Chapter 3
REVIEW OF LITERATURE
3.1 Norethindrone
Norethindrone is 17α-ethinyl-17β-hydroxy-4-estren-3-one. It is also known by its chemical names like norpregneninolone and norethisterone. It is commonly used in some combined oral contraceptive pills, progestogen only pills and is also available as a stand-alone drug. It is white to creamy white in color, odorless, non-hygroscopic, crystalline powder. Norethindrone was the first orally highly active progestin to be synthesized. It was first synthesized by chemists namely Luis Miramontes, Carl Djerassi, and George Rosenkranz at Syntex in Mexico City in 1951 It was the progestin used in one of the first three oral contraceptives. Its related ester, norethisterone acetate, is used for the same indications.
3.2 Norethindrone acetate
This medication is used to prevent pregnancy. It is often referred as “mini-pill” because it does not contain any estrogen. It prevents pregnancy by making vaginal fluid thicker and prevent s sperm from reaching to egg fertilize and also changes the lining of the uterus to prevent the attachment of a fertilized egg. It also stops the release of ovum ( ovulation ) in about half of a woman’s menstrual cycles. While the “mini-pill” is more effective than other methods of birth control such as condoms, cervical cap, diaphragm etc. It is less effective than combination hormone i.e. estrogen and progestin birth control pills because it does not consistently prevent ovulation. It is usually used by women who cannot take estrogen.
It’s a synthetic progestogen which can be used to treat premenstrual syndrome, painful periods, irregular periods, abnormal heavy bleeding, menopausal syndrome (in combination with estrogen), or to postpone period (Lester et al., 2004). It is also commonly used to help prevent uterine hemorrhage in complicated non-surgical or pre-surgical gynecologic cases and also to prevent uterine hemorrhage in pre-surgical gynecologic cases. It also prevents ovulation when administered from fifth to twenty-fifth day of the cycle.
The endocrine disrupting phenomenon was brought to scientific community during the 1980s when deformities in fish were observed across the European rivers. Among the group of endocrine disrupting compounds, natural and synthetic estrogens are considered the most potent estrogenic compounds [1]. Even at concentrations as low as1 ng/L, steroidal hormones can have endocrine-disrupting effects on aquatic organisms, such as decreased fertility, feminization, and hermaphroditism [2–4]. Other recent studies have shown the influence of these substances on the reproductive and immune systems of humans [5, 6]. Special attention has been given to the natural estrogens like estradiol and estrone, as well as to the synthetic estrogen 17-alpha-ethinylestradiol, because of the strong endocrine-disrupting potency of these molecules (they are actually designed to be strongly estrogenic). There is no information available in the literature on the potential adverse environmental effects of the synthetic progestins (norethindrone, levonorgestrel, and medroxyprogesterone) used in various hormone therapies (contraception, the treatment of endometriosis and infertility, and hormone
Replacement therapy (HRT)) [7]. Synthetically produced progestagens are also called gestagens or progestins and are derived from the C21-steroid pregnane.
It is estimated that 128 million birth control pills and 107 million hormone replacement therapy doses are consumed in the province of Quebec, Canada (7.7 million people) each year [8]. Human and animal excretion is considered the main source of estrogen and progestogen (natural and synthetic) occurrence in aquatic ecosystems [9, 10]. Steroids, which are continually being discharged into the environment, are also present at concentrations of toxicological concern, and have now been shown to reach drinking water sources [11–19]. The potential effects to human health and the ecological impacts of these natural and synthetic hormones are a growing concern, and have recently become the focus of scientific research [20–26].
Progesterone is the only naturally occurring progestagen, secreted by the corpus luteum in the ovary and also by the placenta during pregnancy. In contraceptive pills, synthetic progestagens, derived from nortestosterone, norethindrone or hydroxyprogesterone (hydroxyP), are used alone or together with estrogens. The doses of progestagens normally administered exceed those of ethinylestradiol (EE2). Furthermore, synthetic progestogens are also used in treatment of other conditions (e.g., menstrual disorder, infertility and endometriosis).
It is crucial to be able to understand and predict the fate of these substances in environmental ecosystems, and consequently to optimize wastewater and drinking water processes, by establishing their occurrence using sensitive and reliable methods. However, because of the low limits of detection (around 1 ng/L) corresponding to the concentrations of these compounds known to affect living organisms, analyzing them remains a significant challenge, and labor-intensive analytical procedures (sample preparation and solid-phase extraction) are usually needed to accurately measure their occurrence.
Hormones are excreted by humans as glucoronides and sulfates which are water-soluble but after treatment in waste water treatment plants most glucoronides are deconjugated due to the presence of natural fecal bacteria which produce large quantities of β-glucoronidase [27]. It has also been suggested that the removal of estrogenic compounds in WWTPs is not by biodegradation but by transfer from the water phase to sludge as they are rather lipophilic [28]. The potential contamination of soil with hormones may be caused by the application of digested sludge from WWTPs on agricultural fields [29].
