Master of $I)Ilof(Opl)P in 2Oolosp

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.

Figure-1.2. Changing of hormone level during menstrual cycle in female 3 body.

Figure-1.3. First oral contraceptive pill "Envoid". 7

Figure-1.4. Chemical structure of Estrogens and Progesterone. 9

Figure-1.5. Some common brand of combined oral contraceptives pill 9 (a) Mala-D, (b) Saheli mostly used in India.

Figure-1.6. Classification of progestins used in making progestin containing 10 only pill.

Figure-1.7. Some common brand of Progestin only oral contraceptives 10 (a) Unwanted 72, (b) Levonorgestrel mostly used in India.

Figure-1.8. Figure shows the vulnerable targets for contraception in the 12 female reproductive system.

Figure-1.9. Aminotransfer reaction between an amino acid and an alpha- 15 keto acid. The amino (NH2) group and the keto (=0) group are exchange

Figure-1.10. Reaction catalyzed by Aspartate aminotransferase 16

Figure-1.11. Reaction catalyzed by alanine aminotransferase 16

Figure-1.12. Mechanism of Lactate dehydrogenase 17

Figure-1.13. Cholesterol formation 17

Figure-4.1. Lipid profile of oral contraceptive users and non-users. 50

Figure-5.1. Comet assay performed in human peripheral blood lymphocytes 60 (a) non users, and (b) users.

Figure-5.2. Histogram of the total comet tail length in control and in women 60 taking oral contraceptives. UST OF ABBREVIATIONS AND SYMBOLS

% Percentage DNA Deoxyribonucleic Acid

°c Degree Celsius DSBs Double Standard Breaks fig Microgram DSG Desogestrel Ethylene Diamine Tetra ^g/ml Microgram per miligram EDTA Acetate Hi Microliter EE2 Ethinylestradiol Food and Drug Micromolar FDA HM Administration Follicle Stimulating a Alpha FSH Hormone GnRH Gonadotropin-Releasing Beta P Hormone

Y Gamma g Gravitational Acceleration

ALP Alkaline Phosphatase HDL High Density Lipoprotein International Agency for ALT Alanine Transaminase lARC Research Cancer ANOVA Analysis of Variance JMA Japan Medical Association Aspartate Amino AST Potassium chloride Transferase KCl ATP Adenosine Triphosphate kDa kiloDalton

CAD Caspase Activated DNAase LDH Lactate Dehydrogenase

CAS Chromosomal Aberrations LDL Low Density Lipoprotein cm Centimeter LH Luteinizing Hormone Combined Oral COC Contraceptive LMPA Low Melting Point Agarose

CVD Cardiovascular disease LNG Levonorgestrel

DDW Double Distilled Water M Molar Depo-Medroxy DMPA Progesterone Acetate mA Milliampere

DMSO Dimethylsulphoxide MC Menstrual Cycle mg Milligram ROS Reactive Oxygen Species

mg/dL Milligram per deciliter rpm Rotation per minute

Sister Chromatid MI Myocardial Infarction SCEs Exchanges min Minute Single Cell Gel SCGE Electrophoresis ml Milliliter SD Standard deviation mM Millimolar SEM Standard Error of Means MNT Micronucleus test Serum Glutamic SCOT Oxaloacetic Transaminase NaCl Sodium chloride Serum Glutamic-Pyruvic SGPT Nicotinamide Adenine Transaminase NAD^ Dinucleotide (oxidized) TC Total Cholesterol NADH Nicotinamide Adenine Dinucleotide (reduced) TCA Trichloro Acetic Acid NaOH Sodium hydroxide TG Triglyceride nm Nanometer Tris CI Tris Chloride

NMA Normal Melting Agarose U/L Unit per Litre

OCs Oral Contraceptives V/cm Volt/centimeter Very Low Density PBS Phosphate Buffered Saline VLDL Lipoprotein pH Potential of Hydrogen WHO World Health Organization

POP Progesterone Only Pills PPFA Planned Parenthood Federation of America

VI J" "^

ERAL ! I ^ '. " INTRODljcno N f r * I

» * ' If* > V 1. GENERAL INTRODUCTION

1.1. Female Reproductive Cycle

The female reproductive system or genital system is a system of sex organs within an organism which work together for the purpose of sexual reproduction. The female reproductive cycle is the process of producing an ovum and preparmg the uterus to receive a fertilized ovum to begin pregnancy. If an ovum is produced but not fertilized the reproductive cycle restarts itself through menstruation cycle. The entire reproductive cycle takes about 28 days on an average, but may short as 24 days or long as 36 days for few women. Under the influence of follicle stimulating hormone (FSH) and luteinizing hormone (LH), the ovaries produce a mature ovum in a process known as ovulation. In about 14 days of female reproductive cycle, an oocyte reaches maturity and release as an owim (Freedman and David, 1992).

The female internal reproductive organs are the vagina, uterus, fallopian tubes, cervix and ovary. The vagina is the place where semen from the male penis is deposited into the female's body at the climax of sexual intercourse, a phenomenon commonly known as ejaculation. The vagina is a canal that joins the cervix to the outside of the body. This is also known as the birth canal. The uterus is a pear-shaped muscular organ. Its major function is to accept a fertilized ovum which becomes implanted in endometrium and derives nourishment from blood vessels. The fertilized ovum becomes an embryo, develops into a foetus and gestates until childbirth. The process by which the ovum is released is called ovulation. The external components include the mons pubis, pudendal cleft, labia majora, labia minora, Bartholin's glands, and clitoris (Figure 1.1.).

There are two principle ovarian hormone i.e. estrogens, which are capable of stimulating female reproductive activity, femaleness and progestogens for the pregnancy regulation. If fertilization does not occur the corpus luteum degenerates and there is a sudden decrease in vascular supply and tissue is dead. The dead tissue with some blood is shed in the process known as menstruation or menstrual cycle (Gorbman et al., 1982). Menstrual cycle is the cycle of changes that occurs in the uterus and ovary for the purpose of sexual reproduction. Each cycle can be divided into three phases based on events in the ovary (ovarian cycle) or in the uterus (uterine cycle) (Sherwood and Laurelee, 2013; Silverthom and Unglaub, 2013). The ovarian General Introduction

cycle consist of the follicular phase, ovulation and luteal phase whereas the uterine cycle is divided into menstruation, proliferative phase, and secretory phase. Both cycles are controlled by the endocrine system and the normal hormonal changes that occur can be interfered with using hormonal contraception to prevent reproduction (Klumpe/a/.,2013).

Menstrual cycles are counted from the first day of menstrual bleeding. Follicles in the ovary begin to develop under the influence of a complex interplay of hormones and after several days one or occasionally two become dominant (non- dominant follicles atrophy and die). After ovulation, the egg only lives for 24 hrs or less without fertilization while the remains of the dominant follicle in the ovary become a corpus luteum responsible for producing large amounts of progesterone. Under the influence of progesterone, the endometrium (uterine lining) changes to prepare for potential implantation of an embryo to establish a pregnancy. If implantation does not occur within approximately two weeks, the corpus luteum will involute, causing sharp drops in levels of both progesterone and estrogen. The hormone drop causes the uterus to shed its lining and egg in a process menstruation (Figure 1.2.). A woman's first menstruation is termed menarche and occurs typically around age 12-13. The end of a woman's reproductive phase is called the menopause, which commonly occurs somewhere between the ages of 45 and 55. 1 I rienernl Introdiirtinn

oTiMra*

OWMfl MQIMMN

Posterior view

Figure 1.1. Figure shows the different parts of the human female genital tract (Source: http://healthfavo.com/female-reproductive-system.html).

"hypothalamu* ^QnRH I I I anterior pituitary mldcycle peak of FSH r-r LH LH (triggers ovuiation) Blood levels oi FSH

FOLLICULAR PHASE OF ' LUTEAL PHASE OF MENSTRUAL CYCLE MENSTRUAL CYCLE

Figure 1.2. Changing of hormone level during menstrual cycle in female body (Source:http://dentistrvandmedicine.blogspot.in/2011/07/menstrualcvclegynecology- lecture.html). General Introduction

Menstrual cycle is under the control of a range of hormones produced in various parts of the female body. > Gonadotropin-releasing hormone (GnRH) is produced in the hypothalamus, and stimulates body to release follicle stimulating hormone and luteinizing hormone. > Follicle stimulating hormone (FSH) produced by the pituitary gland, makes the eggs in the ovaries to ripen. > Luteinizing hormone (LH) stimulates the ovaries to release eggs. > Estrogen is produced in the ovaries. It has many roles in female body, including the changes in the body during puberty that takes female from girl to womanhood. > Progesterone is also produced in the ovaries. It works with estrogen to keep the reproductive cycle regular and control pregnancy.

Estrogens have a significant impact on a female's body shape. Its levels are significantly higher in women, especially in reproductive age. Besides other fianctions, estrogens promote the development of female secondary sexual characteristics such as breasts and hips (Hess et al., 1997). The whole process of the menstrual cycle starts in the brain after hypothalamus produces GnRH. This hormone stimulates pituitary gland which releases FSH. FSH then in the bloodstream stimulates the ovaries to start ripening some eggs. FSH also stimulates the ovaries to produce estrogen. This encourages the eggs to mature and starts to thicken the lining of the uterus so that it is prepared for implantation.

1.2. History of oral contraceptives

The Pill (hormonal contraception taken by mouth) fulfilled a dream of activists Margaret Sanger and Katherine Dexter McCormick. Over time, the Pill helped millions of women to avoid unplanned pregnancies. The birth control pill was introduced to the public in the early 1960s. Birth control pills are synthetic hormones that mimic the way real estrogen and progestin works in women's body. During the 1930s, it was discovered that hormones prevents ovulation in rabbits. Frank Colton was the inventor of Enovid, the first oral contraceptive pill. Carl Djerassi was the inventor of modem oral contraceptives or the pill. Ludwig Haberlandt, an Austrian physiologist is known as grandfather of the pill. Indeed, in 1921 he found that rabbits General Introduction and guinea pigs became temporarily sterile after transplantation of ovaries from pregnant animals. These experiments paved the way for pharmacological studies on the effect of progesterone on ovulation. The anti-ovulatory effect of progesterone was demonstrated by A.W. Makepeace and his co-workers in 1937 by injecting progesterone in mated female rabbits. Large-scale experiments with progesterone became possible after Russell E. Marker, a professor of organic chemistry found that progesterone could be manufactured from a substance named diosgenin, extracted from the root of a plant {Dioscorea mexicana) of Mexican jungles (Dhont, 2010).

The impetus for converting findings of animal experiments into human hormonal contraception was given by Margaret Sanger, founder of the Plaimed Parenthood Federation of America (PPFA). She approached Pincus in 1951 and provided a small grant to begin hormonal contraceptive research. In the same period, John Rock, an expert in the treatment of infertility, was experimenting with the oral administration of high doses of estrogen (diethylstilboestrol) and progesterone to induce pseudo-pregnancy in infertile women. He suggested that high dose of sex steroids promotes the growth of the uterus and the fallopian tubes and restored fertility but he found that this treatment suppressed ovulation. The biologist Pincus and the gynaecologist Rock shared their experiences and their intention to develop a hormonal oral contraceptive (Diczfalusy, 1982; Davis, 1978; Speroff, 2009; Gladwell, 2000). With these two compounds, Rock continued his experiments to induce pseudo- pregnancy in infertile patients and could prove that ovulation was effectively suppressed in all women and that no breakthrough bleeding occurred with a daily dose of 10 mg of norethynodrel. This set the stage for the first contraceptive trials in woman using norethynodrel as the only compound of the pill. The first pill Enovid containing 10 mg of norethynodrel and 150 mg of mestranol. Clinical trials with a contraceptive could not be done in the United States because dispensing contraception was still a criminal offence at that time. The initial trials therefore were carried out in Puerto Rico. In 1957, Food and Drug Administration (FDA) approved the use of Enovid 10 mg for the treatment of menstrual disorders and approved it to include as contraception in 1960. Enovid containing either 5 mg or 2.5 mg of norethynodrel, which in trials had proved to be equally effective, were initially not approved by the FDA (Dhont, 2010; Speroff, 2009; Gladwell, 2000; Benagiano etal., 2006). General Introduction

The first oral contraceptive, Enovid was manufactured by G.D. Searle and supplied free to clinical trials in Puerto Rico (Figure 1.3.)- It contained 10 mg of norethynodrel (a synthetic form of the hormone progesterone) and 0.15 gm of synthetic estrogen. Estrogen was a by product of the manufacturing process, but removing it caused more breakthrough bleeding. By 1958 the Pill had been tested on 830 women, mostly in Puerto Rico. Many questions arose due to the ethics of such testing, but Puerto Rican women actively sought this new way to control family size. In 1960, the FDA announces its approval of Enovid as a birth control pill. Some 100 million women around the world use the Pill. In early sixties, clinical trials with another oral contraceptive containing 4 mg norethisterone and 50 mg ethinylestradiol were initiated by the Schering Company (Berlin, Germany) in United Kingdom and Belgium. This formula was brought on the market under the name of Anovlar. One year later the Organon company, in the Netherlands, followed it with another brand, Lyndiol, whose tablets contained 2.5 mg lynestrenol, a 3-desoxo-derivate of norethisterone and 75 mg mestranol. Other researchers focused on the effect of estrogens on ovulation. Although the mechanism was not fully understood at the time, it was clear that estrogens alone also were effective in suppressing ovulation. Ethinylestradiol was synthesised in 1938, by Hans Herloff Inhoffen and Walter Hohlweg at Schering (Maisel, 1965; Snyder et al., 2001). The natural estradiol is poorly absorbed when taken orally and is also quickly inactivated by the liver. Substitution at Cn with an ethinyl group results in ethinylestradiol, which is much more resistant to degradation. The latter estrogen has subsequently replaced mestranol, a 3-methylether which is a pre drug of ethinylestradiol, as the estrogenic component of all other contraceptive pills. Several new progestins have emerged over the last ten years of which some have been incorporated in novel pill formulas. Drospirenone is a spironolactone analogue with anti-mineralocorticoid and moderate anti-androgenic effects. Nomegestrol acetate is a 19 norprogesterone derivative which binds almost exclusively to the progesterone receptor and therefore is referred to as a pure progestational compound (Sitruk-Ware, 2006; Wiegratz and Kuhl, 2006). In 2010, a new study of 46,000 women conducted over 40 years found that women on the pill live longer and are less likely to die prematurely of all causes, including cancerandheart disease (Williams and Stancel, 1996). . - I I (General Infrndnrtinn

"(•.-.V,!!r

Figure. 1.3. First oral contraceptive pill "Envoid" (Source:http://w\v\v.cleveland.com/healthFit/index.ssf/2010/05/the_birth_control_pill tums_ 5.html).

1.2.i. Orai contraceptives

Oral contraceptives (OCs) are the most popular type of birth control pills. The pills stop ovulatiou, preventing the ovaries riorn releasing eggs and they also tliicken cervical mucus by making it harder for sperm to enter the uterus (Feminist Women's ixkâiLii x^viiivî, ^vv/oy. vyiai v\Jiiiiav^vpii v c- 13 a \^vjiiiuiiiaLiv;ii KJI voiiugwii:! ciiiva progesterone. When taken orally and regularly it acts as the most effective method to r»r*=»\/i=>nt nrofTr»or»r*\/ J)r\nV rârr-ia Pir»r»iio frr*Tn T TQ A ftrct TMr*n*^*^ri=»r1 in fV»*^ r»linir»al iic«=» Lî^'vvxjtv L^ t. ^^ Â ±*^± xt^ J . ivv^îv^ v^v«i^^xv«^ X xxAVV«u xiv/ixi v^ i^^x ». xtxi^t ^XV./AXV^'X ^'V« xxi vxx^' vxxx«x^-c«.x VAIJ^^ of the pill as contraceptive in 1957. The birth control pill works primarily by blocking ovulation. Moreover the pil! also wôrks bv making cervical mucus thick and unreceptive to sperm, slowing down the tubal function and by making the lining of the endometrium unreceptive to implantation of a fertilized egg (Kiley and Hamm.ond, 2007). In general, women do not ovulate until at least 10 days after stopping birth viîiiicfi piiij. iinîc an- iiiaiiuy lv\\j i}jjvj Oi Oiai COiiuaCCpiiVCS. LOniumCu Ofai contraceptives aiiu Progestui only coiitiaceptives.

1.2.2. Combined oral contraceptives

Combined oral contraceptives contain both estrogen and progesterone (Figure 1.4.). Combined hormonal contraceptives consist of an estrogen and a progestogen which act primarily by preventing ovulation through the inhibition of the follicle- stimulating hormone and luteinizing hormone. The progestogen component also renders the cervical mucus relatively impenetrable to spemi and reduces the receptivity of endometrium for implantation (lARC, 2007; National Women's Health xvesource Centie Inc, 2009). A vaiiety of imiovations have been developed since General Introduction

combined hormonal contraceptives were first available in the late 1950s, including changes in drug components, doses used and the temporal sequencing of exposure to drugs. These mechanisms render combined hormonal contraceptives very effective in the prevention of pregnancy. Annual failure rates vary between 0.02% (two per 10,000 women/year) when full adherence to instructions for use is assumed (Ketting, 1988) and 5% for typical use (Fu et ah, 1999). Estrogens are a group of compounds named for their importance in both menstrual and estrous reproductive cycles. In combined hormonal contraception, ethinylestradiol is the most common estrogen. A variety of progestogens are available and these differ in their properties with regard to progestogenic and androgenic characteristics. The estrogens and progestogen contained in combined hormonal contraceptives are usually given in a monthly cycle. In general, estrogen and progestogen are taken in combination for 21 days followed by 7 drug-free days (often placebo tablets) during which time withdrawal bleeding usually occurs. Other cyclic schedules may be used to reduce or eliminate menses.

A constant combination of estrogen and progestogen doses may be used (monophasic) or the doses of progestogen and (less often) estrogen may vary in two (biphasic) or three (triphasic) phases. The most common estrogen in combined hormonal contraceptives is ethinylestradiol. Over time, other estrogens have been used, including initially mestranol (a pre-drug of ethinylestradiol) and more recently, estradiol. In the early development of combined hormonal contraceptives, doses of estrogen in the range of 100-150 ^g were commonly used. Contemporary combined hormonal contraceptives may be classified by estrogens dose into 'high-dose' (> 50 |ig), 'moderate-dose' (30-35 jug) and 'low-dose' (15-20 ^g). Three estrogens were identified in the late 1920s and 1930s estrone, estriol and estradiol. Currently, they are often distinguished as 'first-generation' estranes (such as norethynodrel or norethisterone), 'second-generation' gonanes (such as levonorgestrel or norgestimate), 'third generation' gonanes (gestodene and desogestrel) and 'fourth- generation' drospirenone. An additional class of progestogens, the pregnanes (e.g. cyproterone and chlormadinone), may also be used. Estranes are highly androgenic, while pregnanes and drospirenone have anti-androgenic activity. The later gonanes are less androgenic than the earlier compounds in that series (Hammond et al, 2001). Progesterones used in OCs are synthetic progesterones, such as norethindrone. 1 ! neneral Intrndnrtinn norgestrel, norethindrone acetate, ethynodiol diacetate, levonorgestrel, norgestimate. desogesirel and drospirenone.

Ethinyl estradiol Levonorgestrel

Figure 1.4. Chemical structure of Estrogens and Progesterone (Source:http://tmedweb.tulane.edu/pharmwiki/lib/exe/detaii.php/oralcontraceptives2.png?id=c ontraceptive pharmacology).

It is important to recognize that many products are relatively new to the market, particularly those that provide newer progestogens. These, together with products that are currently under development, create a challenge for the evaluation of long-term risk from this class of pharmaceuticals. The vast array of products available allows combined hormonal contraception to be tailored to the specific needs and preferences of individual women. While some of the newer products may offer advantages over the older ones. Some very common products available in India are shown in Figure 1.5. (a & b).

iJS^L (a) (b)

Figure 1.5. Some common brand of combined oral contraceptives pill (a) Mala-D, (Source: http://trade.indiamart.com/details.mp?offer=3291188930) (b) Saheli mostly used in India (Source: http://www.indiamart.com/hindustan-latex/products.html).

1.2.3. Progestin only oral contraceptives

This type of pill contains no estrogen, called the progesteron-only pill, or "mini-pill, it's for breastfeeding women because estrogen red uv;<;3 mux prouuction (National Women's Health Resource Centre Inc, 2009). Figure 1.6. shows classification of progestin by which progestin containing pill is manufactured. Minipills are slightly less effective thaji regular pills and often cause irregular I I (General Intrnrliictinn

menstrual patterns. Minipills prevents pregnancies mainly by making the cervical mucus impermeable to sperm and by making ii more difficult for an egg to attach to the uterus lining. Some common minipill brands available in India are shown in

"IgUXC 1./. (a Ot u).

