProximity to \tsources.............................................................................. 80 \n2.3.6.1.2 Levels of metals \tin dietary items \t............................................................ 80 \n2.3.6.1.3 Food frequency questionnaire................................................................. 80 \n2.3.6.2 Measurennt in biological fluids ..................................................................81 \n2.3.6.2.1 \tSample collection and preparation ..........................................................81 \n2.3.6.2.2 Methods for quantitative determination ...................................................82 \n2.3.6.3 Evaluation of symptoms due to chronic heavy metal exposure ....................83 \n2.3.7 \tSummary ...........................................................................................................86 \n2.4. In vitro models for investigating endornetriosis .......................................................... 87 \n2.4.1 \tChicken chorioallantotic membrane..................................................................... 87 \n2.4.2 Human eutopic endometrial explants ................................................................... 88 \n2.4.3 \tInvasion chambers..............................................................................................90 \n2.4.4 Monolayer cultures.............................................................................................91 \n2.4.4.1 Early attempts to culture human endometrialcells .......................................... 91 \n2.4.4.2 \tSources of endometrial cells \t......................................................................... 92 \n2.4.4.3 Monolayer cell cultures in endometriosis....................................................... 93 \n2.4.4.3.1 \tDiversity of cell types ............................................................................ 94 \n2.4.4.3.2 Applications of monolayer cell cultures in endometriosis ......................... 95 \n2.4.5 \tSummary ......................................................................................................... 100 \n3. METHODS .............................................................................................................. 104 \n3.1 \tPhase! ................................................................................................................... 104 \n3.1.1 \tStudy \tdesign........................................................................................................ 104 \n3.1.2 \tStudy \tsetting........................................................................................................ 104 \n3.1.3 \tStudy population.................................................................................................. 104 \niv \n\n3.1.4 \tSample \tsize \t......................................................................................................... 105 \n3.1.5 \tData collection..................................................................................................... 106 \n3.1.6 Collection of blood and tissue samples .................................................................. 108 \n3.1.7 Chemical digestion of blood and tissue samples ..................................................... 110 \n3.1.7.1 \tPrinciple \t....................................................................................................... 110 \n3.1.7.2 \tProcedure...................................................................................................... 110 \n3.1.7.3 Measures to minimize metal contamination during the chemical digestion ............ 111 \n3.1.8 Determination of metal \tlevels ............................................................................... 112 \n3.1.8.1 Determination of metal levels using Total-reflection X-ray Fluorescence .......... 112 \n3.1.8.2 Determination of cadmium levels \t................................................................... 115 \n3.1.8.3 Quality control and validation......................................................................... 117 \n3.1.9 Data analysis \t....................................................................................................... 119 \n3.1.9.1 \tStatistical packages........................................................................................ 119 \n3.1.9.2 Data analysis ................................................................................................. 119 \n3.1.9.3 Analysis of blood and tissue metal levels......................................................... 120 \n3.1.10 \tEthical clearance................................................................................................ 120 \n3.2 \tPhase \tII .................................................................................................................. 121 \n3.2. 1 \tCollection of samples ....................................................................................... 121 \n3.2.1.1 \tStudy \tparticipants \t...................................................................................... 121 \n3.2.1.2 \tEthical considerations \t................................................................................ 122 \n3.2.1.3 Collection of eutopic endometrial tissue samples ......................................... 