Association between Endometriosis and Heavy Metals in a Group of Sri Lankan Women of Reproductive Age

Work described in this thesis constitutes support by many individuals in at least five different institutions where financial assistance was provided by four separate funding sources. The original research question on the association ofheavy metals and endometriosis was first posed by Prof Hemantha Senanayake of University of Colombo. Thus I would like to acknowledge Prof Hemantha Senanayke for supervising me during this research project that was based on his original idea. Dr. Roshini Peiris-John who was always well tuned to conduct quality research inspired me in many ways. She opted to supervise me even after her resignation from the University of Sri Jayewardenepura despite a busy schedule. Meeting Prof Rajitha Wickramasinghe and being supervised by him were indeed life time experiences for me. I would like to thank him for his input not only as a statistician but also as an internationally recognized researcher. The opportunity to work under Prof Kamani Tennekoon as a clinical research assistant back in 2005 was a turning point in my professional life. Prof Tennekoon was a pillar of strength for me during this project. I will eternally cherish the memories of Prof Tennekoon correcting my thesis in her usual methodical manner despite physical constraints. I am indebted to Prof Sharaine Fernando for stepping in as the internal supervisor at a very difficult stage of this project. Her assistance helped me in numerous ways to concentrate on the completion of the bench work and writing of the thesis. I deeply appreciate the personal interest taken by Prof. Fernando that enabled me to complete my PhD. xvii Senior collegues at the Departnnt of Physiology Prof. Sivithri Wimalasekara, Prof Priyadarshika Hettiarchchi, Dr. D.K. Ruberu, Dr. Chandana Hewage and Dr Himasu Waidyaseka helped me in many ways towards the successful completion of this study. I would like to thank them, especillly for allowing me to take study leave for one and a half years. I would like to thank the present Dean of Faculty of Medical Sciences Prof Mohan Dc Silva for the assisstance. Prof Jayantha Jayawardana former Dean of Faculty of Medical Sciences is acknowledged for the support given during the initial stages of this study. In addition to granting me study leave, the University of Sri Jayewardenepura was the first funding source whose research grant (ASPI6/Re12008/06) that enabled the initiation of this study. Dr. Narada Warsasuriya, Former Vice Chancellor of University of Sri Jayewardenepura is acknowledged for approving the above grant. I would like to express my deep sense of gratitude to Dr. N.L.A. Karunaratne, Vice Chancellor of University of Sri Jayewardenepura who never hesitated whenever a request was made for his assistance. University Grants Commission (UGC) is acknowledged for providing the grant that was instrumental in upgrading my MPhil to a PhD. I would like to thank the staff at the International Coorperation division of the UGC for all the support. I wish to express sincere thanks to the National Coordinating Committee on Reproductive Health Research of Sri Lanka (NCC-HRP/ WHO) for providing the necessary fmancial assisstance that enabled the establishment of endometrial stromal cell cultures. xviii International Training and research on environmental and occupational health (ITREOH) programme (NIH grant-Two5497-07) of the University of Alabama, Birmingham, USA is acknowledged for the financial assistance and training during this project. Staff at the Professorial Gynacology unit of the National Hospital and staff of Ward 16 of the De Soyza Maternity Hospital Colombo are acknowledged for the cooperation during data collection. I wish to thank the staff of the Operating Theatre A of the National Hospital Colombo for the excellent support during sample collection. Mr. Vajira Waduge and the staff at the Atomic Energy Authority are acknowledged for all the support during prepartion and analysis of samples. Dr. M.S.M Iqbal and Mrs. D Aluthpatabendiat the Institute ofFundamental Studies, Kandy are acknoeledged for the assistance in cadmium analysis. I wish to thank Staff and colleagues at the Institute of Biochemistry, Molecular Biology and Biotechnology of University of Colombo, especially Mr. Sameera Samarakoon, Scientific Assisstant (Cell culture) who made my stay at the IBMBB indeed a pleasurable one. My parents are fondly remembered for providing me love, care and a good education without which none of this would have been possible. Last but not the least; I would like to thank all the women who volunteered to participate in this study without whose support this thesis would not have been a reality. xix Association between endometriosis and heavy metals in a group of Sri Lankan women of reproductive age Wedikara Arachehige Nalinda Yasanga Silva