Classification of Progestins Progestins

Noretliynodiel 19-nor testosterones Spironolactcaie

C-21 progestins ' DrospiieiToiie Estraiies Gonanes Pregnanes • Noretliindroiie • Norgestrel • Medroxyfffogesteroiie acetate • Noretluiidroae acetate • Levonorgesbel • Megestroi acetate • Etliyiiodiol diacetate • Noi^estiinate • Lynestrenol • Desogestrel • CyprotercHie acetate ' Noretliynodrel • Gestodene

Figure. 1.6. Llassitication of progestins used in making progestin containing only pill to u»*„.// ,, J„ „—/..: f:„i„/ccni i /: c\

tSC^SK? Mi>:J Levonorgesfrel Tablets BP \ Levonoreestrel ?^-^~ UNWANTEI^'72 v.. Tiil)lels0.75ni2

Emergency Contraceptive Tablet S

MankmdH^

(a) (b)

Figure 1.7. Some common brand of Progestin only oral contraceptives (a) Unwanted 72, (Source: http://www.perfectpriceindia.com/price/mankind-unwanted-72-pricc-28039.asp). (b) Levonorgestrel (Source: http://ec.princeton.edu/pills/Levonorgestrel.htmn mostly used in India.

i.Z.4. ! liai «natuiugn.ai i-iassiin-anuii

First generation OCs contain high dose (>50 mg) of ethinyl estradiol (EE). Second generation OCs contain levonorgestrel, norgestimate, norethindrone fam.ily and 30-35 mg EE. Third generation OCs contain desogestrel or gestodene and <30 mg EE (Jessica and Lalley, 2000).

10 General Introduction

1.3. Mechanism of action of oral contraceptives

Combined oral contraceptives are the most widely used oral contraceptives that prevent pregnancy mainly by suppressing ovulation. Both progestogen and estrogen components suppress luteinizing hormone secretion, which prevents ovulation. In addition, progestogen thickens cervical mucus so that sperm cannot penetrate the uterus and produces an endometrium that is unreceptive to ovum implantation. It may also contribute to contraception by interfering with secretory and peristaltic functions inside the fallopian tube (Hatcher et al, 1994). Estrogens and progestogens suppress the secretion of follicle-stimulating hormone, which prevents the selection and emergence of a dominant follicle. They also minimize breakthrough bleeding by stabilizing the endometrium so that irregular shedding is prevented. Because the estrogen component potentiates the contraceptive action of the progestogen component (probably by increasing the level of intracellular progestogen receptors), only a minimal dose of estrogen is needed to maintain the efficacy of the combined oral contraceptive (Speroff and Damey, 1996).

As the progestogens themselves do not always suppress gonadotropins, women who take progestin-only oral contraceptives do not always ovulate. The contraceptive efficacy of the progestin-only oral contraceptive is therefore dependent mostly on its effects on cervical mucus, on the endometrium, and possibly also on the fallopian tube (Speroff and Damey, 1996). The progestin-only minipill must be taken every day at the same time because its circulating progestogen level is about 75% lower than the level resulting from a combination oral contraceptive. Use of the minipill may result in irregular menstrual bleeding and the development of functional ovarian follicular cysts. Minipills containing levonorgestrel have been associated with acne. The acne is caused by the androgenic activity of the unopposed progestogen, despite its very low dose, which decreases the level of circulating sex hormone binding globulin. As a result, biologically available levels of levonorgestrel and testosterone are increased. Low-dose combination oral contraceptives do not produce acne because estrogen, which increases sex hormone binding globulin, counteracts the effect of progestogen (Speroff and Damey, 1996). The minipill does not significantly affect lipid levels, carbohydrate metabolism, or blood coagulation. When it is discontinued, fertility retums promptly (Hatcher et al, 1994).

n General Introduction

Development of new contraceptives for the female depends on a thorough knowledge of anatomy and physiology of the reproductive tract. The female have five major roles in reproduction: (1) developing a gamete (ovum); (2) receiving the male gametes (spermatozoa) and transporting them from the vagina to the distal portion of the oviducts; (3) supplying a suitable milieu for fertilization; (4) directing the fertilized ovum across the fallopian tubes to the uterine cavity; and (5) providing a uterine environment conducive for implantation and maintenance of gestation. These functions provide an excellent opportunity for biologic and pharmacologic manipulation of the female reproductive process to achieve fertility control (Rivera et al., 1999) (Fig. 1.8.). In recent years, much research has been done for the identification and possible function of non-steroidal ovarian factors (Adashi, 1992).

Hypothalamus

6n HH Analogs ^E ^P Progestogens

E ppd/or P - Luteolysfs A1t«Tdtian» of (PCs) NSOF -GnRH (OMI. f=S, LI, t5) ^ PG Synthft tas s Antlzofia Antkbcdics Inhibitors

Llgatron Sclerosing agents Plugs Alteretlon of lubBl 1unc|]on hi OCa, P or PGj r-OCs P •CD -Ar>Usp*rm AntlbodJfts ^Cyclic mucus Chgngs8 (NFP)

Figure. 1.8, Figure shows the vulnerable targets for contraception in the female reproductive system [GnRH, gonadotropin-releasing hormone; E, estrogen; FS, folliculostatin (inhibin); ICD, intracervical device; lUD, intrauterine device; LI, luteinization inhibition factor; LS, luteinization stimulation factor; NFP, natural family planning; NSOF, nonsteroidal ovarian factor; OC, oral contraceptive; OMI, oocyte maturation mhibitor; P, progestins; PG, prostaglandin]. (Source: Moghissi, K and Archer, D, Glob libr. women's med., (ISSN 1756- 2228) 2014).

12 General Introduction

1.4. Scenario in India and abroad

By the end of the 20* century, oral contraceptives (OCs) had become a feature of everyday life, with women reaching for their pill on a daily basis around the globe. In United States on June 10, 1957, the Food and Drug Administration (FDA) approved Enovid 10 mg (9.85 mg norethynodrel and 150 \ig mestranol) for menstrual disorders, based on data from its use by more than 600 women. Numerous additional contraceptive trials showed Enovid at 10, 5, and 2.5 mg doses to be highly effective. In 1961, G.D. Searle and Co. marketed Enovid 5 mg as an OC (Dhont, 2010). FDA refused to consider the application until Searle agreed to withdraw the lower dosage forms. On May 9, 1960, the FDA armounced it would approve Enovid 10 mg for contraceptive use and did so on June 23, 1960 (Marks, 2001; Junod and Marks, 2002; Winter, 1970). The first oral contraceptive introduced outside the United States was Schering's Anovlar (norethindrone acetate 4 mg + ethinyl estradiol 50 |ag) on January 1, 1961 in Australia. The first oral contraceptive introduced in Europe was Schering's Anovlar on June 1, 1961 in West Germany (History of Schering, 2008). On December 28, 1967, the Neuwirth Law legalized contraception in France, including the pill (Science Actualities, 2000). The pill is the most popular form of contraception m France, especially among young women. It accounts for 60% of the birth control used in France. In Japan, Japan Medical Association (JMA) prevented the Pill from being approved for nearly 40 years. The Pill was approved in June 1999. According to estimates, only 1.3 percent of 28 million Japanese females use the Pill (Dhont, 2010).

India's national family program has succeeded in lowering the nation's total fertility rate to 2.97 from a high of 5.97 in 1950 (United Nations, 2001). The most prevalent form of contraception is female sterilization, which accounts for 76% of all use among women (Pathak et al, 1998). Research suggests, however, that female sterilization is unlikely to contribute to ftirther fertility decline (Pathak et al, 1998; Bongaarts and Greenhalgh, 1985). Previous studies have suggested that the general concern toward OCs is low due to the social stigma of using contraceptives in an unsupportive setting (Lewis, 1986; Janowitz and Bratt, 1996; Bongaarts and Bruce, 1995; Nag, 1984), lack of knowledge (Basu, 1984; Chaudhury, 2001) desire for more children (Athavale and Athavale, 2003; Kumar et al, 1999; Santhya, 2004), the costs of acquiring additional information (Pritchett, 1994), worry over possible side effects

13 General Introduction

and fears that reversible methods are ineffective (Bongaarts and Bruce, 1995; Athavale and Athavale, 2003; Rajaretnam and Deshpande, 1994; Chaudhury, 2001).

1.5. Blood serum markers

1.5.1. Serum Enzymes

1.5. i. 1. Alkaline phosphatase [EC 3.1.3.1]

Alkaline phosphatase (ALP, ALKP) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation (Kim and Wyckoff, 1991). ALP is found in many organs, including the placenta, ileal mucosa, kidney, bone, leukocytes, and liver (Lazo et ah, 2008; Manolio et al., 1992; Bayer et al., 1980; Gordon, 1993; Dufour, 1998; McGarrity et al., 1987). In addition to hepatobiliary disease, several additional factors can affect ALP serum levels. High-fat food intake can increase AP levels by 30 U/L; rise in BMI can increase ALP levels by 25%; tobacco use can lead to 10% rise in ALP levels; during the third trimester of pregnancy, ALP levels can increase by 2 to 3 fold and oral contraceptives can increase AP levels by 20. Chronic kidney disease has also been reported to raise serum levels of the intestinal AP Isoenzyme (Manolio et al, 1992; Bayer etal., 1980).

1.5.1.2. Gamma glutamyl transferase [EC 2.3.2.2]

Gamma-glutamyl transferase or gamma-glutamyl transpeptidase (also y- glutamyl transferase, GGT) is an enzyme that transfers gamma-glutamyl functional groups. It is found in many tissues, the most notable one being the liver, and has significance in medicine as a diagnostic marker. GGT catalyzes the transfer of the gamma-glutamyl moiety of glutathione to an acceptor that may be an amino acid, a peptide or water (forming glutamate) (Tate and Meister, 1985). GGT plays a key role in the gamma-glutamyl cycle, a pathway for the synthesis and degradation of glutathione and drug and xenobiotic detoxification (Siest et al, 1992).This general reaction is:

(5-L-glutamyl)-peptide + an amino acid peptide ^ 5-L-glutamyl amino acid

14 General Introduction

1.5.1.3. Amino transferases [EC 2.6]

Aminotransferases or transaminase is an enzyme that catalyzes a type of reaction is an enzyme that catalyzes a type of reaction between an amino acid and a- keto acid. An amino acid contains an amine (NH2) group. A keto acid contains a keto (=0) group. In transamination, the NH2 group on one molecule is exchanged with the =0 group on the other molecule. The amino acid becomes a keto acid, and the keto acid becomes an amino acid (Figure 1.9.). Two important transaminase enzymes are AST (SGOT) and ALT (SGPT), the presence of elevated transaminases can be an indicator of liver damage (Vroon and Israili, 1990).

HO HO'

Transam inase.

HO HO NH,

Figure. 1.9. Aminotransfer reaction between an amino acid and an alpha-keto acid. The amino (NH2) group and the keto (=0) group are exchange (Source: http://en.wikipedia.0rg/wiki/File:Transaminierung.svg').

Aspartate transaminase (AST) or aspartate aminotransferase (EC 2.6.1.1) also known as AspAT/ASAT/AAT or serum glutamic oxaloacetic transaminase (SGOT), is a pyridoxal phosphate (PLP)-dependent transaminase enzyme. AST catalyzes the reversible transfer of an a-amino group between aspartate and glutamate and as such, is an important enzyme in amino acid metabolism. AST is found in the liver, heart, skeletal muscle, kidneys, brain, and red blood cells and it is commonly measured clinically as a marker for liver health. Aspartate transaminase catalyzes the interconversion of aspartate and a-ketoglutarate to oxaloacetate and glutamate (Figure 1.10.) (Kirsch et al, 1984; Berg et ai, 2006).

Aspartate (Asp) + a-ketoglutarate ^ oxaloacetate + glutamate (Glu)

15 General Introduction

aspartate Q transaminase + + O' O' NH,

Figure. 1,10. Reaction catalyzed by Aspartate aminotransferase Source: (http://en.wikipedia.Org/wiki/File:Aspartate aminotransferase reaction.png)

Alanine transaminase (EC 2.6.1.2) or ALT is a transaminase enzyme It is also called serum glutamic-pyruvic transaminase (SGPT). ALT is found in plasma and in various, but is most commonly associated w^ith the liver. It catalyzes the two parts of the alanine cycle It catalyzes the transfer of an amino group from L-alanine to a- ketoglutarate, the products of this reversible transamination reaction being pyruvate and L-glutamate (Figure 1.11.) (Aach et al., 1981).

L-glutamate + pyruvate ?^ a-ketoglutarate + L-alanine

Alanine -NH2 amino transferase HOOC NH2 H3C—C—COOH ;: S^ HOOC c—c II \ ~H COO^"^H COOH COOH O

Figure. 1.11. Reaction catalyzed by alanine aminotransferase (Source: (http://en.wikipedia.0rg/wiki/File:Alanine amino transf%C3%A9rase.png).

1.5.1.4. Lactate dehydrogenase [EC 1.1.1.27]

Lactate dehydrogenase (LDH) is an enzyme found in animals, plants, and prokaryotes. Lactate dehydrogenase is of medical significance because it is found extensively in body tissues, such as blood cells and heart muscle. As it is released during tissue damage, it is a marker of common injuries and disease. A dehydrogenase is an enzyme that transfers a hydride from one molecule to another. Lactate dehydrogenase catalyzes the conversion of pyruvate to lactate and back, as it converts NADH to NAD^ and back (Figure 1.12.) (Markert, 1984).

16 General Introduction

H—O H ^

p>ruva

-NH,

-n^ NADH NAIH

Figure. 1.12, Mechanism of Lactate dehydrogenase (Source. (http://en.wikipedia.org/wiki/File:LDH_mechanism.ipg).

1.5.2. Lipid profile

1.5.2.1. Total Cholesterol (TC)

Cholesterol (C27H46O) a sterol and an essential structural component of animal cell membranes that is required to establish proper membrane permeability and fluidity. Cholesterol is thus considered within the class of lipid molecules (Figure 1.13.). In 1769, Francois Poulletier de la Salle was the first who identified cholesterol in solid form in gallstones. Cholesterol also serves as a precursor for the biosynthesis of steroid hormones, bile acids, and vitamin D (TuUy, 1970; Hanukoglu, 1992).

Figure 1.13. Cholesterol formation (Source: http://en.wikipedia.org/wiki/File:CholestgLol.svgy

1.5.2.2: High Density Lipoprotein Cholesterol (HDL-C)

High-density lipoprotein (HDL) is one of the five major groups of lipoproteins, which in order of molecular size, largest to smallest, are chylomicrons, very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low- density lipoprotein (LDL) and HDL. Lipoprotein molecules enable the transportation of lipids (fats), such as cholesterol, phospholipids and triglycerides within the water around cells (extracellular fluid) including the bloodstream. In healthy individuals

17 General Introduction

about 30% of blood cholesterol along with other fats, is carried by HDL (American Heart Association, 2009).

HDL particles remove fats and cholesterol from cells, including within artery wall atheroma and transport it back to the liver for excretion or re-utilization, the reason why the cholesterol carried within HDL particles (HDL-C) is sometimes called "good cholesterol". HDL is the smallest of the lipoprotein particles. Its most abundant apolipoproteins are apo A-I and apo A-IL The liver synthesizes these lipoproteins as complexes of apolipoproteins and phospholipid which resemble cholesterol-free flattened spherical lipoprotein particles; the complexes are capable of picking up cholesterol, carried internally, from cells by interaction with the ATP-binding cassette transporter A1 (ABCAl). A plasma enzyme called lecithin-cholesterol acyltransferase (LCAT) converts the free cholesterol into cholesteryl ester (a more hydrophobic form of cholesterol), which is then sequestered into the core of the lipoprotein particle, eventually causing the newly synthesized HDL to assume a spherical shape. HDL particles increase in size as they circulate through the bloodstream and incorporate more cholesterol and phospholipid molecules from cells and other lipoproteins. The cholesterol delivered to the liver is excreted into the bile and hence intestine either directly or indirectly after conversion into bile acids. Delivery of HDL cholesterol to adrenals, ovaries, and testes is important for the synthesis of steroid hormones (Toth, 2005; Sirtori, 2006).

1.5.2.3: Low Density Lipoprotein Cholesterol (LDL-C)

Low-density lipoprotein (LDL) is one of the five major groups of lipoproteins, which, in order of molecular size, largest to smallest, are chylomicrons, very low- density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), LDL and high- density lipoprotein (HDL). Lipoprotein molecules enable the transportation of lipids (fats) such as cholesterol, phospholipids, and triglycerides, within the water around cells (extracellular fluid) including the bloodstream. Studies have shown that higher levels of type-B LDL particles (as opposed to type-A LDL particles) are associated with health problems including cardiovascular disease. LDL molecules are often informally called bad cholesterol because they can transport their content of many fat molecules into artery walls, attract macrophages, and thus drive atherosclerosis. In contrast, HDL molecules are frequently referred to as good cholesterol or healthy General Introduction cholesterol, because they can remove fat molecules from macrophages in the wall of arteries (Dashti et al, 2011). Each native LDL particle enables emulsification, i.e surrounding/packaging all fatty acids being carried, enabling these fats to move around the body within the water outside cells. Each LDL has a highly hydrophobic core consisting of polyunsaturated fatty acid known as linoleate and hundreds to thousands (Brunzell et al, 2008). LDL particles contain a variable and changmg number of fatty acid molecules, there is a distribution of LDL particle mass and size. LDL particles appear harmless until they are within the blood vessel walls and oxidized by free radicals it is postulated that ingesting antioxidants and minimizing free radical exposure may reduce LDL's contribution to atherosclerosis (Teissedre et al, 1996).

1.5.2.4: Triglycerides (TC)

Triglyceride (TG or triacylglyceride) is an ester derived from glycerol and three fatty acids. As a blood lipid, it enable the bi-directional transference of adipose fat and blood glucose from the liver. Saturated compounds are "saturated" with hydrogen all available places where hydrogen atoms could be bonded to carbon atoms are occupied. Unsaturated compounds have double bonds (C=C) between carbon atoms, reducing the number of places where hydrogen atoms can bind to carbon atoms. Triglycerides are the main constituents of vegetable oil (typically more unsaturated) and animal fats (typically more saturated) (Nelson and Cox, 2000). Triglycerides are a major component of human skin oils (Lampe et al., 1983). Triglycerides are formed by combining glycerol with three molecules of fatty acid. Alcohols have a hydroxy! (H0-) group. Organic acids have a carboxyl (-COOH) group. Alcohols and organic acids join to form esters. The glycerol molecule has three hydroxyl (HO-j groups. Each fatty acid has a carboxyl group (-COOH). In triglycerides, the hydroxyl groups of the glycerol join the carboxyl groups of the fatty acid to form ester bonds:

HOCH2CH(OH)CH20H + RCOjH + R'COjH + RTO^H i RC02CH2CH(02CR')CH2C02R"+ 3H2O

19 General Introduction

1.5.3. Genotoxicity tests

1.5.3.1. Chromosomal aberration, Sister chromatid exchanges, DNA fragmentation

Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. There are many types of chromosome anomalies. They can be organized into two basic groups numerical and structural anomalies (National Human Genome Research Institute, 2006). The numerical anomilies includes aneuploidy (an abnormal number of chromosomes), and occurs when an individual either is missing a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (trisomy, tetrasomy, etc.).

The structural anomalies results due to alteration in the structure of chromosome for e.g: > Deletions: A portion of the chromosome is missing or deleted. > Duplications: A portion of the chromosome is duplicated, resulting in extra genetic material. > Translocations: A portion of one chromosome is transferred to another chromosome. There are two main types of translocations: • Reciprocal translocation: Segments from two different chromosomes have been exchanged. • Robertsonian translocation: An entire chromosome has attached to another at the centromeres, in humans these only occur with chromosomes 13,14,15,21 and 22 > Inversions: A portion of the chromosome has broken off, turned upside down, and reattached, therefore the genetic material is inverted. > Insertions: A portion of one chromosome has been deleted from its normal place and inserted into another chromosome (Paux et al., 2008).

Sister chromatid exchange (SCE) is the exchange of genetic material between two identical sister chromatids. It was first discovered by using the Giemsa staining method on one chromatid belonging to the sister chromatid complex before anaphase in mitosis. Four to five sister chromatid exchange per chromosome pair, per mitosis is in the normal distribution, 14-100 exchanges is not normal and presents a danger to the organism (Langlois et at., 1989).