122 \n3.2.2 Isolation of endometrial stromal cells................................................................. 123 \n3.2.2.1 \tCollagenase digestion................................................................................. 123 \n3.2.2.1.1 \tPreration of collagenase solution....................................................... 123 \n3.2.2.1.2 Incubation with Collagenase ................................................................ 123 \n3.2.2.2 Isolation of stromal cells with filtration and centrifugation............................ 124 \nV \n\n3.2.2.3 Determination of cell number \t . 124 \n3.2.2.4 Determination of cell viability \t.................................................................... 125 \n3.2.3 Establishment of primary endometrial stromal cell (ESC) cultures....................... 125 \n3.2.4 Establishment of sub cultures ............................................................................ 126 \n3.2.5 Treatment of ESC cultures with metal compounds.............................................. 127 \n3.2.5.1 \tPreration of metal solutions..................................................................... 127 \n3.2.5.1.1 \tMetal compounds ................................................................................ 127 \n3.2.5.1.2 Preration of cadmium solutions......................................................... 127 \n3.2.5.1.3 Preration of nickel solution ............................................................... 128 \n3.2.5.1.4 Preç&ation of lead solution.................................................................. 128 \n3.2.5.2 Treatment of the cell monolayers with metal compounds .............................. 129 \n3.2.6 Determination of relative cell proliferation ......................................................... 130 \n3.2.7 Sulforhodamine B \t(SRB) assay......................................................................... 131 \n3.2.7.1 \tPrinciple.................................................................................................... 131 \n3.2.7.2 Assay procedure........................................................................................ 131 \n3.2.8 3-(4,5-d imethy Ith iazoly I- 2)-2 ,5-d iphenyltetrazo hum bromide (MiT) assay. ........ 132 \n3.2.8.1 \tPrinciple.................................................................................................... 132 \n3.2.8.2 Assay \tprocedure ........................................................................................ 132 \n3.2.9 Imniunohistochemistry \t..................................................................................... 133 \n3.2.9.1 \tPrinciple.................................................................................................... 133 \n3.2.9.2 Preparation of cell monolayer for imrnunohistochemistry ............................. 134 \n3.2.9.3 Immunohistochemistry for vimentin and cytokeratin .................................... 134 \n3.2.9.4 Immunohistochemistry for ER and PR........................................................ 135 \n3.2.9. 5 Microscopic assessment ofimmunohistochemical staining .......................... 136 \n3.2.10 \tStatistical analysis \t.......................................................................................... 136 \n3.2.11 \tMaintenance \tof sterility \t.................................................................................. 136 \nvi \n\n4. RESULTS \t .138 \n4.1. \tResults \tof the \tphase \tI............................................................................................. 138 \n4.1.1 \tDemographical characteristics........................................................................... 138 \n4.1.2 \tBiological characteristics \t................................................................................. 140 \n4.1.3 Multivariate analysis of demographic and biological characteristics ..................... 142 \n4.1.4 Characteristics of women with endometriosis ..................................................... 142 \n4.1.5 Association of endometriosis with sources of heavy metal exposure .................... 144 \n4.1.5.1 Proximity to sources of environmental pollution .......................................... 144 \n4.1.5.2 Association of endometriosis with dietary items........................................... 147 \n4.1.5.3 Association of endometriosis with drinking water and cooking utensils ......... 147 \n4.1.6 Symptoms of chronic heavy metal exposure....................................................... 150 \n4.1.7 Assessment of quality control and validation...................................................... 152 \n4.1.8 Heavy metal concentrations \tin blood ................................................................. 154 \n4.1.9 Heavy metal levels in ectopic endometrial tissue ................................................ 156 \n4.2 \tResults \tof Phase \tII  .................................................................................................. 160 \n4.2.1 \tCharacteristics of the participants ...................................................................... 160 \n4.2.2 Characteristics of isolated endometrial stromal cells ........................................... 