BIOCHEMICAL AND CYTOGENETIC EFFECTS OF ORAL CONTRACEPTIVES AMONG WOMEN DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF /^ Master of $I)ilof(opl)p in 2oolosp w By FALAQ NAZ o^ssf^^^-^''^io*--* ^ Under the Supervision of DR. YASIR HASAN SIDDIQUE SECTION OF GENETICS DEPARTMENT OF ZOOLOGY ALIGARH MUSLIM UNIVERSITY ALIGARH 2014 A iSyo sP:^ ^ <30 2 4 ;.CV 2014 DS4398 DROSOPHILA TRANSGENIC Dr. Yasir Hasan Siddique RESEARCH LABORATORY (Assistant Professor) E-mail: [email protected] CERTinCATE Certified that the thesis entitled ^Biochemical and Cytogenetic Effects of Oral Contraceptives among Women'' by Ms. Falaq naz for the partial fulfillment of the degree of Master of Philosophy (Zoology, Genetics) of the Aligarh Muslim University f Aligarh. The subject matter of dissertation as presented by Ms. Falaq Naz is the actual record of work done by her under my supen^sion and guidance, and the same does not form the part of any other course or degree presented to or taken by her. Dr./Yasir./Yasir Hasan SiSiddiquc e (Supervisor) Section of Genetics, Department of Zoology Aligarh Muslim University, Aligarh-202002, U.P., India DEDICATION TO THE LOVEUEST PARENTS IN THE WORLD WHO SUPPORT ME WITH AU. THEIR . HEARTS I? ' ^ TO TO MV DEAREST FRIENDS FOR THEIR h SUPPORT AND DUA IN HARD CIRCUMSTANCES TO MV UNIVERSITY WHICH IS WORKING TO IMPROVE THE RESEARCH TABLE OF COOTEFTS ACKNOWLEDGEMENTS 1-11 UST OF TABLES iii UST OF FIGURES iv ABBREVIATIONS v-vi 1. GENERAL INTRODUCTION 1-22 1.1. Female reproductive cycle 1 1.2. History of oral contraceptives 4 1.2.1. Oral contraceptives 7 1.2.2. Combined oral contraceptives 7 1.2.3. Progestin only oral contraceptives 9 1.2.4. Pharmacological classification 10 1.3. Mechanism of action of oral contraceptives 11 1.4. Scenario in India and abroad 13 1.5. Blood serum markers 14 1.5.1. Serum Enzymes 14 1.5.1.1. Alkaline phosphatase 14 1.5.1.2. Gamma glutamyl transferase 14 1.5.1.3. Amino transferases 15 1.5.1.4. Lactate dehydrogenase 16 1.5.2. Lipid profile 17 1.5.2.1. Total Cholesterol (PC) 17 1.5.2.2. High Density Lipoprotein Cholesterol (HDL-C) 17 1.5.2.3. Low Density Lipoprotein Cholesterol (LDL-C) 18 1.5.2.4. Triglycerides (PC) 19 1.5.3. Genotoxicity tests 20 1.5.3.1. Chromosomal aberration, Sister chromatid exchanges, DNA 20 fragmentation 1.5.3.2. Comet assay or Single cell gel electrophoresis assay 21 2. REVIEW OF UTERATURE 23-36 3. GENOTOXIC DAMAGE IN CULTURED HUMAN 37-43 PERIPHERAL BLOOD LVMPHOCVTES OF ORAL CONTRACEPTIVE USERS 3.1. Introduction 37 3.2. Materials and Methods 37 3.2.1. Chemicals 37 3.2.2. Human lymphocyte culture 37 3.2.3. Chromosomal aberration analysis 38 3.2.4. Sister chromatid exchanges 38 3.2.5. Quantitative assay for DNA fragmentation 39 3.2.6. Statistical analysis 39 3.3. Results and Discussion 39 3.4. Conclusion 43 4. UPID PROFILE Of WOMEN USING ORAL 45-51 CONTRACEPTIVE PILLS 4.1. Introduction 45 4.2. Materials and Methods 46 4.2.1. Sample Size 46 4.2.2. Blood sampling and processing 46 4.2.3. Blood parameters 46 4.2.4. Lipidprofile 46 4.2.5. Statistical analysis 48 4.3. Results and Discussion 48 4.4. Conclusion 50-51 EFFECT OF ORAL CONTRACEPTIVE PILLS ON THE 53-65 BLOOD SERUM ENZVMES AND DNA DAMAGE IN LVMPHOCVTES AMONG USERS 5.1. Introduction 53 5.2. Material and Methods 53 5.2.1. Sample size 53 5.2.2. Blood sampling and processing 54 5.2.3. Blood serum analysis 54 5.2.4. Enzyme profile 54 5.2.5. Single cell gel electrophoresis (SCGE) 56 5.2.5.1. Blood sampling for comet assay 56 5.2.5.2. Slide preparation and Electrophoresis 56 5.2.5.3. Staining 57 5.2.5.4. Slide scoring 57 5.2.6. Statistical analysis 57 5.3. Results and Discussion 58 5.4. Conclusion 64 6. BIBUOQRAPHY 67-93 ACKNOlfLEDaEMENTS Praise be to Allah, the lord of the worlds, who says in his glorious book, "There has come to you from Allah a light and a plain book" and peace and blessing of Allah be upon the noblest of the prophets and messengers, our Prophet Mohammad who has said, "you should insist on acquiring knowledge even if you have to travel up to China. " And "seek knowledge from cradle to grave. " In the name of Almighty Allah, who favored me to achieve this work I thought unattainable. To whom belong might and majesty, I bring all the praises and commendation for providing me with strength during the work of my research which helped me to overcome the troubles and difficulties in the toilsome journey and who well fixed me whenever I declined in vigor or vitality. Praise is to him. I feel deeply obliged to express my profound gratitude to my esteemed supervisor Dr. Yasir Hasan Siddique, Department of Zoology, Aligarh Muslim