20 General Introduction

DNA fragmentation is the separation or breaking of DNA strands into pieces. Spontaneous or accidental DNA fragmentation is fragmentation that gradually accumulates in a cell. DNA fragmentation was first observed by Williamson in 1970 when he observed discrete oligomeric firagments occurring during cell death in primary neonatal liver cultures. He described the cytoplasmic DNA isolated from mouse liver cells after culture was characterized by DNA fragments with a molecular weight consisting of multiples of 135 kDa. This finding was consistent with the hypothesis that these DNA fragments were a specific degradation product of nuclear DNA. Apoptotic DNA fragmentation is a natural fragmentation that cells perform in apoptosis (programmed cell death) (Zhang et al., 2010; Simon et al, 2010). DNA fragmentation is a biochemical hallmark of apoptosis. In dying cells, DNA is cleaved by an endonuclease that fragments the chromatin into nucleosomal units, which are multiples of about 180 bp oligomers and appear as a DNA ladder when run on an agarose gel. The enzyme responsible for apoptotic DNA fragmentation is the Caspase-activated DNase. CAD is normally inhibited by another protein, the Inhibitor of Caspase Activated DNase (ICAD). During apoptosis, the apoptotic effector caspase, caspase 3, cleaves ICAD and thus causes CAD to become activated. DNA Fragmentation plays an important part in forensics, especially that of DNA profiling (Speyerg^a/., 2010).

1.5.3.2. Comet assay or Single cell gel electrophoresis assay (SCGE)

Comet Assay or Single Cell Gel Electrophoresis assay (SCGE) is a sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell. It was first described by Singh et al., 1988. It has gained popularity as a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. It involves the encapsulation of cells in a low-melting-point agarose suspension, lysis of the cells in neutral or alkaline (pH>13) conditions, and electrophoresis of the suspended lysed cells. The comet assay is an extremely sensitive DNA damage assay (Nandhakumar et al., 2011). This sensitivity needs to be handled carefully as it is also vulnerable to physical changes which can affect the reproducibility of results. Essentially, anything that can cause DNA damage. This is followed by visual analysis with staining of DNA and scoring comets to determine the

21 >«n».

S 3 it / ^ 2. REVIEW OF i f '' LITERATURE

»* 2. EEVIEWOF UTERATURE

2.1. Review of literature

By the early twentieth century, scientists were eager to isolate and wanted to determine the formulation and structural properties of synthetic hormones and they found that the high doses of these synthetic hormone inhibited ovulation. The synthetic progesterone compounds that are used as oral contraceptives are norethisterone and norethynodrel. These compounds were first tested in woman in 1956 in the United States, Puerto Rico and Haiti. The first pill that was launched as an oral contraceptive for use contains the combination of nor-19 progestm with a small amount of synthetic estrogen as a trade name Enovoid (Junod and Marks, 2002). Now-a-days, millions of women of reproductive age around the world use oral contraceptives (Burkman, 2001). Over the decades, use of oral contraceptive pills is increasing in India (Sharma et al., 2001). In recent years, it is reported that the use of oral contraceptive pills by women is increasing as the government and various organizations are campaigning for its use in order to avoid pregnancies especially in developing countries like Nigeria (Emokpae et al., 2010). According to Bukvic et a! oral contraceptives pills are synthesized chemically which can be carcinogenic (Bukvic et al., 2000). For avoiding unwanted pregnancy the combined oral contraceptives should be taken by users consisting of the steroid hormone estrogens in combinafion with a progestogen (lARC, 1979; lARC, WGECRH and WHO, 1987). They are currently available in monophasic, biphasic and triphasic preparations, depending on the number of different doses of progestogen. Monophasic pills maintain a constant dose of estrogens and progestogen, while multiphasic pills allow a lower total dose of progestogen to be given by reducing the amount of progestogen early in the 20-22 day period of exposure. Progestin-only contraception is an option for women that contain only progestin. The most common side-effects of progestin only contraception is complication in pregnancy or eccysis and irregularity in menstrual cycle (Kaunitz, 1997). Oral contraceptives have been shown abrupt increase in the risk of non-fetal myocardial infarction (Ory, 1977; Mann et al.. 1975). Both single progestins and combined oral contraceptive shows the genotoxic damage and risk of cancers. Study stated that steroids shows genotoxic damage at higher doses (Siddique and Afzal 2004a; Siddique et al., 2005a; Siddique et al., 2006a). The

23 Review of Literature therapeutic doses are safe, but care should be taken with regard to their concentration as they may be genotoxic in the long term use in humans (Siddique and Afzal, 2005b; Siddique et ai, 2007a; Siddique and Afzal, 2008). The International Agency for Research on Cancer (lARC) concluded that Progestins (progestogens) are very much prone to carcinogenic to humans. This risk is probably due to hormonal steroids, since these steroids are very much carcinogenic to humans (Brambilla and Martelli, 2002). This review gives a brief account of the studies carried out on the biochemical effects and DNA damage of oral contraceptives among users.

2.2. Biochemical Effects and DNA damage

Estrogen increases the serum high density lipoprotein cholesterol (HDL-C) levels and decrease the levels of low-density lipoprotein cholesterol (LDL-C), where as progestogens have a reverse effect i.e., they reduce high-density lipoprotein cholesterol (HDL-C) and raise low-density lipoprotein cholesterol (LDL-C) levels. The lipoprotein profile resulting from the use of oral contraceptive is therefore, dependent on the balance between the potencies of the estrogen and progestogen components. Although, the high-dose progestogens contained in the older oral contraceptives did raise low-density lipoprotein cholesterol (LDL-C) and reduce high density lipoprotein cholesterol level (HDL-C), modem low-dose progestogens do not appear to affect the lipid profile in users. The increased risk of cardiovascular disease in oral contraceptive users is due to venous or arterial thrombosis (Speroff and Damey, 1996; Brerman et al., 1997).

Several authors have observed that the use of oral contraceptive pills (OCPs) may increase the risk of cardiovascular disease by increasing the levels of triglycerides (WHO, 1995), but the position over the cholesterol is not clear. Wynn et al. (1966) have reported a significant difference in cholesterol levels in young women using OCPs. Other authors observed a significant increase in all lipid fractions in women using OCPs (Donde and Virkar, 1975). Another study showed that serum total cholesterol levels were higher among oral contraceptive users. There was an increase in total cholesterol due to the increased in P-lipoprotein cholesterol. A decrease in a- lipoprotein cholesterol and an increase in triglycerides were also observed (Webber et al., 1982). OC users experienced an increase in the levels of triglycerides, total cholesterol, VLDL, and HDL that were greater than those experienced by non-

24 Review of Literature hormonal contraceptive users (Berenson et ah, 2009). One of the long-range potential concerns with the use of oral contraceptive agents (OCA) is the increase in serum lipids. A study carried out on Nigerian oral contraceptive users revealing the level of serum cholesterol was significantly higher compared to controls. The elevation of serum cholesterol levels may be due to the estrogen content in oral contraceptives. In conclusion, this study has demonstrated the need to periodically reassess the biochemical parameters of oral contraceptive users, especially those who have been on the steroids for a long time. This is necessary in view of significant biochemical changes such as increase of total protein, albumin and cholesterol levels (Obisesan et ai, 2002). Another study on biochemical changes on oral contraceptive have shown the higher levels of triglycerides, total cholesterol, LDL-cholesterol and VLDL- cholesterol in OCs users women. The increase in OCP triglycerides is due to the increase in the synthesis. OCP intake produce changes in lipid metabolism in women, but such changes may not necessarily lead to pathogenic concentration resulting in a cardiovascular disease with the prolonged use of not more than 4 years (Emokpae et al., 2010). Since, the effects caused by OCP intake are short lived, it could be said that the effects of these hormonal preparations may be physiologic rather than pathogenic (Karam, 2001). Many studies have analyzed the relation between cardiovascular risk factors and oral contraceptive use in adult women. Elevated blood levels of lipids are probably the most important biochemical risk factor for atherosclerosis. In the liver, triglycerides synthesis is enhanced by estrogen and inhibited by androgen and these triglycerides are partly brought into the circulation as low-density lipoproteins. In another study, it was observed that in adolescent girls serum total cholesterol was significantly higher among oral contraceptive users compared to non-users (Nawrot et al., 2003).

The effects of oral contraceptive agents (OCAs) on lipid metabolism were reviewed recently by Beck in 1973 who points out that the estrogen-induced rise in serum triglyceride levels is dose regulated and is similar to the general increase in serum triglycerides found in postmenopausal women. Oral Contraceptives have been reported to affect all serum lipids, but their effect on the triglycerides and VLDL is most consistent and striking (Gereshberg et al., 1968; Molitch et al., 1974). Some data also showed a small, non significant inverse relationship between OC users and HDL cholesterol. A recent report showed that different OC preparations had varying effects

25 Review of Literature

on HDL cholesterol. HDL cholesterol levels appeared to be directly related to estrogen dose and inversely related to progestin dose (Bradley et al., 1978).

Progestins (progestogens) have been classified by the International Agency for Research on Cancer (lARC) as possibly carcinogenic to humans on the basis of sufficient or limited evidence for carcinogenicity in experimental animals and inadequate evidence for carcinogenicity in humans (Bauman et al, 1973; Peters et al, 1986; Schwarz and Weiss, 1986; Chilvers et al, 1987).The higher rate of cellular genetic damage in terms of increased frequencies of SCEs and chromosome aberrations have been reported in women during pregnancy (Imao, 1983; Tanigawa, 1989). The studies on the mutagenic effects of OC drugs are limited. Studies on cultured lymphocytes derived from contraceptive users have demonstrated both positive and negative outcomes in terms of genotoxic effects. However, these contradictory results date back to the 1970's (McQuarrie et al, 1970; Bishun et al, 1972; Shapiro et al, 1972) and more recent studies show that estrogens induce the formation of endogenous DNA adducts both in humans and animals and suggest that these could ultimately lead to cancer development (Hundal et al, 1997; Dhillon and Dhillon, 1994; Liehr, 1990). Studies carried out on biochemical and DNA damaging effects of oral contraceptives is shown in Table 2.1.

2.3. Conclusion

The studies performed on oral contraceptive users clearly shows the marked effects on enzymes, carbohydrates and serum lipids. High level of serum lipid invites many problems among contraceptive users i.e. cardiovascular risk by increasing in the cholesterol level, but the full impact of oral contraceptive on cardiovascular risk factors may not be fully concluded from short duration studies done so far. The duration of using oral contraceptives also have importance in studying the effects among women.

26 2. REVlElf OF UTERATURE

2.1. Review of literature

By the early twentieth century, scientists were eager to isolate and wanted to determine the formulation and structural properties of synthetic hormones and they found that the high doses of these synthetic hormone inhibited ovulation. The synthetic progesterone compounds that are used as oral contraceptives are norethisterone and norethynodrel. These compounds were first tested in woman in 1956 in the United States, Puerto Rico and Haiti. The first pill that was launched as an oral contraceptive for use contains the combination of nor-19 progestin with a small amount of synthetic estrogen as a trade name Enovoid (Junod and Marks. 2002). Now-a-days, millions of women of reproductive age around the world use oral contraceptives (Burkman, 2001). Over the decades, use of oral contraceptive pills is increasing in India (Sharma et al., 2001). In recent years, it is reported that the use of oral contraceptive pills by women is increasing as the government and various organizations are campaigning for its use in order to avoid pregnancies especially in developing countries like Nigeria (Emokpae et al., 2010). According to Bukvic et al oral contraceptives pills are synthesized chemically which can be carcinogenic (Bukvic et al., 2000). For avoiding unwanted pregnancy the combined oral contraceptives should be taken by users consisting of the steroid hormone estrogens in combination with a progestogen (lARC, 1979; lARC, WGECRH and WHO, 1987). They are currently available in monophasic, biphasic and triphasic preparations, depending on the number of different doses of progestogen. Monophasic pills maintain a constant dose of estrogens and progestogen, while multiphasic pills allow a lower total dose of progestogen to be given by reducing the amount of progestogen early in the 20-22 day period of exposure. Progestin-only contraception is an option for women that contain only progestin. The most common side-effects of progestin only contraception is complication in pregnancy or eccysis and irregularity in menstrual cycle (Kaunitz, 1997). Oral contraceptives have been shown abrupt increase in the risk of non-fetal myocardial infarction (Ory, 1977; Mann et al., 1975). Both single progestins and combined oral contraceptive shows the genotoxic damage and risk of cancers. Study stated that steroids shows genotoxic damage at higher doses (Siddique and Afzal 2004a; Siddique et al, 2005a; Siddique et al, 2006a). The

23 Review of Literature therapeutic doses are safe, but care should be taken with regard to their concentration as they may be genotoxic in the long term use in humans (Siddique and Afzal, 2005b; Siddique et al., 2007a; Siddique and Afzal, 2008). The International Agency for Research on Cancer (lARC) concluded that Progestins (progestogens) are very much prone to carcinogenic to humans. This risk is probably due to hormonal steroids, since these steroids are very much carcinogenic to humans (Brambilla and Martelli, 2002). This review gives a brief account of the studies carried out on the biochemical effects and DNA damage of oral contraceptives among users.

2.2. Biochemical Effects and DNA damage

24 Review of Literature hormonal contraceptive users (Berenson et al, 2009). One of the long-range potential concerns with the use of oral contraceptive agents (OCA) is the increase in serum lipids. A study carried out on Nigerian oral contraceptive users revealing the level of serum cholesterol was significantly higher compared to controls. The elevation of serum cholesterol levels may be due to the estrogen content in oral contraceptives. In conclusion, this study has demonstrated the need to periodically reassess the biochemical parameters of oral contraceptive users, especially those who have been on the steroids for a long time. This is necessary in view of significant biochemical changes such as increase of total protein, albumin and cholesterol levels (Obisesan et al, 2002). Another study on biochemical changes on oral contraceptive have shown the higher levels of triglycerides, total cholesterol, LDL-cholesterol and VLDL- cholesterol in OCs users women. The increase in OCP triglycerides is due to the increase in the synthesis. OCP intake produce changes in lipid metabolism in women, but such changes may not necessarily lead to pathogenic concentration resulting in a cardiovascular disease with the prolonged use of not more than 4 years (Emokpae et al., 2010). Since, the effects caused by OCP intake are short lived, it could be said that the effects of these hormonal preparations may be physiologic rather than pathogenic (Karam, 2001). Many studies have analyzed the relation between cardiovascular risk factors and oral contraceptive use in adult women. Elevated blood levels of lipids are probably the most important biochemical risk factor for atherosclerosis. In the liver, triglycerides synthesis is enhanced by estrogen and inhibited by androgen and these triglycerides are partly brought into the circulation as low-density lipoproteins. In another study, it was observed that in adolescent girls serum total cholesterol was significantly higher among oral contraceptive users compared to non-users (Nawrot et ah, 2003).

The effects of oral contraceptive agents (OCAs) on lipid metabolism were reviewed recently by Beck in 1973 who points out that the estrogen-induced rise in serum triglyceride levels is dose regulated and is similar to the general increase in serum triglycerides found in postmenopausal women. Oral Contraceptives have been reported to affect all serum lipids, but their effect on the triglycerides and VLDL is most consistent and striking (Gereshberg et al, 1968; Molitch et al, 1974). Some data also showed a small, non significant inverse relationship between OC users and HDL cholesterol. A recent report showed that different OC preparations had varying effects

25 Review of Literature on HDL cholesterol. HDL cholesterol levels appeared to be directly related to estrogen dose and inversely related to progestin dose (Bradley et al., 1978).

Progestins (progestogens) have been classified by the International Agency for Research on Cancer (lARC) as possibly carcinogenic to humans on the basis of sufficient or limited evidence for carcinogenicity in experimental animals and inadequate evidence for carcinogenicity in humans (Bauman et al., 1973; Peters et al., 1986; Schwarz and Weiss, 1986; Chilvers et al., 1987).The higher rate of cellular genetic damage in terms of increased frequencies of SCEs and chromosome aberrations have been reported in women during pregnancy (Imao, 1983; Tanigawa, 1989). The studies on the mutagenic effects of OC drugs are limited. Studies on cultured lymphocytes derived from contraceptive users have demonstrated both positive and negative outcomes in terms of genotoxic effects. However, these contradictory results date back to the 1970's (McQuarrie et al., 1970; Bishun et al, 1972; Shapiro et al., 1972) and more recent studies show that estrogens induce the formation of endogenous DNA adducts both in humans and animals and suggest that these could ultimately lead to cancer development (Hundal et ah, 1997; Dhillon and Dhillon, 1994; Liehr, 1990). Studies carried out on biochemical and DNA damaging effects of oral contraceptives is shown in Table 2.1.

2.3. Conclusion

26 2 I Review of Litemtiire

Table-2.1 Studies carried out on biochemical and DNA damaging effects of oral contraceptives.

S.No. Oral Contraceptive Parameter Studied Conclusion Author 1. Estrogen containing oral Blood pressure and Higher level of Webber et al.. contraceptive serum lipids serum lipid and (1982) lower blood pressure

2. Oral pill (Triphasic OC), Lipid soluble Low lipid serum Pa Ian etai. Vaginal ring (Nuva antioxidant and Serum level, decrease (2010) ring) levels of enzyme coenzyme BK, Transdermal patch and total anti (Ortho Evra) oxidant capacity assay

3. Injectable (DMPA- Serum lipids Elevate lipid Berenson et depot medroxy level in oral pill al., (2009) }jr>jgv,Sn..ruiiv users and low acetate),Oral pill- HDL and high ethinyl estradiol and LDL level in

4, Oral contraceptives Serum protein and Higher serum Obisesan et cholesterol protein, al., (2002) increased cholesterol levels

5. Low dose Effect on deep venous Deep venous Tzankova et contraceptives thrombosis and thrombosis in a/., (2010) (erhinyiesrradiol) arterial by iipid users (aneriai combined with abnormalities thrombosis progestin component increases) dienogest or drospirenon

6. Biphasic lofeminal Serum lipids and Total lipid Emokpae et tablets (norgestrel + blood pressure increases a/., (2010) ethinyl estradioi) reduced blood pressure level

7. Ethinyl estradiol and Blood pressure and Higher systolic Nawrot et al., progestin serum total lipids blood pressure (2003) and higiicf level of serum total lipids

8. Low dose oral Lipid level and blood Normal level of Giribela et al., contraceptive (ethinyl pressure lipids, normal (2007) estradiol/gestodene) Utr 2 Review of Literature

9. Monophasic pills (ethinyl Blood sample (Total Basal free Kirschbaum et estradiol) plasma Cortisol was Cortisol level a/., (1999) measured with a increases, No radioimmunoassay), effect on and first saliva sample Cortisol level (Basal free Cortisol level)

10. Combined oral Serum lipids and Serum lipids is Abdel Barry et contraceptive (estrogen lipoproteins higher a/., (2011) ethinyl estradiol + progestogen)

11. Lyndiol pill (lynoestrenol SGOT levels (serum Increase in Stoll,(1967) and progestin) glutamic oxaloacetic SGOT (serum transaminase) glutamic oxaloacetic transaminase)

12. Oral contraceptive agents Concentration of Decrease plasma McLeroy and combination type plasma ascorbic acid ascorbic acid Schendel, (Progestogen + Estrogen) in leukocytes (1973)

13. Norinyl (1 mgof Vitamin level and Decrease in Smith et al., norethindrone + 80 ng of lipid levels in serum serum vitamin B (1975) mestranol) 12, Lipid level in serum higher

14. Oral contraceptive Serum lipids Increase level of Kannel,(1979) serum lipids

15. Oral contraceptive pill Serum lipids Higher levels of Hennekens et (norethindrone + mestranol) total lipids a/., (1979)

16. Contraceptive pill Serum lipids Decrease serum Demacker et (levenorgestrel + ethinyl lipids a/., (1982) estradiol)

17. Contraceptive pill Plasma vitamins (A, Increase level of Yeung, (1976) E, C) plasma vitamin, plasma triglycerides and triglycerides, cholesterol, cholesterol, leukocytes leukocytes that makes the risk of myocardial infraction

18. Norethindrone, Lipoproteins and Higher Walsh and OrthoNovum (1/35 and777) Serum lipids lipoprotein, high Sacks, (1993) ethinodiol diacetate Serum lipids

28 2 Review of Literature

19. Combined oral contraceptive Histopathology Risk of Weiderpass ei estrogen + progestin endometrial a/., (1999) (desogestrel, ievonorgestrel and cancer is norethisterone) decreased