161 \n4.2.3 Establishment of endometrial stromal cell cultures.............................................. 161 \n4.2.3.1 Characteristics of primary endometrial cell cultures ..................................... 161 \n4.2.3.2 Characteristics of individual endometrial stromal cells ................................. 162 \n4.2.4 Subcultures! passages ofendometrial stromal cells ............................................. 162 \n4.2.5 \tRelative cell proliferation.................................................................................. 166 \n4.2.6 \tSRB \tassay. ....................................................................................................... 168 \n4.2.7 \tMTT \tassay....................................................................................................... 169 \n4.2.8 Immunohistochem ical analysis of endometria I stroma I cells................................ 171 \n4.2.8.1 \tIrnmunohistochemistry for oestrogen receptor.............................................. 171 \nVII \n\n4.2.8.2 Immunohistochemistry for progesterone receptor \t . 171 \n5. DISCUSSION .......................................................................................................... 180 \n5.1 \tPhase \tI................................................................................................................... 180 \n5.1.1 \tDemographic characteristics.............................................................................. 180 \n5. 1.2 \tBiological characteristics.................................................................................. 184 \n5.1.3 \tDietary chamcteristics \t...................................................................................... 189 \n5.1 .4 Association between heavy metal exposure and endometrios is ............................ 192 \n5.1.5 Whole blood heavy metal levels ........................................................................ 193 \n5.1.6 Heavy metal levels in ectopic endometrial tissue ................................................ 196 \n5.2 \tPhase \tII .................................................................................................................. 200 \n5.2.1 Establishment of primary endornetrial stromal cell cultures................................. 200 \n5.2.2 Effect of heavy metals on primary endometrial stromal cell cultures .................... 207 \n5.2.2.1 \tRelative cell proliferation of ESC................................................................ 207 \n5.2.2.2 \tSRB \tand MTfassays ................................................................................. 208 \n5.2.2.3 Expression of ER and PR........................................................................... 212 \n6. CONCLUSIONS AND RECOMMENDATIONS ................................................ 216 \n6.1 \tConclusions \t........................................................................................................... 216 \n6.2 \tRecommendations .................................................................................................. 217 \n7. \t REFERENCES ..................................................................................................... 218 \n8. APPENDICES ......................................................................................................... 265 \nAppendix1 \t.................................................................................................................. 266 \nAppendix2.................................................................................................................. 269 \nAppendix3.................................................................................................................. 282 \nAppendix4 .................................................................................................................. 284 \nAppendix5.................................................................................................................. 287 \nVIII \n\nLIST OF TABLES \nPage number \nTable 2.1: Revised classification of endometriosis of the American \t 30 \nSociety for Reproductive Medicine ....................................................... \nTable 2.2: Characteristics of the main methods used in heavy metal \t 84 \nanalysis in biological samples ................................................................. \nTable 2.3: Features of chronic exposure to heavy metals .......................85 \nTable 2.4: In vitro effects of currently used or novel compounds in \t 101 \nendometriosis.......................................................................................... \nTable 4.1: Demgraphic characteristics of women with or without \t 139 \nendometriosis.......................................................................................... \nTable 4.2: Menstrual and gynaecological characteristics of women \t 141 \nwith or without endometriosis ................................................................ \nTable 4.3: Summary of conditional logistic regression analysis ............142 \nTable 4.4: Stages of endometriosis anng women with endometriosis \t 143 \nTable 4.5: Modes of treatment received by women with endometriosis \t 143 \nTable 4.6: Prevalence of symptoms associated with endometriosis in \t 145 \nwomen with or without endometriosis .................................................... \nTable 4.7: Assoc iation between close proximity (< lOOm) to sources of \t 146 \nenvironmental pollution and endometriosis ............................................ \nTable 4.8: Association between food items and endometriosis \t 148 \nTable 4.9: Sources of drinking water, utensils used for storage of \ndrinking water and cooking in households of women with and without \nendometriosis.......................................................................................... 149  \nTable 4.10: Prevalence of symptoms due to chronic heavy metal \t 151 \nexposure in women with or without endometriosis .............................. \nTable 4.11: Comparison between certified and obtained values for the \t 153 \nreferencestandards ................................................................................. \nTable 4.12: Concentrations of heavy metals in whole blood (ig/L) of \t 154 \nwomen with or without endometriosis .................................................... \nix \n\nTable 4.13: Risk of endometriosis according to the tertile values of \t 155 \ncadmium, lead, iron and zinc .................................................................. \nTable 4.14: Levels of heavy metals inectopic e ndo me trial tissue \t 157 \n(jtg/Kg) and in whole blood (pgIL) of women with endometriosis ........ \nTable 4.15: Metal levels Q.tg/Kg) ofectopic endometrial tissue in \t 158 \ndifferent sites in women with endometriosis .......................................... \nTable 4.16: Correlation between metal levels (pg/Kg) in ectopic \t 159 \nendometrialtissue ................................................................................. \nTable 4.17: Stages of endometriosis in women with endometriosis .......160 \nTable 4.18: Cell number and viability of isolated endometrial stromal \t 161 \ncells in women with and without endometriosis ..................................... \nTable 5.1: Comparison of the demographic and biological \t 188 \ncharacteristics of women with endometriosis ......................................... \nx \n\nLIST OF FIGURES \nPage number \nFigure 1 .1 Gaps in scientific knowledge addressed by the present study 8 \nFigure 2.1 Organizational siructure of the literature review...................... 10 \nFigure 2.2 Female reproductive system.................................................... 11 \nFigure 2.3 Structures of naturally occurring oestrogens............................ 12 \nFigure 2.4 Synthesis of naturally occurring oestrogens............................ 13 \nFigure 2.5 Structure of the oestrogen receptor \t......................................... 14 \nFigure 2.6 Diversity of the ligands that activate the oestrogen 17 \nreceptor........................................................................................................ \nFigure 2.7 Mechanism of oestrogen receptor activation ............................ 18 \nFigure 2.8 Current concepts on the aetiopathology of endometriosis 36 \nFigure 2.9 Complex interplay of endogenous aetiological factors in the 49 \npathogenesis ofendometriosis................................................................... \nFigure 2.10 Sources of heavy metals and their routes of entry into 65 \nhumans....................................................................................................... \nFigure 2.11 In vitro metalloestrogenic effects ofcadmium ..................... 74 \nFigure 3.1 Summary of the methodology for the data and sample \t 109 \ncollection in phase I.................................................................................... \nFigure 3.2 Sample preparation and metal analysis procedure...................112 \nFigure 3.3 Principle of TXRF....................................................................113 \nFigure 3.4 A typical spectrum obtained using the TXRF.........................115 \nFigure 3.5 Principle of GFASS................................................................ 117 \nFigure 3.6 Serial dilution ofcadmium solutions (Ato B).........................128 \nFigure 3.7 Summary of the methodology in phase II.................................137 \nFigure 4.1 Primary endometrial stromal cell cultures under the inverted \t 163 \nphase contrast microscope........................................................ \nxi \n\nFigure 4.2 Endometrial stromal cells in long term cultures under the \t 164 \nphase contrast microscope.............................................. \nFigure 4.3 Endometrial stromal cells following immune histochemical \nstaining using primary antibodies for (A) vimentin (B) cytokeratin and \n(C) universal negative control under the binocular microscope............ 164  \nFigure 4.4 Endometrial stromal cells under the phase contrast \t 165 \nmicroscope................................................................................................. \nFigure 4.5 Endometrial stromal cell culture from a control at 90 days \nafter the initial culture at the 17th  passage (subculture) following \nimmuno histo chemical staining using primary antibodies for (A) vimetin \n(B) cytokeratin and (C) universal negative control; under the binocular \t 165 \nmicroscope............................................................ \nFigure 4.6 Endometrial stromal cell culture from a patient at 60 days \nafter the initial culture at the 101h  passage (subculture) following \nimmunohistochemical staining using primary antibodies for (A) vimetin \ncytokeratin and (C) universal negative control; under the binocular \nmicroscope ............................. ................................. 165 \nFigure 4.7 Relative proliferation of Endometrial Stromal Cells from \nwomen without endometriosis and women with endometriosis after 24 \nhours and 48 hours of treatment with (A) 106  M Cd (b) 10 M Ni and \t 167 \n10 \t M Pb............................................................................................. \nFigure 4.8 Relative proliferation of endometrial Siromal Cells from \nwomen without endometriosis and women with endometriosis after 24 \nhours and 48 hours of treatment with a combination of 106  M Cd, 10 M \nNiand lO 9 MPb ............................................................................... 168  \nFigure 4.9 Results ofSRB assay following 48 hours of incubation with \ndifferent concentrations of Cd (108  M to 10 M) and a combination of \nmetal compounds (106  M Cd, 10 M Ni and 10 M Pb) .........................169 \nFigure 4.10 Results MTT assay following 48 hours of incubation with \ndifferent concentrations of Cd (108  M to 10 M) and combination of \nmetal compounds (10-6  M Cd, 10 M Ni and 10 9  M Pb) .........................170 \nFigure 4.11 Immunohistochemical staining of endometrial stromal cells \nwith primary antibody for oestrogen receptor, following treatment with \n10 6M Cd for 24 hours and 48 hours in (A) women with endometriosis \t 172 \n(B) women without endometriosis ............................................................ \nXII \n\nFigure 4.12 Immunohistochemical staining of endometrial stromal cells \nwith primary antibody for progesterone receptor, following treatment \nwith 10 6M Cd for 24 hours and 48 hours in (A) women with \t 173 \nendometriosis (B) women without endometriosis ...................................... \nFigure 4.13 Immunohistochemical staining of endometrial stromal cells \nwith primary antibody for oestrogen receptor, following treatment with \niO M Ni for 24 hours and 48 hours in (A) women with endometriosis \n(B) women without endometriosis.......................................................... 174 \nFigure 4.14 Immunohistochemical staining of endometrial stromal cells \nwith primary antibody for progesterone receptor, following treatment \nwith 10 9M Ni for 24 hours and 48 hours in (A) women with \t 175 \nendometriosis (B) women without endornetriosis...................................... \nFigure 4,15 Immunohistochemical staining of endometrial stromal cells \nwith primary antibody for oestrogen receptor, following treatment with \n10 9M Pb for 24 hours and 48 hours in (A) women with endometriosis \t 176 \n(B) women without endometriosis............................................................. \nFigure 4.16 Immunohistochemical staining of endometrial stromal cells \nwith primary antibody for progesterone receptor, following treatment \nwith 10 9M Pb for 24 hours and 48 hours in (A) women with \t 177 \nendometriosis (B) women without endometriosis ..................................... \nFigure 4.17 Immunohistochemical staining of endometrial stromal cells \nwith primary antibody for oestrogen receptor, following treatment with a \ncombination of 10 6M Cd, 10 9M Ni and 10 9M Pb for 24 hours and 48 \nhours in (A) women with endometriosis (B) women without \nendometriosis............................................................................................ 178  \nFigure 4.18 Immunohistochemical staining of endometrial stromal cells \nwith primary antibody for progesterone receptor, following treatment \nwith a combination of 10 6M Cd, 10 9M Ni and 10 9M Pb for 24 hours \nand 48 hours in (A) women with endometriosis (B) women without \nendometriosis............................................................................... 179  \nFigure 5.1 Interplay of demographic and biological characteristics in the \t 192 \npresent study \n\nLIST OF ABBREVIATIONS \nAKT - \t Threonine/serine kinases \nAP-1 - \t Activating Protein-i \nAs - \t Arsenic \nAu - \t Gold \nBct-2 - \t B- Cell lymphoma/leukae mia-2 \nBLyS - \t B lymphocyte stimulator \nCa - \t Calcium \nCAM - \t Chicken chorioallantoic membrane \nCd - \t Cadmium \nCdCl2  - \t Cadmium chloride \nCEA - \t Central Environmental Authority \nCo - \t Cobalt \nCr - \t Chromium \nCu - \t Copper \nE1  - \t Oestrone \nE2  - \t 170-oestradiol \nE3  - \t Oestriol \nECM - \t Extracellular mairix \nEGFR - \t Epidermal growth factor receptor \nER - \t Oestrogen receptor \nERa - \t Oestrogen receptor alpha \nERJ - \t Oestrogen receptor beta \nERE - \t Oestrogen response elements \nERK - \t Extra cellular signal- regulated kinases \nxlv \n\nFe - \t Iron \nFSH - \t Follicular stimulating hormone \nGnRH - \t Gonadotropin releasing hormone \nHg - \t Mercury \nIIPO - \t Hypothalamo-p ituitary- ovarian axis \nIHC - \t Immonohistochemisiry \nIL - \t Interleukin \nK - \t Potassium \nmER - \t Membrane oestrogen receptor \nMAPK - \t Mitogen-activated protein kinase \nMMP - \t Matrix metalloproteinase \nNK - \t Natural killer \nNi - \t Nickel \nNF-K - \t Nuclear Facto r-Kf3 \nNSAID - \t Nonsteroidal antiinflammatory drug \nOD - \t Optical density \nLi CAM - \t Li cell adhesion molecule \nLH - \t Luteinizing hormone \nP - \t Phosphorus \nPb -Lead \nPF - \t Peritoneal fluid \nPMC - \t Peritoneal mesothelial cell \nPR - \t Progesterone receptor \nPTWI - \t Provisional tolerable weekly intake \nS - \t Sulphur \nxv \n\nSERM - \t Selective oestrogen receptor modulator \nSn -Tin \nTFF 1 - \t Trefoil factor 1 \nTIMP - \t Tissue inhibitors of matrix metalloproteinase \nTNFa - \t Tumournecrosis factor a \nTSP-i - \t Thrombospondin-1 \nTXRF - \t Total-reflection X-ray Fluorescence \nWHO - \t World Health Organization \nuPA - \t Urokinase plasminogen activator \nUK - \t United Kingdom \nUS - \t United States \nliv - \t Ultraviolet \nVEGF - \t Vascular endothelial growth factor \nZn -Zinc \nxvi \n\nACKNOWLEDGEMENTS \nWork described in this thesis constitutes support by many individuals in at least five \ndifferent institutions where financial assistance was provided by four separate \nfunding sources. \nThe original research question on the association ofheavy metals and endometriosis \nwas first posed by Prof Hemantha Senanayake of University of Colombo. Thus I \nwould like to acknowledge Prof Hemantha Senanayke for supervising me during \nthis research project that was based on his original idea. \nDr. Roshini Peiris-John who was always well tuned to conduct quality research \ninspired me in many ways. She opted to supervise me even after her resignation \nfrom the University of Sri Jayewardenepura despite a busy schedule. \nMeeting Prof Rajitha Wickramasinghe and being supervised by him were indeed \nlife time experiences for me. I would like to thank him for his input not only as a \nstatistician but also as an internationally recognized researcher. \nThe opportunity to work under Prof Kamani Tennekoon as a clinical research \nassistant back in 2005 was a turning point in my professional life. Prof Tennekoon \nwas a pillar of strength for me during this project. I will eternally cherish the \nmemories of Prof Tennekoon correcting my thesis in her usual methodical manner \ndespite physical constraints. \nI am indebted to Prof Sharaine Fernando for stepping in as the internal supervisor at \na very difficult stage of this project. Her assistance helped me in numerous ways to \nconcentrate on the completion of the bench work and writing of the thesis. I deeply \nappreciate the personal interest taken by Prof. Fernando that enabled me to complete \nmy PhD. \nxvii \n\nSenior collegues at the Departnnt of Physiology Prof. Sivithri Wimalasekara, Prof \nPriyadarshika Hettiarchchi, Dr. D.K. Ruberu, Dr. Chandana Hewage and Dr Himasu \nWaidyaseka helped me in many ways towards the successful completion of this \nstudy. I would like to thank them, especillly for allowing me to take study leave for \none and a half years. \nI would like to thank the present Dean of Faculty of Medical Sciences Prof Mohan \nDc Silva for the assisstance. \nProf Jayantha Jayawardana former Dean of Faculty of Medical Sciences is \nacknowledged for the support given during the initial stages of this study. \nIn addition to granting me study leave, the University of Sri Jayewardenepura was \nthe first funding source whose research grant (ASPI6/Re12008/06) that enabled the \ninitiation of this study. Dr. Narada Warsasuriya, Former Vice Chancellor of \nUniversity of Sri Jayewardenepura is acknowledged for approving the above grant. \nI would like to express my deep sense of gratitude to Dr. N.L.A. Karunaratne, Vice \nChancellor of University of Sri Jayewardenepura who never hesitated whenever a \nrequest was made for his assistance. \nUniversity Grants Commission (UGC) is acknowledged for providing the grant that \nwas instrumental in upgrading my MPhil to a PhD. I would like to thank the staff at \nthe International Coorperation division of the UGC for all the support. \nI wish to express sincere thanks to the National Coordinating Committee on \nReproductive Health Research of Sri Lanka (NCC-HRP/ WHO) for providing the \nnecessary fmancial assisstance that enabled the establishment of endometrial \nstromal cell cultures. \nxviii \n\nInternational Training and research on environmental and occupational health \n(ITREOH) programme (NIH grant-Two5497-07) of the University of Alabama, \nBirmingham, USA is acknowledged for the financial assistance and training during \nthis project. \nStaff at the Professorial Gynacology unit of the National Hospital and staff of Ward \n16 of the De Soyza Maternity Hospital Colombo are acknowledged for the \ncooperation during data collection. \nI wish to thank the staff of the Operating Theatre A of the National Hospital \nColombo for the excellent support during sample collection. \nMr. Vajira Waduge and the staff at the Atomic Energy Authority are acknowledged \nfor all the support during prepartion and analysis of samples. \nDr. M.S.M Iqbal and Mrs. D Aluthpatabendiat the Institute ofFundamental Studies, \nKandy are acknoeledged for the assistance in cadmium analysis. \nI wish to thank Staff and colleagues at the Institute of Biochemistry, Molecular \nBiology and Biotechnology