University, Aligarh for his scholastic guidance, persistence inspiration, and vital encouragement throughout the course of this research work. His predicted advices as a think tanker, imagination towards the target help have been invaluable at every stage of this work. He has indeed been a source of inspiration to me. I will always remain thankful to him. I would like to express my thanks and gratitude to Dr. (Mrs.) Nishat Akhtar, Department of Obstetrics and Gynaecology, Jawahar lal Nehru Medical College, Aligarh Muslim University, Aligarh for her consistent help and guidance and help me in drawing the blood sample from the women in my research tenure. Her guidance rendered to me with a dedication far beyond the call of any duty. At this juncture of my life I wish to thank my beloved parents and my brother Mr. Abdul Sheeraz, and my dearest friends Ms. Mahino Fatimafor their inspiration and the innumerable sacrifices, the unmatched love, sincerity and for encouraging me to accomplish this task and provided me the self-confidence. The biggest and unforgettable thanks belong to Professor (Dr.) Mohammad Afzal who gives sound advice and numerous fruitful discussions regarding fundamental of research and Dr. (Mrs.) Gulshan Arafor their cheerful and helping moment. I sincerely thank to her suggestions from time to time that helped me to explore innovativeness and tackling the experimental difficulties. She always willingly spared much of her precious time to discuss my problems. Words are inadequate to express my indebtedness to her, who made this intricate and difficult work, easy and fruitful to me. / would like to express my special appreciation for my lab colleague Ms. Smita Jyoti, Mr. Rahul, Mr. Fahad AH, Ms. Ambreen Fatima and Ms. Saba Khanam, for their sound advice, good company, long drive and fruitful discussions and of course to the wonderful moments we had together. I will always remain thankful to Mahino Fatima, Shagufi Fatima, Nabila Abbas, Mr. Kamal Hussain, Ms. Huma Haider, Mr. Irfanullah Siddique, Mr. Rahul, for being my financial strength through all this long process no matter at what time or day of the week to making this journey of mine as stress free as possible. Life is incomplete without friends. I am so privileged to have friends like Vignesh Venkataswamy, Sharad Behal, Barkha Shakya, Vibhor Singh, Rafi Alam, Krishna Kumar, Sakshi Saxena, Yusra Zaidi, Areeba Ahmad, Nazia Nazam, Umme Asma, Salman Khalil, Friyanka Gaur, Abu Hussain, Rashid Nayeem, Nazia Kamal, Afzal Khan, Ahsana Shah, Rashmi Medhavifor always inspiring me to step ahead to achieve my goals and taught me to smile in every circumstances. I would like to thanks my seniors Dr. Subuhi Abidi, Dr. Zeba Khan, Ms. Ruqaiyya Hussain, Dr. Jyoti Gupta, Dr. Nuzhat Perveen, Iram Khan, for their constant encouragement with prayers and support. I would like to thank Chairman Professor (Dr.) Iqbal Pervez, Department of Zoology, Aligarh Muslim University, for the facilities provided to me by the department during my study. I was very fortunate to be in contact with Professor (Dr.) Waseem Ahmad Faridi, Professor (Dr.) Irfan Ahmad and (Dr.) Nazura Usmani. My special thanks to University Grant Commission (UGC), New Delhi for the award of project entitled: "Biochemical and Cytogenetic effects of Oral Contraceptives among women of different reproductive histories" fF.No. 39-582/2010(SR)J to Dr. Yasir Hasan Siddique, Department of Zoology, Aligarh Muslim University, Aligarh in which I worked as project fellow and take financial support for my work. FftLAQ NAZ UST OF TABLES Table-2.1. Studies carried out on biochemical and DNA damaging effects of oral 27-36 contraceptives. Table-3.L Chromosomal aberration in cultured human peripheral blood lymphocytes of 42 (OC) users and non-users (control). Table-3.2. Distribution of Sister chromatid exchanges (SCEs) among women using oral 43 contraceptives (OCs). Table-3.3. Estimation of DNA damage among Oral contraceptives (OCs) users and non- 43 users. Table- 4.1. Lipid profile of oral contraceptive (OC) users and non-users women. 50 TabIe-5.1. Age distribution of oral contraceptives non-users and users. 57 TabIe-5.2, Distribution of users on the basis of using oral contraceptives. 57 Table-5.3. Oral contraceptive formulations used by Women. 57 TabIe-5.4. Enzyme profile in the blood serum of oral contraceptive users and non users 58 in different age groups Tabie-5.5. Enzyme profile in blood serum on the basis of duration (months) of using 59 oral contraceptives. Ill UST OF FIGURES Figure-1.1. Figure shows the different parts of the human female genital 3 tract.