20. Combined pills (ethinyl Bone markers, serum Decreased bone Garnero el al., estradiol) biochemistry. Bone formation; (1995) mineral measurement serum albumin is lower in oral contraceptive users. Normal bone mineral

21. Oral contraceptive pill Serum levels of lipid- Lower level of Palan ei al., soluble antioxidants serum levels of (2006) lipid soluble antioxidants

22. Ievonorgestrel (90 ^g) Demographic data Use of this Bamhart et a/., Ethinyl estradiol (20 \ig) composition (2009) does not delay the return to fertility

23. Monophasic oral contraceptive Follicle diameters and Suppression of Coney and (Ievonorgestrel (100 ]ig) + serum progesterone ovarian activity DelConte, ethinylestradiol 20 \ig) and 17p-estradiol in users (1999)

24. Ortho Nuvum 7/7/7, Triphasil Serum concentration Serum Sinofsky and (combination of norethindrone, of ethinyl estradiol by concentration of Pasquale, ethinyl estradiol) Radioimmunoassay ethinyl estradiol (1998) increases

25. Cyproterone acetate + ethinyl Lipid metabolism Lipid Mastorakos et estradiol, Desogestrel + ethinyl phosphatase, hirsutism metabolism al., (2002) estradiol of adolescents with increased, polycystic ovary decrease syndrome (PCOS) hirsutism

26. Mifepristone (contraceptive Biochemical Increase Agarwal etal., pill) parameter (serum biochemical (2009) alkaline phosphatase parameters and serum glutamyl (serum alkaline pyruvate phosphatase and transaminase) serum glutamyl pyruvate transaminase)

27. Ethinyl estradiol and Assessment of ovarian Inhibition of Spona et al..

29 2 Review of Literature

chlormadinone acetate function, thickness of ovulation and (2010) endometrium, cervical unfavorable reaction sex, hormone condition for level and overall fertilization. tolerability pregnancy

28. Depot medroxyprogesterone Menstrual symptoms No adverse Berenson et Oral contraceptive pill (20 |j,g) and Physiologic effects al., (2008) symptoms

29. Oral contraceptive (ethinyl Body Mass Index The White et al. estradiol + norgestimate), (BMI) and serum contraceptive (2005) Contraceptive patch (ethinyl androgen levels patch had an estradiol + norelgestromin) effect comparable, to the OC on several key androgenic potential as a therapeutic agent for disorders of androgen excess

30. Ethinyl estradiol + progestin Pelvic pain, bleeding. Pelvic pain. Sulak et al.. headaches, breast bleeding and (2000) tenderness breast tenderness shown, headaches problem

31. Oral contraceptive (estrogen), Demographic data Oral Schaffir e/a/.. DMPA (progestin) form, serum contraceptives (2010) testosterone and have low level estradiol level of testosterone compared to DMPA and high level of estradiol

32. Oral contraceptive (30 \ig Survey based Study Slight delay in Wiegratz and ethinyl estradiol + 2 mg regaining Kuhl, (2006) dienogest) fertility after the use of this oral contraceptive

33. Mircette (desogestrel / ethinyl Serum lipids. Increase serum Berga,(1998) estradiol) carbohydrate lipids, no metabolism, endocrine changes in parameters carbohydrate metabolism, effect on

30 2 Review of Literature

endocrine parameter

34. Oral contraceptive pill Standard Liver carcinoma Palmer et al.. questionnaire was found in oral (1989) used to record contraceptive descriptive users characteristics and medical and reproductive history

35. Oral contraceptive pill Total serum Total serum Yesmin ei ai., cholesterol, high- cholesterol and (2013) density lipoprotein LDL-cholesterol cholesterol. level increased Triglycerides and and HDL level LDL-cholesterol decreased

36. Monophasic combined oral Total serum Total serum Mohammad ei contraceptive contain 0.3 mg cholesterol, cholesterol, a/., (2013) norgestrel+0.03 ethinyl triglycerides, LDL triglycerides, estradiol cholesterol and Very LDL cholesterol low density increased lipoproteins (VLDL)

37. Levonorgestrel with ethinyl Total cholesterol, Changes found Young and estradiol high-density in the lipid DelConte, lipoprotein profile (1999) cholesterol, high- density lipoprotein subfraction 2, and apolipoprotein A-I concentrations

38. Levonorgestrel in low (150 ^g, Serum total Progestins may Godsland et high (250 |ig) and triphasic cholesterol and have unwanted a/., (1990) (50-125 ng) doses, triglycerides metabolic side Norethindrone in low (500 ng), effects when high (1000 |ig) and triphasic combined with (500-1000 ^g) and desogestrel estrogen (150 ng) with 30-40 \ig ethinyl estradiol

39. Oral contraceptive pills Total cholesterol, Different Thane et al., LDL-cholesterol and types of OC (2002) triacylglycerol may have concentrations different impacts on blood analytes due to variable estrogen and progestogen

31 Review of Literature

content

40. Estrogen-dominant oral Triglycerides Total Khoo et al., contraceptives (Ortho Novum triglycerides (1999) 1-35, Ortho were Novum 777, Desogen, significantly orModicon) containing 0.035 higher in the mg ethinyl estradiol. users compared to the nonusers

41. Ethinyl estradiol and LDL cholesterol, No negative Machado et drospirenone HDL cholesterol effects were a/., (2010) found

42. Ovral (75 ^g norgestrel and 7.5 Total serum Oral Parks et al., |xg ethinyl estradiol); Demulen cholesterol, contraceptive (1989) (150 \ig ethynodiol diacetate triglycerides, HDL treatment was and 7.5 ng ethinyl estradiol) cholesterol and low associated with density lipoproteins an increase in (LDL) plasma triglycerides (TG) and a decrease in low density lipoproteins (LDL) size

43. Oral Contraceptives (Estrogens High-density- Level varied Bradley et al.. and Progestins) lipoprotein cholesterol with the type (1978) and dose of the component steroids, in general increasing with increasing dose of estrogen and decreasing with increasing dose or potency of progestin

44. Estrogen/Progestin Low-density and high- Oral Wahl et al., density lipoproteins contraceptives (1983) that are high in estrogen and low in progestin (Enovid or Oracon) had significantly higher concentrations of high density Review of Literature

lipoprotein cholesterol as compared to non-users

45. Oral contraceptives Total cholesterol, Increased high- Vaziri et aL, triglycerides, high- density (1993) density lipoprotein lipoprotein cholesterol and cholesterol and apolipoprotein decreased low- density lipoprotein cholesterol were also observed

46. A transdermal patch delivering Total cholesterol, No Effect Brynhildsen 0.025 mg estradiol and 0.125 VLDL cholesterol and and Hammar, mg norethisterone acetate LDL cholesterol (2002)

47 Norethisterone 1 mg/ethinyl HDL and LDL HDL and LDL Loke et aL, estradiol 35 ^g (NET/EE) or cholesterol and cholesterol is (1992) levonorgestrel 150 jAg/ethinyl alkaline phosphatase. reduced and estradiol 30 ng (LNG/EE) bilirubin and aspartate alkaline transaminase (SGOT) phosphatase, bilirubin and aspartate transaminase (SGOT) are suppressed

48. Oral contraceptives Serum Triglyceride No Effects Wynn et al.. levels and (1966) cholesterol levels

49. Oral Contraceptive Alkaline phosphatase. Slightly raised Thulin and SGOT, SGPT alkaline Nermark, phosphatase (1966) value and high transaminase levels

50. Oral contraceptives High-density Increase in High- Campesi et aL, lipoprotein, Low- density (2012) density lipoprotein, low- lipoprotein, density Triglycerides, lipoprotein. Alkaline Triglycerides, phosphatase. Aspartate Aspartate aminotransferase. aminotransferase. Alanine Alanine aminotransferase. aminotransferase. Y Glutamyl

33 2 Review of Literature

y Glutamyl transpeptidase transpeptidase and decreased in alkaline Phosphatase decreased

51. Oral contraceptives Alkaline phosphatase, Multiple Shaaban et al.. SCOT, SGPT and changes in (1982) serum albumin and serum levels do globulin not necessary indicate liver damage

52 Oral contraceptives Alkaline phosphatase, y Glutamyl Schiele et al.. and serum transpeptidase (1998) triglycerides albumin increased and and globulin y Alkaline Glutamyl phosphatase, transpeptidase serum triglycerides, albumin globulin decreased

53. 150 ^ig desogesterel and 30 ng Chromosomal Frequency of Mahrous, ethinyl estradiol, 250 jug aberration, sister cells with (2008) Norgestamine and 35 |ig chromatid exchanges aberrations ethinyl estradiol, 75ng found, gestodene and 30 ^ig ethinyl chromatid gaps estradiol. and breaks were predominantly observed

54. 150 ng desogestrel and 20 or Sister chromatid OC users Biri et al.. 30 ng ethinylestradiol exchanges, Comet showed (2002) assay significantly higher damage by both cytogenetic methodologies compared with untreated control women

55. 20, 40 and 75 |ag/ml for Sister chromatid The use of Ahmad et al.. norethindrone and 10, 25 and exchanges, norgestrel is (2001) 50 fig/ml for norgestrel) Chromosomal associated with aberrations a significant increase in the incidence of chromosomal abnormalities

34 2 Review of Literature

per cell as well as with an increase in the number of abnormal cells

56. Lyndiol 2.5 Chromosome Lyndiol 2.5 may Timson. abnormalities have a slight (1969) anti-mitotic effect but do not provide any evidence of steroids causing structural chromosomal abnormalities

57. Estrogen and Sister-chromatid SCE frequencies Kayikciogiu et medroxyprogesterone acetate exchange were ai,(2000) and IVP-estradiol only significantly elevated during the third month

58. Norethynodrel Chromosomal An ascorbic acid Siddique et al.. aberrations and sister potent enough to (2007a) chromatid exchanges reduced the genotoxic damage of norethynodrel in the presence of metabolic activation and may reduce the possible risk of carcinogenesis

59. Norgestrel Chromosomal Treatment of Siddique et al.. aberration and sister peripheral blood (2007b) chromatid exchanges lymphocytes with vitamins C or E, either separately or in combination, decreases the genotoxic damage caused by norgestrel

60. Oral contraceptive Chromosomal Gaps and breaks Naz et ai. aberration analysis, of chromosome (2012a)

35 2 Review of Literature

Sister chromatid as well as exchanges, DNA chromatid were fragmentation observed, but dicentric, exchanges and rearrangements were not observed

61. 17 a Ethynylestradiol and Sister chromatid Hormones do Bukvic et al.. Norgestrel exchange (SCE), not induce (2000) micronucleus structural test (MN), and chromosome chromosome aberrations in aberrations (CA), lymphocytes FISH procedure and indicate that and in vitro anaphase ethynilestradiol and metaphase and norgestrel preparation have an analyses aneugenic effect on fibroblast and lymphocyte cultures

62. 17 P-estradiol Comet assay Comet assay to Baj payee et assess al., (2005) genotoxicity in female human populations

63. Oral contraceptives Comet assay Lymphocytes Braz and from women at Salvadori, leuteal phase (2007) with high level of progestone and estradiol showed DNA damage

Oral contraceptives Sister chromatid Increase risk of Landi and exchanges (SCEs), chromosomal Barale, (1999) micronucleus test imbalances (MN), and chromosome aberrations (CAs)

36 ^ 1

% I 3. 6EII0T0X1C DAMAGE IN . CULTURED HUMAN PERIPHERAL BLOOD - • «»^i. . LYMPHOCVtES OF ORAL ^ ^ CONTRACEPTIVE USERS ^

/ 3, QENOTOXIC DAMAGE IN CULTURED HUMAN PERIPHERAL BLOOD LYMPHOCVtES OF ORAL CONTRACEPTIVE USERS

3.1. Introduction

Oral contraceptives are the combination of estrogen and progesterone. They are used to treat various hormonal disturbances, premenstrual syndrome and ovarian cysts (Speroff e? a/., 1999). The composition of pills varies from country to country due to the responsiveness variation from individual to individual or one population lo another (Goldzieher, 1990). Earlier reports suggest the genotoxic effects of synthetic progestins in cultured human peripheral blood lymphocytes (Kochhar, 1988; Shyama and Rahiman, 1996; Siddique and Afzal, 2005c; Siddique et al., 2006b; Joosten et al; 2004). In these studies the genotoxic effects were observed at a very high dose and the reactive oxygen species was (ROS) suggested as a causative agent for the genotoxic damage (Siddique and Afzal, 2004b; Siddique et al., 2005a). The conversion of estrogen into catechol estrogens and quinines via redox reactions cause oxidative damage to DNA (Siddique and Afzal, 2005; Siddique et al, 2005b). There are both positive as well as negative reports regarding the genotoxic effects of estrogens and synthetic progestins (Littlefield and Mailhes, 1975; Hundal et al, 1997; Dhillon et al, 1994). In the present study an attempt has been made to investigate the possible genotoxic effects among oral contraceptives (OCs) users (females) by using chromosomal aberrations, sister chromatid exchanges and DNA damage as a parameters.

3.2. Materials and Methods

3.2.1. Chemicals

RPMI 1640, fetal calf serum, phytohaemagglutinin-M, antibiotic-antimycotic mixture (Invitrogen); 5-bromo-2-deoxyuridine, Giemsa stain (Merck, India), Tris.HCl, EDTA, Triton-X, Trichloro acetic acid and diphenylamine, colchicine (SRL, India).

3.2.2. Human Lymphocyte Culture

A sample of heparinized venous blood was obtained from 25 women taking an OCs preparation containing levonorgestrel and ethinylestradiol as a content for 12

37 Genotoxic damage in cultured human peripheral blood months as a means of contraception following their first pregnancy. Age matched healthy 25 controls were evaluated along with the above subjects. A written informed consent was obtained from all subjects. The mean age of 25 women using OCs was 28.5 years and of the 25 controls was 27.5 years. Individuals answered a questionnaire relating to lifestyle factors. The OC users were not having any other addictions such as smoking, alcohol drinking etc nor have any history of disease. Briefly, heparinised blood sample (0.5 ml), was obtained from each female and was placed in a sterile cultured tube containing 7 ml of RPMI 1640 medium, supplemented with fetal calf serum (1.0 ml), antibiotic-antimycotic mixture (l.O ml) and phyto-haemagglutinin-M (0.1 ml). The culture tubes were placed in the incubator at 37°C for 48 hr (Carballo et al, 1993).

3.2.3. Chromosomal aberration analysis

After 47 hr, an amount of 0.2 ml of colchicine (0.2 |ig/ml) was added to the culture tubes. Cells were centrifiaged at 800 g for 10 min. The supernatant was

o removed and 8 ml of pre warmed (37 C) 0.075 M KCl (hypotonic solution) was added. Cells were re-suspended and incubated at 37 C for 15 min. The supernatant was removed by centrifugation at 800 g for 10 min and subsequently 5 ml of chilled fixative (methanol:glacial acetic acid; 3:1) was added. The fixative was removed by centrifiigation and the procedure was repeated twice. The cells were stained in 3% Giemsa solution in phosphate buffer (pH 6.8) for 15 min. About 300 metaphases were examined for the occurrence of different types of abnormality. Criteria to classify different types of aberrations were in accordance with the recommendation of Environment Health Criteria 48 for Environment Monitoring of Human Population (IPCS, 1985).

3.2.4. Sister chromatid exchanges

For sister chromatid exchange analysis, bromo deoxyuridine (10 |ag/ml) was added at the beginning of the culture and the mitotic arrest was attempted 1 hr prior to harvesting by adding 0.2 ml of colchicine (0.2 |ag/ml). Hypotonic treatment and fixation were performed as described for chromosomal aberration analysis. The sister chromatid average was taken from an analysis of metaphase during second cycle of division. A total of 25 well spread metaphases were scored for individual (Perry and Wolff, 1974).

38 Genotoxic damage in cultured human peripheral blood

3.2.5. Quantitative assay for DNA fragmentation

Quantitative assay for DNA fragmentation was performed according to protocol of Burton (1956). After incubation of 48 hr, the cell suspension containing 1- 10x10* cells in a 1 ml volume was prepared for each donor. About, 0.8 ml of cell suspension was transferred to a micro centrifuge tube and 0.7 ml of ice cold lysis buffer (5mM Tris CI, pH 8.0/20mM EDTA/0.5% (v/v Triton X-100) was added. The

o tubes were vortexed and allowed to lyse for 30 mm at 4 C. The tubes were centrifuged for 15 min at 15,000g (4 C) and the supernatant was transferred to a labelled conical glass tube. About 0.65 ml of 5% trichloroacetic acid (TCA) was added to the pellet in a micro centrifuge tube and 1.5 ml of 10% TCA to the sample in a labelled glass tube. o The sample was allowed to precipitate overnight at 4 C. The tubes were then centriftiged for 10 min at 2500 g at room temperature and the supernatant was removed and about 0.65 ml of 5% TCA was added to the pellet. The tubes were boiled for 15 min at 1OOC in a water bath. After cooling down to room temperature the samples was centriftiged at 2500 g for 5 min. About 0.5 ml of each supernatant (from both glass and micro centriftige tubes) was added to the labelled glass tube. About, 1 ml of diphenylamine reagent was added to each tube and was incubated for 4 hr at 37 C (Siddique et al., 2010). Finally the absorbance were noted at 600 nm in a spectrophotometer and the results were expressed in percentage of DNA fragmented as follows:

% Fragmented DNA = absorbance of supernatant absorbance of supematant+pellet 3.2.6. Statistical analysis

Student's t-test was used for the analysis of data obtained for chromosomal aberrations and sister chromatid exchanges and ^ (Chi-square) was applied for the analysis of DNA damage.

3.3. Results and Discussion

The frequency of cells with aberration in OC users was almost similar to their age matched controls. Gaps and breaks of chromosome as well as chromatid were observed, but dicentric, exchanges and rearrangements were not observed. The mean values obtained were not statistically significant (Table-3.1.). A significant difference

39 Genotoxic damage in cultured human peripheral blood

(p > 0.05) in the number of SCEs/cell was observed between OC users (3.9 ± 0.076) and their age matched control (2.62 ± 0.068) (Table-3.2.). The mean value for the DNA damage was (0.1172 ± 0.0020) in OC users and (0.1148 ± 0.0025) in controls. The values were not statistically significant (Table-3.3.).

The results of the present study suggest that the OC users have a significantly higher frequencies of SCEs/cell, but the CAs and DNA damage were not significant. An increase in the frequencies of SCEs and CAs has been reported during pregnancy (Murthy and Prema, 1983; Ghosh and Ghosh, 1988). The concentration of the estrogens is increased during pregnancy, which may be the possible reason of an increase in SCEs (Sharma and Das, 1983). Earlier studies performed on the genotoxic potential of steroids have shown to cause chromosomal damage, induction of SCEs and formation of endogenous adducts (Blakey and White, 1985; Shimomura et ai, 1992; Yager and Fifield, 1982; Joosten et al, 2004). The results of the present study shows that the OCs are not potent in inducing DNA damage/chromosomal aberrations, but are able to increase the number of SCEs/cell. The study performed by Biri et al, (2002) also showed the increase in SCEs/Cell in OC users. The SCEs are the cytological manifestation of interchanges between DNA replication products at apparently homologous loci. It is more sensitive indicator of genotoxic effects than structural aberrations (Tucker and Preston, 1996; Tucker et al., 1993). The induction of SCE is also correlated with the induction of cancer (Gebhart, 1981). Besides various reports on the genotoxicity of steroids some of the progestogens have been studied in short and long term toxicology studies in rodents, dogs and monkeys for their carcinogenic activity, no significant or unusual toxicities were reported (Jordan, 2002). In some cases the oral contraceptives have also been suggested to be having a role in preventing ovarian cancer by both suppressing ovulation and altering the tumour promoting milieu (Siskind et al, 2000).

The results obtained in the present study are contrary to the studies performed by other workers (Fridrichova, 1990; Pinto, 1986; Dhillon et al, 1994). This may be due to the new hormonal formulations and preparations developed in an attempt to reduce the adverse effects of OCs, such as the reduction in the estrogen content. Estrogen such as ethinylestradiol, in the liver undergoes aromatic hydroxylation and the product, 4-hydroxyestrone, 3, 4-dihydroxy 1,3,5 (10)-oestratien-17-one (4-OHE) is carcinogenic in male Syrian golden hamster kidney tumor model (Liehr et al, 1986

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Table 3.2. Distribution of Sister ciiromatid exchanges (SCEs) among women using oral coniracepiives (OCs). SCE per cell 1 Subjects OC users Control 1 42 23 2 4.1 2.5 3 4.3 2.9 4 3.9 2.7 5 3.5 2.4 6 3.6 2.3 7 3.7 2.6 8 3.3 3.1 9 3.7 2.2 10 3.8 2.3 11 4.1 2.4 12 4.0 2.5 13 3.2 3.2 14 3.2 2.9 15 3.3 2.8 16 4.2 27 17 4.1 3.2 18 4.3 2.2 19 4.4 2.8 20 4.0 2.7 21 4.3 2.4 22 4.2 2.3 23 4.1 2.8 24 3.9 2 I 25 44 3.3 Mean ± SE 3.91 ±0.076* 2.62 ± 0.068

'pXJ.U!) (Signiticant with respect to control).