of University of Colombo, especially Mr. Sameera \nSamarakoon, Scientific Assisstant (Cell culture) who made my stay at the IBMBB \nindeed a pleasurable one. \nMy parents are fondly remembered for providing me love, care and a good \neducation without which none of this would have been possible. \nLast but not the least; I would like to thank all the women who volunteered to \nparticipate in this study without whose support this thesis would not have been a \nreality. \nxix \n\nAssociation between endometriosis and heavy metals in a group of Sri \nLankan women of reproductive age \nWedikara Arachehige Nalinda Yasanga Silva \nABSTRACT \nHeavy metals such as cadmium, nickel and lead activate the oestrogen receptor (ER) to \nexert oestrogenic effects; thus implicated in the aetiology ofendometriosis an oestrogen \ndependant disease. A purpoted role of heavy metals in the continuation of ectopic \nendometrial tissue could be hypothesized. \nThe objectives of this study were to compare the demographic, biological, exposure \nstatus of heavy metals, blood levels of heavy metals as well as the effects of heavy \nmetals on endometrial stromal cell cultures between women with endometriosis and \nwomen with no evidence of the disease. In addition, presence of heavy metal in ectopic \nendometrial tissue was investigated. \nIn this case-control study women with endometriosis (n=150) were compared with age-\nmatched controls (n150) with no evidence of endometriosis, both confirmed by \nlaparoscopy or laparotomy. A pre tested interviewer-administered questionnaire was \nused for data collection. In a sub population of women (n=50 in each group), venous \nblood samples and ectopic endometrial tissue samples were obtained and digested with \nsupra pure 65% HNO 3. Blood and tissue samples were analyzed for heavy metals using \nTotal-reflection X-ray Fluorescence and graphite furnace atomic absorption \nspectroscopy. From both groups (n=10 in each group) eutopic endometrial samples \nwere obtained to isolate endometrial stromal cells (ESC). Primary ESC cultures were \nestablished in RPMI medium and the cultures in the third passage were treated with Cd, \nPb and Ni at concentrations of 106  M, lO M and 10 9M respectively. At 24 hand 48 h \nxx \n\nthe relative cell proliferation was calculated using the Neubauer haemocytometer, while \nProgesterone receptor (PR) and ER expression were assessed by immunohistochemistry. \nSRB and MTT assays were performed following 48 hours of exposure of ESC cultures \nof both groups to Cd at concentrations of 108  M to 10 M. \nChi square tests and odds ratios (OR) (McNemar's) were used to determine associations \nbetween dichotomous variables. Conditional logistic regression analysis was done to \nadjust for potential confounding variables. Log transformed blood levels of metals were \ncompared using paired t-test while blood and tissue metal levels were correlated using \nthe spearman rank correlation. ANOVA was used to analyze relative cell proliferation. \nSRB and MTT results. \nThe risk of endometriosis was 2.084 times greater among those with an education above \nAdvanced Level as compared to those having an education below Advanced Level \n(p=0.021) after adjusting for confounding lhctors. Endometriosis was commoner aimng \nwomen living close to a main road (OR=1.694; 95 % CI=1.037-2.767) (unadjusted) as \ncompared to the respective reference group. Women with endometriosis had \nsignificantly higher (P=0'01 1) geometric mean (95% Cl) blood nickel levels [2.65 \n(1.94-3.38) jtg/L] as compared to women without endometriosis [0.80 (0.70-0.90) \ntg/L]. Blood levels [women with vs. without] of cadmium [0.84 (0.17-1.98) vs 0.85 \n(0.26-1.45) [tg/L] and lead [11.09 (0.67-13.36) vs 6.90 (1.72-8.09) .igIL] were similar in \nthe two groups p0289 and p=0123 respectively). The geometric mean (95% Cl) \nlevels of cadmium, nickel and lead detected in the ectopic endometrial tissue of women \nwith endometriosis were 2.86 (2.13-3.60), 17.55 (13.15-21.94) and 25.78 (18.49-33.08) \njig/Kg respectively with no significant correlation between blood and tissue levels of \nxxi \n\ncadmium, nickel or lead. Cd treatment increased the relative proliferation in ESC and at \n48 4 cell proliferation was higher in cultures form women with endometriosis (p=0.02) \nthan in women with no evidence of disease. Treatment with Cd reduced expression of \nER and increased expression of PR in the ESC from women with endometriosis when \ncompared to normal women. Relative cell proliferation and alterations in ER and PR \nlevels were most prominent in ESC of women with endometriosis at 48 Ii. In the MTT \nassay, while significant differences were noted at 106  M of Cd there was no dose \ndependent response. \nIn this group of Sri Lankan women in the reproductive age, endometriosis is commoner \namong those who are more educated. Cadmium, nickel and lead were present in ectopic \nendometrial tissue and higher levels of nickel in blood were detected in women with \nendometriosis compared to women without endometriosis. Cadmium was capable of \ninducing oestrogenic effects in cultured endometrial stromal cells that were more \nprominent in women with endometriosis.","source_license":"CC0","license_restricted":false}