Heavy metals such as cadmium, nickel and lead activate the oestrogen receptor (ER) to exert oestrogenic effects; thus implicated in the aetiology ofendometriosis an oestrogen dependant disease. A purpoted role of heavy metals in the continuation of ectopic endometrial tissue could be hypothesized. The objectives of this study were to compare the demographic, biological, exposure status of heavy metals, blood levels of heavy metals as well as the effects of heavy metals on endometrial stromal cell cultures between women with endometriosis and women with no evidence of the disease. In addition, presence of heavy metal in ectopic endometrial tissue was investigated. In this case-control study women with endometriosis (n=150) were compared with age- matched controls (n150) with no evidence of endometriosis, both confirmed by laparoscopy or laparotomy. A pre tested interviewer-administered questionnaire was used for data collection. In a sub population of women (n=50 in each group), venous blood samples and ectopic endometrial tissue samples were obtained and digested with supra pure 65% HNO 3. Blood and tissue samples were analyzed for heavy metals using Total-reflection X-ray Fluorescence and graphite furnace atomic absorption spectroscopy. From both groups (n=10 in each group) eutopic endometrial samples were obtained to isolate endometrial stromal cells (ESC). Primary ESC cultures were established in RPMI medium and the cultures in the third passage were treated with Cd, Pb and Ni at concentrations of 106 M, lO M and 10 9M respectively. At 24 hand 48 h xx the relative cell proliferation was calculated using the Neubauer haemocytometer, while Progesterone receptor (PR) and ER expression were assessed by immunohistochemistry. SRB and MTT assays were performed following 48 hours of exposure of ESC cultures of both groups to Cd at concentrations of 108 M to 10 M. Chi square tests and odds ratios (OR) (McNemar's) were used to determine associations between dichotomous variables. Conditional logistic regression analysis was done to adjust for potential confounding variables. Log transformed blood levels of metals were compared using paired t-test while blood and tissue metal levels were correlated using the spearman rank correlation. ANOVA was used to analyze relative cell proliferation. SRB and MTT results. The risk of endometriosis was 2.084 times greater among those with an education above Advanced Level as compared to those having an education below Advanced Level (p=0.021) after adjusting for confounding lhctors. Endometriosis was commoner aimng women living close to a main road (OR=1.694; 95 % CI=1.037-2.767) (unadjusted) as compared to the respective reference group. Women with endometriosis had significantly higher (P=0'01 1) geometric mean (95% Cl) blood nickel levels [2.65 (1.94-3.38) jtg/L] as compared to women without endometriosis [0.80 (0.70-0.90) tg/L]. Blood levels [women with vs. without] of cadmium [0.84 (0.17-1.98) vs 0.85 (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 the two groups p0289 and p=0123 respectively). The geometric mean (95% Cl) levels of cadmium, nickel and lead detected in the ectopic endometrial tissue of women with endometriosis were 2.86 (2.13-3.60), 17.55 (13.15-21.94) and 25.78 (18.49-33.08) jig/Kg respectively with no significant correlation between blood and tissue levels of xxi cadmium, nickel or lead. Cd treatment increased the relative proliferation in ESC and at 48 4 cell proliferation was higher in cultures form women with endometriosis (p=0.02) than in women with no evidence of disease. Treatment with Cd reduced expression of ER and increased expression of PR in the ESC from women with endometriosis when compared to normal women. Relative cell proliferation and alterations in ER and PR levels were most prominent in ESC of women with endometriosis at 48 Ii. In the MTT assay, while significant differences were noted at 106 M of Cd there was no dose dependent response. In this group of Sri Lankan women in the reproductive age, endometriosis is commoner among those who are more educated. Cadmium, nickel and lead were present in ectopic endometrial tissue and higher levels of nickel in blood were detected in women with endometriosis compared to women without endometriosis. Cadmium was capable of inducing oestrogenic effects in cultured endometrial stromal cells that were more prominent in women with endometriosis.