Table: 3.3. Estimation of DNA damage among Oral contraceptives (OCs) users and non-users.

Subjects OC users Controls 1 0 11 0.10 2 0.12 0.11 3 0.12 0.12 4 0.13 0.11 5 0.12 0.12 6 0.10 0.13 7 0.11 0.13 8 0.13 0.12 9 0.14 0.11 10 0.12 0.09 II 0.11 0.08 12 0.10 0.11 13 0.11 0.12 14 0.12 0.11 15 0.11 0.12 16 0.13 0.13 17 0.13 0.11 18 0.12 0.12 19 0.12 0.12 20 0.12 0.13 21 0.11 0.12 22 0.12 0.11 23 0.11 0.10 24 0.10 0.12 25 0.12 0.13 vfesR ± SE 0.!! 72 ± 0.0*^20 0. II48 ± 0.00252

p>0.05 NS (Non significant) 42 Genotoxic damage in cultured human peripheral

Li and Li, 1987). The genotoxic potential of steroids is determined by the metabolic conditions in the test system and the human body. The extrapolation from the experimental data to humans is not only difficult but also complex. The most of the studies conducted for genotoxic potential of steroids involve the concentration m microgram per ml range whereas the therapeutic plasma concentration ranges from nanogram or picogram per ml (Joosten et al, 2004).

3.4. Conclusion

The present study was conducted on the women taking OCs for one year and the increase in SCEs may be due to the variable hormonal profile among them. Further, the occupation and work place, polymorphic metabolizing genes and efficiency of DNA repair can also affects the response against OCs.

43 Genotoxic damage in cultured human peripheral

44 * ^

4, UPID PROFILE OF WOMEN USING ORAL CONTRACEPTIVE PILLS 4. UPID PROFILE OF WOMEl USINQ OEAL CONTRACEFTIVE PILLS

4.1. Introduction

From the last few decades, world is facing the most serious problem of population explosion. Contraception method is used worldwide for over birth control or unwanted pregnancies. Many workers have suggested that the use of contraceptives is beneficial, but it may also have some side effects too. The researchers believe that the widespread use of hormonal contraceptive provides an opportunity for assessing the influence of estrogens and progesterone on various biochemical parameters among users. Synthetic progestins not only have genotoxic potential (Siddique and Afzal, 2004a; Siddique and Afzal, 2005a; Siddique et al., 2005a,b,c; 2006a,b) but are also vulnerable to various types of cancer (Siddique and Afzal, 2008; Siddique and Afzal, 2005b). It was found that the bone formation was significantly decreased in women taking oral contraceptive pills (Gamero et al., 1995). Some remarkable findings are well documented by workers which showed that oral contraceptives are involved in many diseases such as, myocardiac infraction, and carcinogenicity (Obisesan et al.. 2002; Tzankova et al., 2010; Naz et al., 2012b). An increase in the risk for ischemic stroke in women taking oral contraceptives has been reported (Thorvaldsen et al.. 1995; Vessey et al., 1984; WHO, 1996) and other studies showed that oral contraceptives also increases the risk for venous thromboembolism (Farmer and Lawrenson, 1998; Weiss, 1999; Westhoff, 1998; Rosing and Tans, 1999; Kemmeren et al., 2001). The relationship between oral contraceptive users and the risk for breast cancer remains controversial. Most studies suggests no overall risk or a slight increased risk of breast cancer in women using oral contraceptives (Anonymous 1986; Marchbanks et al., 2002; Brinton et al., 1998; Hankinson et al., 1997). It has been reported that the serum total cholesterol is significantly higher among oral contraceptive users as compared to non users (Nawrot et al., 2003). The mode of action depends upon the formulation of pills. The pills possibly inhibits ovulation by the formation of pituitary luteinizing hormone that renders the cervical mucus hostile to sperm penetration. The oral contraceptive pills (OCPs) are the combination of different concentrations of estrogen and progestin. In our present study the composition of the oral contraceptive taken by the women was: Ethinyl Estradiol (EE2) (0.03 mg) and Levonorgestrel (0.15 mg). In the present study an attempt has

45 Lipid profile of women using oral contraceptive pills been made to investigate the possible effects of oral contraceptives (OCs) among users on high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), total cholesterol (TC) and triglycerides (TG).

4.2. Materials and Methods

4.2.1. Sample Size

The subjects for the study were married women of low income group to a total of 100 participants from Malkhan Singh hospital and Jawahar lal Nehru Medical College of Aligarh city, who were of 21 to 40 years of age, among which 55 women were taking combined oral contraceptives preferring after 8-10 days of withdrawl period and 45 non-users healthy married women who were not taking any oral contraceptive. A random sampling were performed and the women taking oral contraceptive continuous for the three months were included for the comparison to non-users. A written consent was obtained from the subjects for taking their blood samples. The detailed questionnaire includes some issues about fasting, age, health history, nature of menstrual cycle, bleeding, disease, etc.

4.2.2. Blood sampling and processing

In morning as overnight fasting of users and non-users women, about 4 ml blood was collected in vacutainer tube with clot activator from each women in study sample. The serum samples were obtained by centriftigation at 3000 rpm for 15 min and the sera was stored at -20°C and analyzed within three days.

4.2.3. Blood parameters

The analysis of lipid profile of cholesterol, triglycerides High-density lipoprotein (HDL-C) and Low-density lipoprotein LDL-C were carried out using a commercially available diagnostic test kits (Crest Biosystems kits, India).

4.2.4. Lipid profile

Estimation of Total serum Cholesterol (TC)

Principle

TC was determined by CHOD/PAP method in which cholesterol is determined after enzymatic hydrolysis and oxidation. Cholesterol esterase hydrolyses esterified

46 Lipid profile of women using oral contraceptive pills cholesterol to free cholesterol. The free cholesterol is oxidized to form hydrogen peroxide which further reacts with pheivol and 4-aminoantipyrine by the catalytic action of peroxidase to form a red coloured quinoneimine dye complex. The mtensity of the colour formed is directly proportional to the amount of cholesterol present in the sample.

^1 , , , TT ^ Cholesterolestrase ^, , ,,^ Cholesterol esters + H2O ->-Cholesterol+ Fatty acid

Cholesterol+ O2 ^ Cholestenone + H2O2

H2O2+4 Aminoantipyrine+Phenol >- Red Quinoneimine dye + H2O

Estimation of Triglycerides (TG)

Principle

TG was determined by GPO/PAP method. In this method, lipoprotein lipase hydrolyses triglycerides to glycerol and free fatty acids. The glycerol formed with ATP in the presence of glycerol kinase forms glycerol- 3- phosphate which is oxidized by the enzyme glycerol phosphate oxidase to form hydrogen peroxide. The hydrogen peroxide further reacts with phenolic compovmd and 4-aminoantipyrine by catalytic action of peroxidase to form a red coloured quinoneimine dye complex. Intensity of the colour formed is directly proportional to the amount of triglycerides present in the sample.

^ . , ., Lipoprotein lipase Triglycerides ». Glycerol Free fatty acids „, ,, Glycerol kinase (jlycerol-r ATP *- QlyceroBphosphate + ADP GlyceroBphosphate +O2 G'ycero'3PQ» Dihydroxyacetone+H2O2 H2O2+ 4 aminoantipyrine Peroxidase^ ^^^ Quinoneimine+H2O2

Estimation of Heavy Density Lipoproteins-Cholesterol (HDL- C)

Principle

(HDL-C) determined by PEG precipitation method. The estimation of HDL-C involved precipitation of LDL and VLDL from serum by polyethylene glycol.

47 4 Lipid profile of women using oral contraceptive pills

Polyethylene glycol Serum — ~—*- HDL+ LDL+VLDL

When serum is reacted with PEG contained in the precipitating reagent all VLDL and LDH were precipitated. The HDL remained in the supernatant and can be assayed as a sample for cholesterol using Cholesterol (CHOD/PAP) reagent.

Estimation of Low-density lipoprotein LDL-C

Estimation of Low Density Lipoproteins-Cholesterol (LDL-C) was estimated by Freidewald's Formula as follows:

LDL Cholestrol (mg/dl) = Total Cholesterol- [Triglycerides] - (HDL Cholesterol) 5 4.2.5. Statistical analysis

The obtained data was analyzed by using a software statistical package SPSS version 16.0. Students t-test was also used to differentiate between two numerical data.

4.3. Results and Discussion

The results of the present study suggest that the oral contraceptive users have higher level of total Cholesterol, HDL-C, LDL-C and triglycerides as compared to non-users. As shown in the Table-4,1., the mean values of Cholesterol (242.92±2.842), HDL-C (58.65± 1.098), LDL-C (I15.84±1.266) and triglycerides (105.56±2.341mg/dL) were significantly higher among users as compared to the non- users (total Cholesterol 218.49±1.762, HDL-C 48.17±0.543, LDL-C 100.32±0.951 and triglycerides 83.77±2.299mg/dL) (Fig.4.1.).

It has been observed that the LDL-C and HDL-C increase is prone to have stroke and myocardial infarction in women taking oral contraceptives (Wynn and Doar, 1966). The present study supports the earlier studies carried out on lipid profiles of women using OCs (Emokpae et al., 2010; Abdel-Barry et al., 2011). Elevated serum levels of lipids are probably the most important biochemical risk factors for atherosclerosis. In the liver triglyceride synthesis is enhanced by estrogen and inhibited by androgen and these triglycerides are partly brought into the circulation as low-density lipoproteins. When a contraceptive pill containing an estrogen and a pro-

48 4 I Lipid Profile of Women Using Oral Contraceptive Pills

labie- 4.1. Lipid profile of oral contraceptive (OC) users and non-users women.

Lipid Profile mg/dl -J OC users (n = 55) Non-users (a=4S) t^noiesieroi 242.92 ± 2.842'" HDL-C 58.65 ±1.098" 48.17 ±0.543 LDL-C 115.84 ±1.266' 100.32 ±0.951 Trielvcerides 105.56 ±2.341^ 83.77 ± 2.299

Values arc as McaniSE, ''p<0.05 (significant Vvith respect to non-users)

300

^ Users 250 -

Non Users 200

150

100

50 - %mm cholesterol HDL- C LDL-C Triglycerides

Figure. 4.1. Lipid profile of oral contraceptive users and non-users.

49 Lipid profile of women using oral contraceptive pills

-gestogen is introduced, the resultant effect is not predictable. The clinical significance of this observation is supported by the fact that atherosclerosis begins early in life and is accelerated in the presence of high cholesterol and serum triglycerides (Newman et ai, 1991). Contraceptive steroids having a combination of an estrogen and progestin or only progestin are effective and reversibly regulate fertility. Combined oral contraceptives affects a variety of metabolic factors including hemostatic variables and estrogen-sensitive liver proteins and these effects can be modulated by the type of estrogen and progestin in a given combination (Sitruk-Ware and Nath, 201 J). The estrogen component of OCs has been reported to increase the production of very low density lipoprotein (VLDL) and high density lipoprotein (HDL) but reduce the level of low density lipoprotein (Sitruk-Ware, 2006). The reduction in the doses of levonorgestrel/ethinyl estradiol containing contraceptives from 30-20 ^g (EE) and that of 150-100 ^g (levonorgestrel) resulted in the less adverse effect on the lipid profile and have been suggested to lower the incidence of thrombosis as compared to other EE based contraceptives (Skouby et al., 2005). OCs containing levonorgestrel was associated with an almost four times higher risk of venous thrombosis as compared to non-users (Van Hylckama Vlieg et al., 2009). The intake of 100 \ig levonorgestrel with 20 |ig ethinyl estradiol to 28 women whom were at the risk of pregnancy shows a little change in lipid profile as compared to higher dose oral contraceptive preparation (Young and DelConte, 1999). Despite the changes in lipid profile among users of higher dose oral contraceptive agents, there is little and no effect on the long term risk artherogenic cardiovascular disease (Stampfer et al., 1990). The status of elevated serum triglyceride level may be an independent predictor of coronary heart disease as ethinyl estradiol increases hepatic secretion of triglyceride rich lipoprotein (Avins et al., 1989; Schaefer et al., 1983). The progestin component of the OC may oppose this increase. The injectable oral contraceptives have also been reported to increase the levels of triglycerides, total cholesterol, high density lipoprotein (HDL) cholesterol in the serum (Godsland et al., 1990).

4.4. Conclusion

The lipid profile depends on the brand of OCs or duration of their use. The users in our present study were taking OCs of ethinyl estradiol (0.03 ^g), levonorgestrel (0.5 |ig). The increase in the lipid profile may be attributed to their

50 Lipid proflle of women using oral contraceptive pill$ responsiveness towards the drug. However the long term effects were not taken into account in the present study, but still there is a need of a detailed study taking the various life style factors into the consideration.

51 5. EFFECT OF ORAL CONTRACEPTIVE FILLS ON THE BLOOD SERUM |f f*^ ENZYMES AND DNA DAMAeE IN I ' LVMPflOCVtES AM0N6

USERS ^ n

I «, m'' ^^ « ' # 5. EFFECT OF ORAL CONTRACEPTIVE PILLS ON THE BLOOD SERUM ENZYMES AND ONA DAMA6E IN LYMPHOCYTES AMONa USERS

5.1. Introduction

Women use exogenous female hormones as oral contraceptives (DCs). Over several decades the side effects of OCs are of major public health interest. Their association with ovarian cancer in particular has occupied an attention of man> epidemiologists (Siskind et al., 2000). Soon after the introduction of OCs pills numerous side effects among users were reported (Weiderpass et al., 1999). The contraceptive containing either a combination of an estrogen and progesterone or progesterone only is being used by millions of women (Palan et al., 2010). The most effective contraceptive works by suppressing the levels of follicle-stimulating hormone and luteinizing hormone i.e. by reducing metabolic activity of ovary, including the suppression of ovulation (Petitti, 2003). Currently there are several hormonal methods available other than oral pills such as transdermal patches, vaginal rings and intrauterine systems (Palan et ah, 2010). Few research evidences suggest that oral contraceptive pills might have negative effects on health of the women rather than the prevention of pregnancies, such as such as coronary atherosclerosis and myocardial infarction (Engel et at., 1983; Jick et al., 1996; Lewis et al, 1997; Dunn et al, 1999; Tanis et al, 2001; Sidney et al, 1998), risk of breast cancer (Grabrick et al, 2000; Rosenberg et al, 2009; Stanford et al, 1989) and hepatocellular carcinoma (Neuberger et al, 1986). In the present study blood serum enzymes such as alkaline phosphatase (ALP), gamma-glutamyltranseferase (GOT), lactate dehydrogenase (LDH), amino transeferases (SGOT and SGPT), serum proteins (albumin and globulin) and DNA damage in the peripheral blood lymphocytes were studied among women of different age groups using OCs for various durations.

5.2. Material and Methods

5.2.1. Sample size

About 600 subjects were included in the study from Jawahar Lai Nehru Medical College of Aligarh Muslim University, Aligarh, who were 16 to above 40 years of age. Among them 300 women were taking oral contraceptives preferring Effect of oral contraceptive pills on the blood serum enzymes continuous months and 300 were non-users healthy women with regular menstrual cycle who were not taking oral contraceptives. The detailed questiormaire includes some issues such as age, health history, type of pill, nature of menstrual cycle, any other disease, smoking habit, alcoholism, bleeding etc. About 64% of women were taking OCs as birth control, 15% for irregular bleeding, 11% for dysmenorrhea/pelvic pain, 6% for premenstrual syndrome and 4% for menorrhagia. A written consent was taken from each subject taking participation in the study.

5.2.2. Blood sampling and processing

After overnight fasting, about 2 ml of blood was collected in a vacutainer tube with a clot activator from women in the study sample. The serum was obtained by centrifugation at 3000 rpm for 15 min and analyzed for various enzyme activities.

5.2.3. Blood Serum analysis

The blood serum was analyzed for enzymes: ALP, GGT, LDH, aminotransferases (SGPT and SOOT) and serum proteins (albumin and globulin) among users and non-users by using commercially available diagnostic test kits (Crest Biosystems kits, India).

5.2.4. Enzyme profile

Alkaline phosphatase (ALP)

Principle

ALP was determined by (Modified Kind & King's Method) in which an alkaline pH hydrolyses di sodium phenylphosphate to form phenol. Further phenol reacts with 4-aminoantipyrine in the presence of potassium ferricyanide, as an oxidising agent, to form a red coloured complex. The intensity of the colour formed is directly proportional to the activity of ALP present in the sample.

di Na Phenylphosphate + H2O ALP >- Phenol + di Na Hydrogen phosphate

. , ^ A • .• • Alkaline Medium „ , , , , Phenol+4-Ammoantipyrme *- Red coloured complex K3Fe(CN)6

54 Effect of oral contraceptive pills on thej^lq^jaeASftm: y-Glutamyl transferases (GGT)

Principle

GGT was determined by (Carboxy substrate Method) in wliidJIl tl' UUlalyzes the transfer of amino group between L- y- Glutamyl-3-carboxy-4nitroaniiide and Glycylglycine to form L- y- Glutamylglycylglycine and 5-amino-2-nitrobenzoate. The rate of formation of 5-amino-2-nitrobenzoate was measured as an increase in absorbance which is proportional to the GGT activity in the sample.

L-Gamma-Glutamyl-3-Carboxy-4-Nitroanilide ^^ • L-GammaGlutamyigiycylglycine 5-amino-2-nitrobenzoate Glycylglycine Lactate dehydrogenase (LD)

Principle

Lactate dehydrogenase (LD) was determined by [lactate dehydrogenase (P- L) modified IFCC method]. Lactate dehydrogenase catalyzes the reduction of pyruvate with NADH to NAD. The rate of oxidation of NADH to NAD was measured as a decrease in absorbance which is proportional to the LDH activity in the sample.

LDH Pyruvate + NADH + H^ *- Lactate + NAD

Aminotransferases (SGOT and SGPT)

Principle of SGOT (ASAT)

SGOT (ASAT) was determined by (Reitman & Frankel's method). SGOT converts L-aspartate and a-ketoglutarate to oxaloacetate and glutamate. The oxaloacetate formed reacts with 2,4 Dinitrophenyl hydrazine to produce a hydrazone derivative, which is in alkaline medium produces a brown coloured complex. The calibration curve was plotted using a pyruvate standard.

L-Aspartate + Ketoglutarate ^^^^ *- Oxaloacetate + L-Glutamate pH7.4 a

Oxaloacetate + 2,4, DNPH -^^ 2, 4, Dinitrophenyl Hydrazone (Brown colour complex)

55 Effect of oral contraceptive pills on the blood serum enzymes

Principle of SGPT (ALAT) SGPT (ALAT) was determined by (Reitman & Frankel's method). SGPT converts L-alanine and a ketoglutarate to pyruvate and glutamate. The pyruvate formed reacts with 2,4 dinitrophenyl hydrazine to produce a hydrazone derivative, which in alkahne medium produces a brown coloured complex whose intensity was measured.

L-Alanine +a Ketoglutarate » Pyruvate + L-Glutamate pH 7.4 •'

AI K3 imp Pyruvate + 2,4, DNPH ^ .. >- 2, 4, Dinitrophenyl Hydrazon (Brown colour complex)

Serum proteins (albumin and globulin)

Principle

Albumin and Globulin were determined by BCG method. Albumin binds with the dye Bromocresol Green in a buffered medium to form a green coloured complex. The intensity of the colour formed is directly proportional to the amovmt of albumin present in the sample.

Albumin + Bromocresol Green >-Green Albumin BCG Complex

5.2.5. Single cell gel electrophoresis (SCGE)

5.2.5.1. Blood sampling for Comet assay

After overnight fasting, about 2 ml of blood was collected in a heparinised vacutainer tube from each woman (user and non-user). The blood lymphocytes were isolated by Ficoll-Histopaque density gradient centrifiigation and washed in PBS.

5.2.5.2. Slide preparation and Electrophoresis

The alkaline SCGE technique of Singh et al. (1988) was followed. The half frosted slides were covered with 1% normal melting agarose (NMA) at about 45°C in PBS. The slides were immediately covered with a coverslip and kept at room temperature for about 5 min to allow the agarose to solidify. This layer was followed

56 Effect of oral contraceptive pills on the blood serum enzymes by the second layer of 0.5% LMA. Five to 10 ^1 of cell suspension was mixed with 75 |j,l of 0.5% LMA for embedding on slides. After gently removing the coverslip the cell suspension was laid onto the first agarose layer, spread out with a coverslip, and maintained on an ice-cold flat tray for 5 min to solidify. After removal of the coverslip the slides were immersed in cold lysing solution (2.5 M NaCl,100 mM EDTA,10 mM Trizma base, 0.2 mM NaOH, pH 10), 1% Triton X-100 and 10% DMSO for overnight at 4°C. The slides were then removed from the lysing solution, drained and placed in a horizontal gel electrophoresis tank near the anode. The tank was filled with fresh electrophoresis buffer (ImM Na2EDTA and 300mM NaOH. pH 13) to a level approximately 0.25 cm above the slides. Before electrophoresis, the slides were left in the buffer for 20 min to allow the unwinding of the DNA. Electrophoresis was conducted at 1.6 V/cm for 20 min (300 mA) at room temperature. All steps were conducted under dimmed light (tank was covered with a black cloth) to prevent the occurrence of additional DNA damage. After electrophoresis, the slides were taken out of the tank. Tris buffer (0.4M Tris, pH 7.5) was gently added drop wise to neutralise the excess alkali. The neutralising procedure was repeated three times.

5.2.5.3. Staining

To each slide, 65^1 ethidium bromide (EtBr-20^g/ml) was added. The slides were covered with a cover-slip, placed in a humidified air-tight container to prevent drying of the gel and analysed within 3-4 hr.

5.2.5.4. Slide scoring

About 50 cells were scored per slide (3 slides/subjects) were scored randomly and analyzed with Cometscore^'^ software (version 1.5, TriTek Corporation, Sumerduck). DNA tail length was measured and expressed as arbitrary unit.

5.2.6. Statistical analysis

The obtained data was analyzed by using a software statistical package SPSS version 16.0. Students t- test was applied for the significant difference among users and non users. One-way analysis of variance (ANOVA) using post hoc tukey test was applied to compare the differences amongst the users of different age groups.

57 Effect of oral contraceptive pills on the blood serum enzymes

5.3. Results and Discussion

Table- 5.1. shows the age distribution of OCs users and non-users. Table-5.2. shows the distribution of users according to the duration of using OCs. The more number of users were of 26-30 years of age group. Table-5.3. describes the composition of OCs used by the women. The highest concentration of estrogens in the combined oral contraceptives was BO^ig. Table 5.4. shows the distribution of various blood serum enzymes in women of different age groups. As is evident from the Table- 5.4., a significant decrease in the ALP (U/L) was observed among the users of all age groups as compared to their respective age group non-users (p<0.05). Concerning the other enzymes such as LDH (U/L), SCOT (U/L), SGPT (U/L), and GOT (U/L), a significant increase in their levels were found among users of all age groups as compared to non-users to their respective age groups (p<0.05). The level of serum albumin was also found to be decreased significantly among users of all age groups compared to non users of their respective age group (p<0.05) (Table-5.4.). The level of serum globulin was found to be significantly higher among users of all age group compared to non users of their respective age group (p<0.05) (Table 5.4.). Table-5.5. shows the enzyme profile according to the duration of using the OCs. A duration dependent significant decrease (r= -0.99) in serum ALP was found as compared to the non users (p<0.05) (Table-5.5.). In other serum enzymes LDH (r= 0.97), SCOT (r=0.98) and SGPT (r=0.99) and GGT (r=0.99) a duration dependent significant increase was found as compared to the non-users (p<0.05) (Table-5.5.). The serum albumin level (r=0.99) was also found to be decrease among users in accordance with increase in the durafion of use of OCs (p<0.05) (Table-5.5.). The serum globulin level was found to be increase among users (r=0.98) with increase in duration of the use of OCs. The results obtained for comet assay performed on the human peripheral blood lymphocytes of users and non-users is shovm in Figure 5.1. (a &b). Among women using OCs for 1-6 months showed 1.73 fold (p<0.05), increase in the mean tail length as compared to control (Fig. 5.2.). The women using OCs for more than six, thirteen and nineteen months showed 2.20. 2.40 & 2.74 folds significant increase respectively, in the mean tail length as compared to non users (p<0.05).

Hormonal contraceptives are in clinical practice for more than 70 years for the prevention of ovulation, implantation and sperm penetration into ovum. Estrogen or progesterone or both are also used in hormonal replacement therapy and are given to

58 5 I EfTect of oral contraceptive pills on the blood serum

Table-5.1. Age distribution of oral contraceptives non-users and users.

Age Groups Non- users (N=300) Users (N=300) (Years) 1 16-20 30 31 21-25 55 60 26-30 89 95 31-35 34 42 36-40 48 30 >41 44 42 MeaniSD 30.61±8.20 30.42±8.4i

Tab!e-5.2. Distribution of users on the basis of using oral contraceptives.

Duration of contraceptive OC users use (months) 1-6 62 7-12 79 13-18 65 19-24 94 MeaniSD 13.44±6.96

f. ^^^.^^ .. •^ k Combined oral contraceptives Progestin Estrogens (Ethinyl Estradiol) (mg) (HR) Desogestrel 0.5 10 Levonorgestrol 0.10 30 Medroxyprogesterone 0.15 20 Drospirenone 0.10 10 Gestodene 0.15 30 Norgestimate 0.75 10 Norethindrone acetate 0.5 10 Desogene 0.10 30 Norgestrel 0.15 10 Progestin only 20 Ethynodiol diacetate 0.5 - Desogestrel plus 0.35 .

Levonorgestrel 0.3 -

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(a) (b)

Figure 5.1. Comet assay performed in human peripheral blood lymphocytes (a) non users, and (b) users.

14 - 12 - 10 - I_ 8 - e 6 -I m i 4

Non 1-6 7-12 13-18 19-24 users months months months months (n= 300) (n=62) (n=79) (n = 65) (n=94)

Figure 5.2. Histogram of the total comet tail length in control and in women taking oral contraceptives. [*Significant difference between oral contraceptive users and non-users (p<0.05)].

62 Effect of oral contraceptive pills on the blood serum enzymes women after menopause (Moore et al, 1999). However the hormonal replacement therapy has been reported to be associated with increased risk of cancer (Speroff et al, 1999; Pike and Ross, 2000). There are several reports on the genotoxic potential of estrogens and synthetic progestins in vivo and in vitro (Siddique and Afzal. 2004a; Siddique and Afzal, 2004b; Dom et al, 2007; Dom et al, 2008; Seraj et a/., 1996; Siddique et al, 2006; Siddique and Afzal, 2008; Yan et al, 2013). Most of the oral formulations have a definite proportion of estrogen and synthetic progesterone (Jordan, 2002). However the composition and salts of the OCs can vary from country to country so as the response toward the drugs that forms the basis of pharmacogenomics (Franconi and Campesi, 2014). The reports on the genotoxic potential in women's using steroid hormones as an oral contraceptives or HRT are little (Ozcagli et al, 2005; Kayikcioglu et al, 2000).

In early 80s it was well documented that the use of oral contraceptives was associated with the benign hepatic adenoma. The association was fiirther confirmed by Palmer et al (1989). The use of OCs have been reported to be associated with the higher plasma retinol, 25-hydroxy vitamin D, total iron binding capacity, total chlolesterol, LDL-cholesterol and triacyglycerol (Thane and Bates, 2002; Naz et al, 2012). Concerning the serum enzymes levels in our present study an increase in LDH, SGPT, SGOT and GOT was found among users. This increase was found to be in correlation with the duration of using OCs. The use of OCs has been reported to alter lipids and enzymes profile among OCs users (Naz et al, 2012a; Machado et al, 2010). As most of the drugs are metabolized by liver hence it is becomes difficult to correlate whether larger doses of drugs can cause adaptative changes in the form of increasing the detoxifying enzymes, or increase in the metabolic functions of liver. DNA damage and increase in the frequency of sister chromatid exchanges in peripheral blood lymphocytes have been reported in OCs users (Naz et al, 2012b). The extent of DNA damage and SCEs frequency were due to the altered hormonal profile of the users as the damage was reported to be non significant compared to pregnant women (Biri et al, 2002). The increase in the mean tail length in our study may also be correlated to the altered hormonal profile. The increased in the DNA damage has been correlated with the induction of various cancers. There are reports on the increase in lipid profile among OCs users and increased risk of atherogenesis and coronary arterial disease (Santos et al, 2008). The mechanism by which

63 Effect of oral contraceptive pills on the blood serum enzymes

progestins induce tumour development is not very clear. It has been suggested that induction of homo oxygenases leads to the conversion of procarcinogenes to carcinogens (Schulte-Hermann et al, 1983), or by stimulation of the genetically altered pre-neoplastic cells (Ochs et al, 1986). The relationship between the carcinogens and sex hormones is not very clear, however a series of or combination of genotoxic and epigenetic changes have been reported to be associated with OC users (Braz and Salvadori, 2007). In our earlier in vitro studies synthetic progestins have been reported to generate reactive oxygen species (ROS) (Jordan et al, 2002). Estrogens have been reported to generation free radicals by metabolic redox cycling between quinone and hydroquinone forms of estrogens (Roy and Liehr, 1999), 17 P- estradiol was repored to be mutagenic and genotoxic effects on the blood cells of Oreochromis niloticus (Sponchiado et al, 2011). In cultured cells northindrone has been reported to induce double stranded breaks (DSBs) (Gallmeier et al., 2005). In our present study the DNA damage was found to correlated with the duration of using the OCs (Y=6.6100+0.24433X; r=0.987; p<0.0047). There were no difference in the level of globulin in both users and non-users. Steroid hormones have been reported to increase aminotransferases among OC users (Siest et al., 1998; Schiele et al., 1998). In our earlier study with triglycerides, HDL and LDL, the level of serum markers was found to be high among users (Naz et al, 2012c). Thus it is concluded that the intake of OC effects the level of various blood serum enzymes and it may depends on the estrogens and progestin dosage or the androgenic activity of the progestin (Schulte- Hermann et al, 1983). Concerning the DNA damage in blood lymphocytes, the damage was found to increase with increase in the duration of using OCs. Comet assay is a very sensitive method to estimate DNA damage at single cell level and the sensitivity of this test is higher than that of any other DNA damage detection method (SCE, MN or chromosomal aberration) (Ozcagli et al, 2005). Earlier studies have revealed association between DNA damage and malignancies (Smith et al, 2003; Colleu-Durel et al, 2001). Nearly all oral contraceptives have some amount of estrogens which is converted in to catecholestrogenes and result in the production of oxygen radicals that may cause DNA damage (Bolton, 2002).

5.4. Conclusion

Although estrogen are present normally in women, but the presence of it in the form of OCs may lead to high concentration in human body. Hence, it is suggested

64 Effect of oral contraceptive pills on the blood serum enzymes that while prescribing OCs to the women various life style factors (cigarette smoking, alcohol consumption etc) that may possibly enhance DNA damage should be taken in consideration. The biochemical parameters (enzymatic profile of various blood serum enzymes) should continuously be monitored as estrogens may also act as tumour promoting agent due to'^heir estrogens-receptor mediated mitogenic activity.

65 Effect of oral contraceptive pills on the blood serum enzymes

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94 UST OF PUBUCATIONS

1. Naz, F., Jyoti, S., Afzal, M. and Siddique, Y.H, (2012). Biochemical effects of oral contraceptive among users: A review. International Journal of Pharmacology 8,314-320.

2. Naz, F., Jyoti, S., Akhtar, N., Afzal, M. and Siddique, Y.H. (2012). Genotoxic damage in cultured human peripheral blood lymphocytes of oral contraceptive users. Egyptian Journal of Medical Human Genetics 13, 301-305.

3. Naz, F., Jyoti, S., Akhtar, N., Afzal, M. and Siddique, Y.H. (2012). Lipid profile of women using oral contraceptive pills. Pakistan Journal of Biological Sciences 15, 947-950. International Journal of Pharmacology, 2012 ISSN 1811-7775 / DOI 10 3923/ijp 2012 © 2012 Asian Network for Scientific Information

Biochemical Effects of Oral Contraceptives among Users: A Review

Falaq Naz, Smita Jyoti, Mohammad Afzal and Yasir Hasan Siddique Human Genetics and Toxicology Laboratory, Department of Zoology, Section of Genetics, Faculty of Life Sciences, Aligarh-202002 (UP), India

Abstract: Oral contraceptives (OC) are widely used to prevent ovulation, implantation and therefore pregnancy The widespread use of the oral contraceptive pills provides an opporturuty for assessing their influenL-e <.JII vanous biochemical parameters i e , enzymatic, serum hpid and proteins among users Recent studies have shown its impLcation m mar^ diseases such as thromboembolic disease, myocaididl infaictioii, ciit-ulatou disorders and carcinogemcity The negative effects on the hver and heart have also been reported due to h^h serum cholesterol levels among OC for their possible biochemical effects

Key words: Oral conlrdceptives pills, biochemical studies, oidl ocgilidoeptive uscis

INTRODUCTION progestogen, while multiphasic piUs allow a lower total dose ot progestogen to be given by reducing the amount By the early twentieth century, scientists were eager of progestogen early m the 20-22 day penod of exposure to isolate and wanted to determine the formulation and Prc^estin-only ccmtraception is an option fcx women that structural properties of synthetic hormones and they contam only progestm The most common side-effects of found that the high doses of these synthetic hormones progestm only c«itracep>tion is complicaUoii in inhibited ovulation The synthetic progesterone pregnancy or eccysis and irregulanty m menstrual cycle compounds that are used as an oral cc«traceptives are (.Kauratz, 1997j Oral contracepliveb have been stiowTi Norethisterone and Norethynodrel These compouiKls abrupt increase m the nsk of ncn-fetal myocardial were first tested m woman m 1956 m the Umted States, infarcUon (,Ory, 1977, Mann ei al, 1975j Both single Puerto Rico and Haiti The first pill that was launched as progestins and combmed oral contraceptive shows the an oral contraceptive ioc use contdins the combination genotoxic damage and risk of Cdiicers Study slated tiidl of nor-19 progestin with a small amount of synthetic steroids shows genotoxic damage at higher doses estrcgen as a trade name Enovoid (Junod and Marks, (Siddique et al, 2006,2005, Siddique and Afzal, 2001, The 2002) Now-a-days mdhon of women of reproductive therapeutic doses are safe, but care should be taken with age around the world use oral contraceptives regard to tlieir concentiatioii as Oiey may be geiioto\ic m (Burkman, 2001) Over the decades, the use of oral the long term use m humans (Siddique and Afzal, 2008 contraceptive pdls is mcreasing m India (^Sharma et al, Siddique et al, 2007, Siddique and Afzal, 2

Correiponding Author: Yasir Hasan Siddique, Human Genetics and Toxicdogy Laboratory, Department of Zoology Section of Geiietits, Faculty of Life Suein.es, Aligcirh-202002 (UP), India 1 Int. J Pharmacol, 2012

Cholesterol (LDLC) levels The lipoprotein profile, levels of tnglycendes, total cholesterol, LDL-cholesterol resultu^ from the use ot cral cxjntracepbve is therefore, and VLDL-diolesterol m UCs users wonen The increase dependent on the balance between the potencies of the in OCP tnglycendes is due to the increase in the ebtrogen and progestogen components Although, the synthesis OCP intake produce changes m lipid high-dose progestogens contained in the older oral metabolism m women, but such changes may not contraceptives did raise Low-density Lipcyrotem necessarily lead to pathogeruc concentration resulting m CholestCTol (LDLC) and reduce High Density Lipoprotein a cardiovascular disease with the prolorêd use of not Cholesftetol level (HDLQ, modem low-dose progestogens more tlian 4 yeais (Einokpae et al, 2010) Smce, tlie effe&tb do not appear to affect the lipid profile in users The caused by OCP intake are shcrt hved, it could be said that increased risk of cardiovascular disease in oral the effects of these hormonal prep)aratic8Ts may be contracef)tive users is due to venous or artenal physiologic rather than pathogenic (Karam, 2001) tlnombosis (Speroff and Darney, 1996, Brennan the androgenic acu\ lu mtiacellular progestogen receptors), only a mmimal dose of the unopposed prcêstogen, despite its very low dose, of estrogen is needed to mauitaui the efficacy of the which (fecreaseb tlie level of ciioulatuig sex hoiuiune combination oral contraceptive (Speroff and binding globulin. As a result, biologically available levels Damey, 1996) of lev onoigestrel and testcsterone are increased Low-dose ccMnbmation oral contracepjtives do not Progestin-only oral contraceptives. As tlie progestogens produce acne because esliugen, wliich iiit-ieas>es ^cx themselves do not always suppress gonadotropins, hormone bmdmg globulin, counteracts the effect of women who take pwogestm-only oral contraceptives do progestogen i^Spieroff and Damey, 1996) The mimpili Joes not always ovulate The contraceptive efficacy of the not significantly affect hpid levels, carbohydrate prcêstin-only oral ccxitraceplive is therefore dependent metabolism, or blood coagulauon When ii ib mostly on its effects on cervical mucus, on the discontinued, fertility returns promptly (Hatclwr et al endometnum and possibly also on the fallopian tube 1994) (Speroff and Damey, 1996) The progestin-only mimpill Studies earned out on the biochemical effects on oral must be taken every day at the same time because its contraceptives are as given in Table 1

Tihle 1 SludCT oo hodwrntil effecte of oat coctnctohvcs Oral coBteicctitivc Pinmtter itndtd Cooduaon Autbor Ee(k(nypiogedcn»e accUte), Oid piU- HDL and high LDL level m DMFA usas ethmyl eatraJol and deaogestrel Oral CTotrioetifcves Senm proteui and cfaoleattrol HJijief seram proteui, inaetsed dwleslerol levtis Ohsesan >»< g. (2002 Loir dOK contnccpfava (etlan^lefiradcd) Effect on deep venous diromboas Deep venous lhrombo'3'!: in user: (aitena! Tzanksna (20")' combmed with progestm component and aitenal by lipid abnonnalitacs thrombosis mcreases) Aeao«eat or itriMBWcnooe Biphase lofenunal taUeta (nargestrd+ Senm bpida and Mood preaaure Total hpid mcreases reduced Uood pressure levd Emokp«e»'(a/ (2010) etfamyleHradol) Edmyiearadoi andprogeann Blood presaure and Senim teal Higher systolic blood pressure and higher level Nawrot-' 2. (2U0J) hpida of aeram total hpids Low dose cral ccntraceptave (ethmyl Lipid levd and Blood pressure Nonnal level of lipids, Nonnal Mood pressure Qinbelac ai (2007) eatraJol/geatodene) Mcnophiac pill< ttlbaM edndiol) Blood (ample (Total plaama cof«»l Basal free Cortisol levd mcreases No effect on Kuschtmum et al (1999) was measured with a corhsollevd radiaunmunouaiQr) and first sahva sample (Baaal fiee Cortisol level) Combined onri contraceptive (estrogen Senm Lipids and bpoprctetns Senan hpitfe is higher Abdd Banye ai (2011) eflmyleah^dlol+progeatowen) Lyndlcl pill (lynoealienol andprogeshn) SOOT levds (aenan glutamic Increase m SOOT (senim glutamic oxaloacetic Stdl (1967) oxaloacetic tranaammaae) transaimnaae) Oral cxMiiaxptive ^cntsconibinafaan Concenlrafaon of plasma Decrease plaana ascoitic aad McLeroy and Schendd type (ProKcatowP-tEatun'Oi) ascoifaic aad m leukocytes 11973; Nonnyl (1 mg of ooretfaui(kt«e 480 pg Vitamm levd and hpid levds m Decrease m senim vitsnm Smith el a( (1975) ofmcstranol'l scnsn BlZ, lipid Ictclm scrum hirfict Oral oonhaceptive Serum hpids Inaease level of saum hpids Kannd (1979) Oral conhacepbve pll (norethmct-ane-^ Senim hpidE Higher In-ds (^total lipids Heanefcen" meatranrt) Ccotraccptive pill (Ievonorgeatiel+ Serum Upids Deaease ^enlm lipid:> Dt^iiacket " a (19oij ettmylestiadol) Contraceptive pill Plasma vitamms (A, E, c| plaana Increase level ot vitanin, plasma, tnglycendes, veung(iv'6) biglycendes and cholesterol, diolesterol, leukocytes that makes the nsk of leukocytes myocariial miection Int. J. Pharmacol., 2012

Table 1 Caotmue Oral ContnctDtive Parameter Studed Concluaoo Author Noretbmdtme, Ottho-Novum (l/3i Lipoproteins and Serum lipids Higher lipoprctem, high Serum hpids Walsh and Sacks (1993) and 777), ediynodiol dacetate Cambued oral contracqitive Histopalhology Risk of endometnal cancer is decreased Wadcrpasseta/ (1999) eslrogai-)frogestu(desogeslrd. ievoncrxestrd and Doretiiistercme) Combined pills (etbuiytMtndlol) Bone markers, Scrun biodionstiy, Decreased bone forauticn, scrum albumm is Qamaoetal (1995) Bone mineral measurement lower m oral contraceptive users Noimal bone mmeral Oral contraceptave pill Senin levels of lipid-aoluble Lowa levd of serum levds of hpid Pdanefoi (2006) anHondants soluble aitfioradants Levonorgeslrel (90 fig), etbuiyl eslradol Demograptac data Use of this compoataon does not dday Bmbtrtetal (2009) (20 W() the return to fotihty Moaophaac oral ccotractptive Foilide dameteii and serum Suppfe&>ioa of ovanan activity m u&cr> Coney and DdContc (levooorgestrel (100 fig) progesterone and 17 P-estndd (1999) 1 oliiovicslradiai 2u m> Oflho-Novum 7/7/7, TYiphaal Serum conccntntion of ethmyl estradiol Scrum concenlrMion of etbmylestradicl mcreascs Smi^ky and Fasifiale (1998) (cambuiataoa of Nordliindrone, ethinyl by Ridoimmunoissay eslradol), Cypniterane acetate. Lipid Lipid mctaboliam mcreased. Lipid metabdian mcreased, deaease hirsuhan Utstonkasetal (2002) metabdism, binutism -l-dbuiyleatradol of adolescents with polycyshc DesoKestrd-t^ttunvl eslradol ovarv syndxime (PCOS) Mifepnstcoe (contraceptive pill) Biocfaeimcal parameter (scrum allcalme Increase biocheimcal [>aramctas Agarwde«a/ (2009) phosphatase and amim glutamyl (scrum alkahne phosphatase and pyruvate transaminase) pyruvate transaminase), serum glutamyl Etbmylcgtndid and Assesantnt of ov>i an fimdioa. Inhibihon of ovulataon and m&vourable Spam eta! (2010) cUonnadnooe acetate tfaidcoets of eadometnum, cervical condibon for fcrtUizAon, pregnancy reaction sex, hormone levd and overall tdcrabibty Dqwt medroocyprogesterone Oral Menstrual symptoms and Physiologic No adverse effects Berenson e; 12/ (2008) conirtceplive ptU (20 UK). symptoms Ural confracepdve (edunyl eslradoi-i- Bo(^ Mass Index (BMI) and serum IIM coHracepnve patch had an ettect White ctoJ (2005) norgestunate) Contraceptive patdi andogco levels comparable, to the OC on several key andogemc (etiiinjiesliadloi-fnofelgeslroiiui) potenbal as a therapeutic agent for dsorders of andxiscn excess Elfainylegtiadol+progestui Pdvic pam, bleedog, headaches. Pdvic pam, faleedng and breast Sulakefoi (2000) breast tenderness tenderness Aown, headadies problem, breaa tenderness shown Oral contraceptive Demographic

CONCLUSION about the affect of oral contraceptive The efforts should continued to produce safer cral contraceptive for tlie The studies perfortn on Oral contraceptive users users clearly shows the marked effects on enzymes, carbohydrates and serum lipids High level of serum lipid ACKNOWLEDGMENT invites many problems to the contraceptive users i e, cardiovascular risk by increase m the cholesterol level but The authors are thankful to Umversity Grant the full impact of oral contraceptive on cardiovascular nsk Commission (UGC), New Delhi for the award of project factors may not be fully concluded from the short entitled Biochemical and Cytogenetic effects of Oral duTdtioii of studies done so fdr Tlie durdlion of oral Coiitidceplives ditiuiig wtsneii of different reproductive contracejAive also have importance in stucfying the effect histories {FNo 39-582/2010(SR)} to Dr Yasir Hasan amoi^ the women We must leam the correct durahcwi of Siddique, Department of Zoology, Aligarh Muslim usage,'dosage more wisely and keep their user aware Um vers it}', Ahgaih Int. J. Phannacol., 2012

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Kirschbaum, C, B M Kudielka, J Gaab, N C Schommer Sharma, RS, M Rajalakshmi, RS Sharma and and D H Heilhammer, 1999 Impact of gender, D A Jeyaraj, 2001 Current status of fertihty control menstrual cycle phase and oral contraceptives on the methods m India J Biosci, 26 391 -405 activity of the hypothalamus-pitiutary-adrenal axis biddique, YH and M Afzal, 20U4 Evaluation of Psychosom Med, 61 154-162 genotoxic potential of synthetic progestm Mann, JI, MP Vessey, M Tliort^ocxl aiul S R Doll, chlormadinone acetate foxicol Lett, 153 221-225 1975 Myocardial infraction m young women with Siddique, Y H and M Afzal, 2005 Genotoxic potential of special reference to oral contraceptive practice cyproterone acetate A possible role of reactive Br Med J, 2 241-245 oxygen species Toxicol In vitro, 19 63-68 Mastorakos, G, C Kohopoulos and G Creatsas, 2002 Siddique, YH and M AfM 2008 A review on the Aixlrogen and lipid profiles m adolescents with genotoxic effects of s«ne synthetic progestins polycystic ovary syndrome who were treated with bit J Pharmacol, 4 410-430 two forms of combmed oral contraceptives Fertil Siddique, Y H, G Ara, T Beg and M Afzal, 2006 Steril,77 919-927 Geiwtoxic potential of medroxyprogesterone acetate McLeroy, VJ and HE Schendel, 1973 Influence of Oral contraceptive on ascorbic acid concentration on m cultured human penpheral blood lymjAiocytes Life healthy, sexually mature women Am J Clm Nutr, Sci,80 212-218 26 191-196 Siddique, Y H, T Beg and M Afzal, 2005 Genotoxic Mohtch, ME, P Oill and WD Odell, 1974 Ivlassive potential of ethmylestradiol in cultured mammalian Ityperlipemia during estrogen therapy J Am Med cells Chem Biol LTteract,151 133-141 Ass, 227 522-555 Siddique, YH, T Beg and M Afzal, 2007 Nawrot, TS, ED Hond, RH Fagard, Anticlastogerac effects of ascorbic acid against the K Hoppenbrouwers and J A Staessen, 2003 Blood genotoxic damage induced by ncTethynodrel Adv pressure, serum total cholesterol and contracepuve Environ Biol, 1 27-32 pill use in 17-year-old girls Eur J Cardiovasc Smofsky, FE and SA Pasquale, 1998 The effect of PieventionReliab,10 438-442 fluconazole on circulatir^ ethinyl estradiol levels m Obisesan, KA, FA Adenaike, MA Okunlola and women taking oral contracepbves Am J Obstet A A Adenaike, 2002 EffectA of oral contraceptives Gynecol, 178 300-304 on total serum proteins, albumin, globulins and Smith,JL,GA Goldsmith and J D Lawrence, 1975 Effect cholcsteiol levels ui Ibadaii, Nigeiia West Afr of oral contraceptive bteroids on vitamm and lipid J Med, 21 197-199 levels mserum Am J Clin Nutr, 28 371-376 Qry, H W, 1977 Assot-ialioii between oral ooiiLrdcepUves Sperott, L and F D Damey, 1996 A Climcal Guide tor and myocardial infarction A review J Am Med Contraception 2nd Ed WiUiam and Wilkms, Ass, 237 2619-2622 Baltimore, USA , Pages 247 Palan, P R, A T Magneson, M Castillo, J Duime and Spona, J , N Bmder, K Hoschen and W Feichtinger, M S Mikhail, 2006 Effects of menstrual cycle and 2010 Suppression of ovanan function by a combmed oral contraceptive use on serum levels of hpid- oral contraceptive caitammg 0 02 mg ethmyl soluble antioxidants Am J Obstet Gynecol, estradiol and 2 mg chlormadinone acetate given m a 194 e35-e38 24/4-day intake regimen over three cycles Fertil Palan, PR, F Strube, J Letko, A Sadikovic and Steril,94 1195-1201 MS Mikhail, 2010 Effects of oral, vagmal and Stoll, B A, 1967 Effect of lyndiol an oral contraceptive on transdermal hormonal contraception on serum levels breast cancer Bnt med J , 1 150-153 of coenzyme q(10), vitamin e and total antioxidant Sulak, PJ, RD Scow, C Preece, MW Riggs and activity Obstet Gynecol hit, 10 1155/2010/925635 Palmer, J R, L Rosenbeig, D W Kaufman, T J Kuehl, 2000 Hormcme withdrawal symptoms m ME Warshauer, P Stolley and S 9iapiro, 1989 Oral oral contraceptive users Obstet Gynecol, contraceptive use and hver cancer Am J Epidemiol, 95 261-266 130 878-882 Tzankova, V, V Petrov and N Danchev, 2010 Impact Schattir J A , M M Isley and M Woodward, 2010 Oral of oral contraceptives and smoking on artened contraceptives Vs injectable progestm m their effect and deep venous thrombosis A retrospective on sexual behavior Am J Obstet Gynecol, case-control study Biotechnol Biotechnol Eq, 203 545 el-545 e5 24 2026-2030 Int. J. Pharmacol, 2012

WHO, 1995 Venous thromboembolic disease and White, T , J K Jam and F Z Stanczyk, 2005 Effect of oral combined ocal contraceptives Results of versus transdermal steroidal contraceptives on mtemational multicentre case-control study World androgemc markers Am J Obstet Gynecol, health orgamzation collaborative stucfy of 192 2055-2059 cardiovascular disease and steroid hormone Wiegratz, I, K Mittmann, H Dietnch, T Zunmermann and contraception Lancet, 346 1575-1582 H Kuhl, 2006 Fertihty after discontinuation of Walsh, B W and F M Sacks, 1993 Effects of low dose treatment with an oral contraceptive containir^ 30 pg oral contraceptives on veiy low density and low of ethmyl estradiol and 2 ing of dienogebl Feibl density lipoprotein metabolism J Clm Invest, Steril,85 1812-1819 91 2126-2132 W>nn, V , J W H Doar and G L Mills, 1966 Some effect-, Webber, L S, S M Hunter, J G Bai^h, S R Srmivasan, of oral ccaitracepbves an serum-hpid and lipoprotein M.C Sklov diidG S Bereiison, 1982 Tlie mleraction levels Lancet, 288 720-723 of cigarette smoking, oral contraceptive use and Yeung, D L, 1976 Relationship between cigarette cardiovascular nsk factor variables m children The Smdcng, oral contraceptives and plasma \ iiamms \ Bogalusa Heart Study Am J Public Health, E, C and Plasma tnglycendes and cholesterol Am J 72 266-274 chn Nutr,29 1216-1221 Weiderpass, E, H O Adami, J A Baron, C Magnusson, A Lm(%ren and 1 Persson, IS'99 Use of oral contraceptives and endometnal cancer nsk (Sweden) Cancer Causes Control, 10 277-284 The Egyptian Journal of Medical Human Genetics (2012) 13 301 105

Ain Shams University The Egyptian Journal of Medical Human Genetics

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ORIGINAL ARTICLE Genotoxic damage in cultured human peripheral blood lymphocytes of oral contraceptive users

Falaq Naz '', Smita Jyoti ^, Nishat Akhtar '', Mohammad Afzal ^, Yasir Hasan Siddique *'*

* Human Genetics and Toxicology Laboratory^ Section of Genetics, Department of Zoology, Aligarh Muslim University, Aligarh 202002 UP. India ^ Department of Obstetrics and Gynecology, J N Medical College, Aligarh Muslim University, Aligarh 202002, UP, India

Received 18 May 2012, accepted 2 June 2012 Available online 29 June 2012

KEYWORDS Abstract Synthetic progestins and estrogens have been reported to be toxic in vanous expenmen Oral contraceptives. tal models Their prolonged use has been reported to induce cancer in humans In the present study Chromosomal aberrations, the effects of oral contraceptives were studied among users using chromosomal aberrations, sister DNA damage chromatid exchanges and DNA damage as a parameter, in cultured human peripheral blood lym Sister chromatid exchanges. phocytes The study was performed on 25 women (users) and 25 age match controls No significant Human lymphocytes difference was observed in chromosomal aberrations and DNA damage A significant increase was observed in sister chromatid exchanges (SCEs) Ceil among users The results obtained and the nsic of oral contraceptives (OCs) genotoxicity have been discussed © 2012 Am Shams University Production and hosting by Elsevier B V All rights reserved

1. Introduction peripheral blood lymphocytes [3 7] In these studies the geno toxic eiTects were observed at very lugh doses and, the reactive Oral contraceptives are tiie combination of estrogen and oxygen species (ROS) was suggested as a causative agent for progesterone They are used to treat vanous liormonal the genotoxic damage [8,9] The conversion of estrogen into disturbances, premenstrual syndrome and ovarian cysts [1] catechol estrogens and quinines, via redox reactions causes Tiie composition of pills vanes from country to country due oxidative damage to DNA [i 10] There are l>oth positive as to the responsiveness variation from individual to individual well as negative reports regarding the genotoxic effects of or one population to another [2] Earlier reports suggest the estrogens and synthetic progestins [11 13] In the present study genotoxic effects of synthetic progestins in cultured human an attempt has t)een made to investigate the possible genotoxic effects among oral contraceptives (OCs) users (females) by Corresponding author using chromosomal aberrations, sister chromatid exchanges E-mail address yasirhdsansiddique'o.redifrmailcom (Y H Siddique) and DNA damage as a parameter Peer review under responsibility of Am Shams University

m oc 0 — 2. Subjects and methods

0 0 -H -H 2 / Chemicals a

0 0 1) RPMI 1640, fetal calf serum, phytohaemdgglutinin-M, antibi g 0 otic antimytotit mixture (Invitrogen), 5-bromo-2-deoxyun- Vi 0 dme, Giemsa stain (Merck, India), Tns HCl, EDTA, Triton- g X, Tnchloro acetic acid and diphenylamine, colchicine (SRL, 2 D. u 0 India) U) t/5 -H 0 0 2 2 Human lymphocy te culture c CO -H -Vi 0 0 u A sample of hepannized venous blood was obtained from 25 women taking an OCs preparation containing levonorgestrel a •3 •g and ethinylestradiol as a content for 12 months as a means of kfl E ^ c 2 n. contraception following their first pregnancy Age matched 0 so ^ 0 0 a healthy 25 controls were evaluated along with the above sub en w jects A written informed consent was obtained from all sub 0 1/3 3 +1 0 0 jects Tlie mean age of 25 women using OCs was 28 5 years C c -H -H and of the 25 controls was 27 5 years Individuals answered a 0 -s ^ r* c a> questionnaire relating to lifestyle factors The OC users were •0 0 0 c :s not having any other addictions such as smoking, alcohol a 73 09 drinking etc nor have any history of disease Briefly, hepann a> s0 GO M 3 0 ized blood sample (0 5 ml), was obtained from each female ID g "> > 0 -^ and was placed in a stenle cultured tube containing 7 m] of u ^ 0. RPMI 1640 medium, supplemented with fetal calf serum •^ T^ u a r- rj (1 0 ml), antibiotic-antimycotic mixture (1 0 ml) and phytohae- UJ 0 0 ^c 1/1 magglutinin-M (0 1 ml) The culture tubes were placed in the 0 0— " 0^ " 0 -H incubator at 37 °C for 48 h [14] +1 -H •5 rN 00 0 r- so w- 0 0 0 V3 •0 2 3 Chromosomal aberration analvsis u >> rt u g •" 0 After 47 h, an amount of 0 2 ml of colchicine (0 2 ng/ml) was ex 00 0 E U Xi — (N added to the culture tubes Cells were centrifuged at 800 g for rv^| <—N 10 min TTie supernatant was removed and 8 ml of pre m •^ f^ -a' 00 warmed (37 °C) 0 075 M KCI (hypotonic solution) was 0 -H 0 0 J03 c -H -H added Cells were re-suspended and incubated at 37 °C for „ "T Tf a «u so sO 15 mm TTie supernatant was removed by centnfugation, at

j= s 800 g for lOmin, and subsequently 5 ml of chilled fixative Q, Ui c (methanol glacial acetic acid, 3 I) was added The hxative a>

2 5 Quantitative assavjor DMA Jragmentation '6 Statiitual analvsis

Quantitative assay for DNA fragmentation was performed Student's / test was used for the analysis ol data obtained for according to the protocol of Burton [17] After mcubation of chromosomal aberrations and sister chromatia excnanges and 48 h, the cell suspension containing 1-10 x 10* cells m a 1 ml X' (Chi-square) was applied for the analysis ol DNA damage volume was prepared for each donor About, 0 8 ml of cell sus pension was transferred to a micro centnfuge tube and 0 7 ml 3. Results and discussion of icc cold lysis buffer (5 mM Tns CI, pH 8 0 20 mM EDTA/ 0 5% (v/v Tnton X-lOO) was added The tubes were vortexed Tlie frequency of cells with aberration in OC users was almost and allowed to lyse for 30 mm at 4 °C The tubes were centri- similar to their age matched controls Gaps and breaks of chro fuged for 15 mm at 15,000 g (4 °C) and the supernatant was mosome as well as chromatid were observed but dicentric ex transferred to a labeled conical glass tube About 0 65 ml of changes and rearrangements were not observed The mean 5% tnchloroacetic aad (TCA) was added to the pellet in a mi values obtained were not statistically significant (Tahle I) A cro centnfuge tube and 1 5 ml of 10% TCA to the sample in a significant difference {P > 0 005) in the number of SCEs cell labeled glass tube The sample was precipitated overnight at was observed between OC users (3 9 J- 0 076) and their agt 4 °C The tubes were centnfuged for 10 min at 2500 g at room matched control (2 62 ± 0 068) (Table 2) The mean value for temperature and the supernatant was removed and about the DNA damage was (0 1172 ± 0 0020) in OC users and 0 65 ml of 5% TCA was added to the pellet The tubes were (0 1148 ± 0 0025) in controls The values were not staiislicallv boiled for 15 mm at 100 °C in a water bath After coohng significant (Table 3) The results of the present study suggest down to room temperature the sample was centnfuged at that the OC users have significantly higher Irequencies of 2500 g for 5 mm About 0 5 ml of each supernatant (from both SCEs/cell, but the CAs and DNA damage wert not significant glass and micro centnfuge tubes) was added to the labeled An increase in the frequencies of SCEs and CAs has been rt glass tube About, 1 ml of diphenylamine reagent was added ported dunng pregnancy [19 20] The concentration ot the to each tube and was incubated for 4 h at 37 °C [18] Finally estrogens is increased dunng pregnancy, which may be the pos the absorbances were noted at 600 nm in a spectrophotometer sible reason of an increase in SCEs [21] Earlier studies per and the results were expressed in the percentage of DNA frag formed on the genotoxic potential of steroids have shown to mented as follows cause chromosomal damage, induction of SCEs and formation absorbance of supernatant of endogenous adducts [7 22 24] The results of the present %Fragmented DNA = dbsorbance of supernatant + pellet study show that the OCs are not potent in inducing DNA

Table 2 Distribution of Sister chromatid exchanges (SCEs) among women using oral contraceptives (OCs) Table 3 Estimation of DNA damage among oral contracep Subjects SCE per cell tives (OCs) users and non-users OC users Control Subjects OC users Control 1 42 23 1 0 11 0 10 2 41 25 2 0 12 0 II 3 43 29 3 0 12 0 12 4 39 27 4 0 13 0 II 5 35 24 5 0 12 0 12 6 36 23 6 0 10 0 13 7 37 26 7 Oil 0 13 8 33 31 8 0 13 0 12 9 37 22 9 0 14 0 11 10 38 23 10 0 12 009 II 41 24 11 Oil 0 08 12 40 25 12 0 10 0 II 13 32 32 13 Oil 0 12 14 32 29 14 0 12 0 II 15 33 28 15 Oil 0 12 16 42 27 16 0 13 0 13 17 4 1 32 17 0 13 0 11 18 43 22 18 0 12 0 12 19 44 28 19 0 12 0 12 20 40 27 20 0 12 0 13 21 43 24 21 Oil 0 12 22 42 23 22 0 12 0 II 23 41 28 23 Oil 0 10 24 39 21 24 0 10 0 12 25 44 33 25 0 12 0 n Mean ± SE 3 91 ± 0 076* 2 62 ± 0 068 Mean ± SE 0 1172 ± 0 0020 0 1148 ± 0 00252 P > 0 005 NS (Non significant) F Na/ ei al

damage/chromosomal aberrations, but are able to increase the [6] Siddique YH, Ara G, Beg T, Afzal M Genotoxic potential ot number of SCEs/cell The study performed by Bin et al [25] also medroxyprogesterone acetate in cultured human peripheral blood showed the mcrease m SCEs/Cell in OC users The SCEs are the lymphocytes Life Sci 2006,80 212-8 cytological manifestation of interchanges between DNA repli [7] Joostcn HFP, van Acker FAA, van den Dobbclstecn DJ, Horbach cation products at apparently homologous loci It is more sen GJMJ, Krajnc EI Genotoxicity of hormonal steroids Toxicol sitive mdicator of genotoxic effects than structural aberrations Lett 2004,151 113^34 [8] Siddique YH, Afzal M EvaluaUon of genotoxic potential of [26,27] The induction of SCE is also correlated with the induc synthetic progestin chlormadinone acetate Toxicol Lett tion of cancer [28] Besides various reports on the genotoxicity 2004,153 221 5 of steroids some of the progestogens have been studied in short [9] Siddique YH, Beg T, Afzal M Genotoxic potential of ethylest- and long term toxicology studies in rodents and dogs or mon radiol in cultured mammalian cells Chem Biol Interact keys for their carcinogenic activity, no significant or unusual 2005,151 141-4 toxicities were reported [29] In some cases the oral contracep [10] Siddique \II, Beg T, Afzal M Aniigenotoxic effects of ascorbic tives have also been suggested to be having a role in prevent acid against megeslrol acetate induced genotoxicity in mice Hum ing ovarian cancer by both suppressing ovulation and altenng Exp Toxicol 2005,24 121-7 the timior promoting milieu [30] The results obtained in the [II] Littlefild LG, Mailhes JB Comparison of chromosome breakage present study are contrary to the studies performed by other in lymphocytes and fibroblasts from control women taking oral workers [13,31,32] This may be due to the new hormonal contraceptives Fertil Stenl 1975,26 828-32 [12] Hundal BS, DhiUon VS, Sidhu IS Genotoxic potential of formulations and preparations developed in an attempt to re estrogens Mutat Res 1997,389 173^81 duce the adverse effects of OCs, such as the reduction in the [13] Dhillon VS, Singh JR, Singh H, Kler RS In vitro and in vivo estrogen content Estrogen such as ethinylestradiol, in the li genotoxicity evaluation of hormonal drugs v mestranol Mutat ver undergoes aromatic hydroxylation and the product, 4- Res 1994,322 173-83 hydroxyestrone, 3,4-dihydroxy 1,3,5 (10)-oestratien-17-one [14] Cdrballo MA, Alvarez S Bove^^ A Cellular stres^ by light and (4-OHE) IS carcinogenic in male Syrian golden hamster kid Rose Bengal in human lymphocytes Mutat Res 1993,288 ney tumor model [33,34] The genotoxic potential of steroids 2d IS determined by the metabolic conditions in the test system [ 15] IPCS International programme in chemical system. Environmen and the human body The extrapolation from the expenmen- tal health criteria 46, guidehnes for the study of genetics effects in tal data to humans is not only difficult but also complex The human populaUons, vol 46 Geneva WHO, 1985 p 25^54 most of the studies conducted for genotoxic potential of ste [16] Perry P, Wolff S New Geimsa method for the differential staining of sister chromatids Nature 1974.251 156-8 roids involve the concentration in microgram per ml range [17] Burton KA Study of the condition and mechanism of the whcred!> the therapeutic plasma concentration ranges from diphenyiamine reaction for the colonmcinc estimation of deoxy nanogram or picogram per ml [7] The present study was con ribonucleic acid Biochcm J 1956,62 315-23 ducted on the women taking OCs for one year and the in [18] Siddique YH, Ara G, Beg T, Afzal M Assessment of cell viability, crease in SCEs may be due to the variable hormonal profile lipid peroxidation and quanufication of DNA fragmentation after among them Further, the occupation and work place, poly the treatment of anticancerous drug Mitomycin C and curcumin morphic metabolizing genes and efficiency of DNA repair can in cultured human blood lymphocytes Exp Toxicol Pathol also affect the resjwnse against OCs 2010,62 503-8 [19] Murthy PBK, Prema K Further studies on sister chromatid exchange frequency in users of hormonal contraceptives Mulat Res 1983.119 351 4 Acknowledgements [20] Ghosh R. Ghosh PK Sister chromatid exchanges in the lympho cytes of control women, pregnant women and women taking oral The authors are thankful to the University Grant Commission contraceptives effects of cell culture temperature Environ Mol (UGC), New Delhi for the award of project entitled "Biochem Mutagen 1988,12 179-83 ical and Cytogenetic effects of Oral Contraceptives among [21] Sharma T, Das BC Higher incidence of spontaneous sister women of different reproductive histones" {FNo 39-582/ chromatid exchanges (SCEs) and X-ray induced chromosome 20I0(SR)} to Dr Yasir Hasan Siddique, Department of aberrations in peripheral blood lymphocytes during pregnancy Zoology, Aligarh Muslim University, Ahgarh Mutat Res 1983,174 27 33 [22] Blakey DC, White INH Unscheduled DNA synthesis caused b> References norethindrone and related contraceptive steroids in short-term male rat hepatocyte culture Carcinogenesis 1985,6 1201 5 [I] SperofT L, Glass RH, Kase NG, editors Oral contraception In [23] Shimomura M, Higashi S, Mizomoto R 32P-postlabeling analysis Clinical gynecologic endocruiology and infertility Lippincott of DNA adducts in rats during estrogen-induced hepatocarcmo- Williams & Wilkins, 1999 p 867-944 genesis and effect of tamoxifen on DNA adduci level Japanese J [2] Goldzieher JW Selected aspects of the pharmacokinetics and Can Res 1992,83 438-44 metabolism of ethinyl estrogens and their clinical implications [24] Yager JD, Fifield DS Lack of hepatogenotoxiaty of oral Am J Obst Gynecol 1990,163 318-22 contraceptive steroids Caranogenesis 1982,3 625-8 [3] Kochhar TS Steroid hormone enhanced sister chromatid [25] Bin A, Civeiek E, Karahahl B, Sardas S Assessment of DNA exchange m cultured CHO cells Expcnentia 1988,44 62-3 damage in women using oral contraceptives Mutat Res [4] Shyama SK, Rahiman MA Genotoxicity of lynoral (ethynilest- 2002.521 113-9 radiol, an oestrogen) in mouse bone marrow cells, in vivo Mutat [26] Tucker JD, Preston RJ Chromosome aberrations, micronuclei, Res 1996,370 175-80 aneuploidy, sister chromatid exchanges, and cancer risk assess [5] Siddique YH, Afzal M Genotoxic potential of cyproterone ment Mutat Res 1996,365 147-59 acetate a possible role of reactive oxygen specnes Toxicol In [27] Tucker JD, Aulleta A, Cimino MC, Dearfield KL, Jacobson vitro 2005,19 63-8 Kram D, Tice RR, et al Sister chromatid exchanges second Genotoxjc damage in cultured human peripheral blood lymphocytes of oral contraceptive useri. 305

report of the gene tox programme Mutat Res 1993,297 [31] Fndnchova I Cytogenetic study of blood in women who had used 101-80 oral contraceptives Neoplasm 1990,37 545 53 [28} Gebhart E Sister chromatid exchange (SCE) and structural [32] Pinto MR Possible effects of hormonal contraceptives on human chromosome aberrations in mutagenicity tcbtmg Hum Genet mitotic chromosomes Mutat Res 1986,169 149 57 1981.58 235-54 [33] Liehr JG, Fen WR, Sirbasku DA, Ari-Ulubelen A Carcinoge [29] Jordan A Toxicology of progestogens of impUnuibic contracep nicity of catechol estrogens in Syrian hamsters J Sicroiu BKx.ni.in tives for women Contraception 2002;65 3-8 1986,24 353-6 [30] Siskmd V, Green A, Bam C, Purdie D SisWmd beyond ovulation [34] Li JJ, Li SA Estrogen cartinObenesi-, m Synao hamster ti-,iue^ oral contraceptives and epitheUal ovanan cancer Epidemiology role of metabolism Fed Proc 1987,46 1858-63 2000,11 106-10 Pakistan Jcnimal of Biological Sciences, 2012 ISSN 1028-8880 / DOI 10 3923/pjbs 2012 © 2012 Asian Network for Scientific Information

Lipid Profile of Women Using Oral Contraceptive Pills

'F Naz,'S Jyoti,^ Akhtar,'M Afzaland'Y H Siddique 'Human Genetics and Toxicology Laboratory, Section of Genetics, Department of Zoology Ahgarh Muslim University, Aligarh-202002 (U P), India 'Department of Obstelricb and Gynecology, J N Medical College, Ahgarh Mubhm Univcrbity Ahgarh- 202002, (U P) India

Abstract: Oral Contraceptives (OCs) are the most popular type of birth control pills The study was designed to examine tlie biochemical charges which occui due to tlie use of oral contiaceptive piUh \,OC^i Tlie ^tudv Wd^ based on the questionnaire for having the information of any reproductive history fasting, age, health, nature of menstrual cycle, bleedu^, disease etc and blood profilir^ for biochemical anaij-sis of the women mcluaes High Density Lipoprotem Cholesterol (HDL-C), Low Daisity Lipoprotem Cholesterol (LDL-C), Total Cholesterol (^TC; and Tnglycendes (TG^ Lipid profiling was earned out by using a commerciall) available diagnostic test kits SPSS was used to analyze the data The results showed statistically significant differenceb among useis of OCs compared to non-users Total cholesterol (242 92i2 842 mg dL" ), HDL-C (58 65± 1 098 mg dL » LDL-C (115 84±1 266 mg dL~') and tnglycaides (105 56tfc2 341 mg dL"') were significantly higher compared to the Non-usere (Total cholesterol 218 4S^1 762, HDL-C 48 17ztO 543, LDL-C 100 32±0 951 and tnglycerideb 8377i2 299iiigdL~') The result suggests that OCs mcrease the level ofHigh Density Lipoprotem Cholesterol (HDL-C), Low Density Lipoprotem Cholesterol (LDL-C), lotal Cholesterol (IC) and Inglycendes (IG)

Key words: Oral cQutracepUves, liealtliy womeii, biochemical pdrdiueterb

INTRODUCTION reported (Thorvaldsen et al, 1995 Vessey et at 1984 WHO, 1996; and other studies showed diat orai From the last few decades, world is facing the most contraceptives also mcreases the nsk for venous senous problem that is population explosion Uiiomboembohbm (Famiei and Ldwieiiboii, 1998, Wei^-, Contraception method IS used worldwide for over birth 1999, Westhoff, 1998, Rosing and Tans, 1999 control or unwanted pregnancies Many workers have Kemmeren ef a/, 2001) The relationship betweer. ural si^gested that the use of contraceptives is beneficial but contraceptive users and the nsk for breeist cancer also have some side effects too The researchers believe remains controversial Moit itudie!> ûggesti, no that the widespread use of hormonal contraceptive oversdl nsk or a shght mcreased nsk of breast cancer m provides an opportunity for assessing the influence of women using oral contraceptiv es (Ânonymous 1986 estrogens and progesterone on vanous biochemical Marchbanks et al, 2002, Bnnton et al, \ 998 parameters among users Synthetic progesUns not only Hankinson et al, 1997; It has been reported thai the have genotoKic potertial (Siddique and Afzal, 2(X)4,2(X)5a, serum total cholesterol is significantly higher among oral Siddiqueefa/,2(XJ5a,b, 2006a, b) but are also vulnerable contraceptive users as conpared to non users to vanous types of cancer (Siddique and Afzal, 2(X)8, (Nawrot et al, 2003) The mocfe of action dep>ends upon Siddique and Afzal, 2005b) It was found that the bone the formulaticm of pills The pills possibly mliibits formation was sigmficantly decreased m women taking ovulabon by the fcxmation of pituitary luteinizing oral contraceptive pdls (Gamero et al, 1995) Some hormone that renders the cervical mucus hostile to sf»mi remarkable findu^s are well documented by woikers penetrabon The oral contraceptive pdls are the which showed that oral contraceptives are mvolved m ccmibination of different concentrations of estrogen and many diseases such as, myocardiac infracbon and prcêstin. In our present study the composition of the carcmogemcity (,Obisesan ef a/, 2002, Tzankova e/^ a2 , oral contraceptive taken by the women was Etiiinyi 2010, Naz et al, 2012) An mcrease m the nsk for ischemic Estradiol (EEj) (0 03 mg) and Levoncogestrel (0 1 5 mg) stroke m women takii^ oral contraceptives has been In the present study an attempt lias been luaut. (u

Corresponding Antfaor Y H Siddique, Human Genetics and Toxicdt^'Laboratory', Section of Genebes Department of Zoc4ogy, Aligarh Mushm Uraversity, Aligarh-202002 (U P), India 1 Pak. J. Biol. Sci., 2012

investigate the piossible effects of Oral Contraceptives It has been observed that the LDL-C and HDL-C (OCs) among users on High Density Lipqsrotein UKrease is prcme to have stroke and myocardial Cholesterol (HDL-C), Low Density Lipoprotein infarction in women taking oral contraceptives Cholesterol (LDL-C), Total Qiolesterol (TC) and (Wynn et al, 1966). The present study supports tlie Triglycerides (TG). earlier studies on lipid profiles on cholesterol (Emd^jae et al, 2010, Abdel-Bany etal, 201V). Elevated MATERIALS AND METHODS serum levels of lipids are probably the most important biochemical risk factors for atherosclerosis. In the liver Sample size: The subjects for the study were married friglyceride synthesis is enhanced by esfrogen and women of low income group to a total of 100 participants inhibited by androgen and these triglycerides are partly from Malkhan Singh Hospital and Jawahar lal Nehru broight into the circulation as low-density lipoproteins. medical college of Aligarh city, who were of 21 to 40 years When a contraceptive pill containing an estrogen and a of age, from which 55 women were taking combined oral progestogen is introduced, the resultant effect is not contraceptives preferring after 8-10 days of withdrawl predictable. The clinical significance of this observation paiod and 45 non-users healthy married women who were not takir^ any oral contraceptive. A random sampling is supported by the fact that atherosclerosis begins early were performed and the women taking the oral in life and is accelerated in the presence of high contraceptive continuous for the three months were cholesterol and serum triglycerides (Newman etal, 1991). included for the comparison to non-users. A written Contraceptive steroids having a combination of an consent was obtained from the subjects for taking their estrogen and progestin or only progestm are effective and blood samples. The detailed questionnaire included some reva^ibly regulate fertility. Combined oral ccxitraceptives issues about fasting, age, health history, nature of afTects a variety of metabolic factors includir^ hemostatic menstrual cycle, bleedir^, disease, etc. variables and esfrogen-sensitive Uver proteins and these effects can be modulated by the type of estrogen and BkMid sampling and processing: In morning as overnight progestin in a given canbination (Sitruk-Ware and Nath, fasting of user and non-user WOTnen, about 4 mL blood 2011). The estrogen component of OCs has been reported was collected in vacutainer tube with clot activator from to increase the production of Very Low Density each women in study sample. The serum samples were Lipop-otein (VTDL) aixi High Density Lipop-otein obtained by centrifugation at 3000 rpm for 15 min and the (HDL) but reduce the level of low density sera were stored at -20^C and analyzed within three days. lipoprotein (Sitruk-Ware, 2006). Tlie reducticm in the doses of Levonoigestrel/ethinyl estradiol containing Blood parameters analysis: The analysis of lipid profile of contraceptives from 30-20 ng (EE) and that of 150-100 )ig High-Density Lipoprotein (HDL-C), Low-Density Lipoprotein LDL-C, cholesterol and triglycerides were TAIcl: Lipid profile of oral contraceptive (OC) users and non-users women carried out using a commercially available diagnostic Lipid profile (ms dL '2L- OC users (n = 55) Non-users (n = 45) system test kits (Crest Biosys terns kits, India). Cholesterol 24292±1842' 2ia49±1.762 HDL-C 58.65±L09ff 48.17i0.543 LIX.-C 115.»4±1.26e 100.32t0.951 Statistical analysis: The obtained data were analyzed by Trialvcerides 105.5

(levonorgestrel) resulted in tiie less adverse effect on Anorônous, 1986 Oral-contraceptive use and the risk of the lipid profile and have been suggested to lower the breast cancer The cancer and steroid hormone btudj incidence of thrombosis as compared to other EE based of the centers for disease control and the national contraceptives (Skouby et al, 2005; OCs containing institute of child health and human development levonorgestrel was associated with an almost four times N Ei^l J Med, 315 405-411 higher nsk of venous thrombosis as compared to non- Avins, A L , R J Haber and S B Ilulley 1989 The .tatu^ usere (VanHylckama Vlieg efa/,2009) The intake of of hypertnglycendemia as a nsk factor for coronary 100 Jig levonorgestrel with 20 \i% ethinyl estradiol to 28 heart disease Clm Lab Med, 9 153-168 women whom were at the nsk of pregnancy shows a httle Brmton, L A, D R Brogan, R J Coates, C A Swanson change in hpid profile as compared to higher dose oral N Pobsdiman and J L Stanford, 1998 Breast cancer contraceptive preparation (Young and DelConte, 1999) nsk amoi^ women under 55 years of age b> joint Despate the changes m lipid profile among users of higher effects of usage of oral contraceptives and hormone replacement therapy Mencpause, 5 14S-151 dose oral contraceptive stents, there is little and no effect on the loi^ term nsk artherogemc cardiovascular disease Emokpae, MA, PO Uadia and HB Osadolar 2010 (Stampfer et al, 1990) The status of elevated seiiim Effect of duration of use of hcrmonal contraceptive pills on total hpid and lipoproteins m Nigenan triglycende level may be an mdependent predictor of women Int J Pharm Bio Sci, 1 1-5 coronary heart disease as ethutyl estradiol mcreases Farmer, R D and R A LawrensMi, 1998 ()ral heptatic secretion of triglyceride nch lipoprotem contraceptives and venous thromboembolic disease (Avins etal,l989; Schaefer etal,\983) The prcêstm The findings from database studies ui the T'nited component of the OC may oppose this increase The Kingdom and Germany Am J Obstet Gynecol mjectable oral contraceptives have also been reported to 179 S78-86 increase the levels of tnglycendes, total cholesterol, High Gamero, P , E Somay-Rendu and P D Dehnas 199^ Density Lqxjprotem (HDL) cholesterol in the serum Decreased bone turnover m oral contraceptive users (Godslande/a/,1990) Bone, 16 499-503 Godsland, I F , D Crook R Simpson, T Proudler and CONCLUSION C Felton et al, 1990 The effect* of different formulations of oral contraceptive ênts on hpid The lipid profile depends on the brand of OCs or and carbol^'dratc metabolism N Eng! J Med duration of their use The users m our present stural Commission (UGC), New Delhi for the award of prqect contraceptives and the nsk of breast cancer 1\ entitled. "Biochemical and C5^togenetic effects of Oral Ei^l J Med, 346 2025-2032 Contraceptives among women of different reproductive NawTot, TS, ED Hond, RH Fagard histories" {FNo 39-582/2010(SR)} to Dr Yasir Hasan K Hoppenbrouwers and J A Staessen, 2CW3 Blood Siddique, Department of Zoology, Aligaih Muslim pressure, serum total cholesterol and contraceptive Umversity, Aligarh. pill use m 17-year-old girls Iiur J Cardiovasc Prevention Rehab , 10 438-442 REFERENCES Naz, F, S Jyoti, M Afzal and YH Siddique 2012 Biochemical effects of oral ccsitraceptiv e aiiiong Abdel-Bany, J A, MS Flafl, LM Al-Namaa and users A review Int J Pharmacol, 8 314-320 Nevwnan, W P, W Bao, W Wattigney and N A Hassan, 2011 Lipoprotem changes m women G S Berenson, 1991 Autopsy studies m United takii^ low-dose combmed oral contraceptive pills A States children and adolescents Relationship ot nsk cross-secbonal study m Basra, Iraq East Mediterr factors to atherosclerotic lesions Ann N Y Acad Health J, 17 684-688 Sci,623 16-25 Pak. J. Biol. Set, 2012

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