3.3 MECHANISM OF HORMONAL ACTION
Norethindrone or Norethindrone acetate mimic’s the endocrine properties of their natural hormonal counterpart progesterone, which is a vital female sex steroid hormones that regulate numerous biosynthetic and metabolic events. Estradiol which is another vital sex hormone in females. The principal function of estradiol is to promote proliferation and growth of specific cells in sex organs and other reproductive tissues. As such, estradiol plays critical roles in female sexual maturation and reproduction by initiating epithelial proliferation of the vagina, uterus, and breast [30]. Progesterone modifies and redirects cellular growth and the biosynthetic activity of estradiol by promoting secretory changes in the endometrium during the latter half of the female sexual cycle, thereby preparing the uterus for implantation of a fertilized ovum [31].
Progesterone also prevents ovulation and stimulates the lobules and alveoli of the breast to proliferate, enlarge, and become secretory in nature. Steroid hormones exert their biological effects by inducing a profound increase in RNA and protein synthesis. Steroid hormones enter most cells by passive diffusion where they bind to specific cytosolic and nuclear receptors. Once bound, the receptor undergoes an allosteric conformational change converting the receptor complex from an inactivated form into an activated form. The activated receptor complex has a high affinity for specific nuclear sequences referred to as steroid response elements, which have characteristics of classic enhancer elements [31]. Binding of the steroid–receptor complex typically results in gene activation (i.e., gene transcription) and increased mRNA synthesis. mRNA is then translated on cytoplasmic ribosomes thereby increasing the expression of hormone responsive proteins, which in turn alters cell function, growth, and/or differentiation [31].
3.4 SPECIES DIFFERENCES IN SENSITIVITY TO SEX STEROID HORMONES
Administration of synthetic steroid hormones into experimental animals yielded considerable amount of interspecies variation in physiological responses due to potency differences between hormonal agents, differential pharmacokinetic patterns, and unique hormonal physiology (sensitivity) of each species. These interspecies differences should be considered when evaluating toxicology data for steroid hormones. Combinatorial oral contraceptives usually contain estrogenic and progestogenic agents within a certain ratio in each species in order to achieve the desired suppression of fertility, yet to still maintain physiological homeostasis. For example, using the decidual response of the uterus as a benchmark, the optimal human estrogen/progestin ratio (E: P ratio) is between 1: 5 and 1: 80, similar to that of the monkey. In contrast, the optimal E: P ratio for dogs is approximately 1: 1000 to 1: 3000, while in the rat it is around 1: 10,000 to 1: 20,000 [32-33]
These species differences are due in part to dissimilar patterns of hormonal cycling in each species. Species with low E: P ratios (i.e., rat) given oral contraceptive formulations experience a greater disruption of hormonal homeostasis than species with a high E: P ratio. Therefore, administration of oral contraceptive formulations to dogs or rats tends to grossly overdose with an estrogenic stimulus, as well as augment the progestin response. In addition, a specific tissue response to exogenous hormones also varies among species. For instance, mammary tissue in dogs is highly sensitive to progestins, while the human uterus is considerably more sensitivity to estrogens. In light of such species differences in hormonal physiology, two toxicological issues emerge: (1) data obtained in rodents and dogs should be viewed conservatively; and (2) the monkey represents the best animal model for predicting human responses to synthetic hormones.
3.5 TOXICOLOGY OF ESTROGENS AND PROGESTINS
3.5.1 Acute toxicological studies
It was found that estrogen and progestins possess low to moderate acute toxicological effects in humans as well as in animals [34]. The acute LD50 of EE and NA in rodents ranges from 0.5 to >5 g/kg (35, 36-37). Deaths of rodents given large acute doses of EE or NA have been attributed to liver and kidney failure. Premonitory clinical signs include apathy, abnormal breathing and gait, emaciation, and eventually, convulsions. Simultaneous exposure to EE and NA does not appear to substantially alter the acute toxicity of either agent. There are very little toxicologically relevant gender or species differences in the acute toxicity of oral contraceptives. The low acute toxicity of these agents in experimental animals is consistent with the lack of toxicity seen in humans, other than occasional vomiting in children who have accidentally ingested large quantities of oral contraceptives [38].
3.5.1.2 Multi-dose Studies
They are also known as Subacute and/or subchronic studies. There are no published studies available that specifically in vestigate the subacute or subchronic toxicity of a combination of EE and NA. However, based on early time points in long-term studies of EE and NA in combination, there appear to be no major adverse effects, other than the expected pharmacological actions. The results of long-term studies are discussed in the
Chronic /carcinogenicity section.
3.5.2 Chronic toxicity and carcinogenicity studies
Given such species differences, the chronic toxicity and carcinogenicity of combinations of EE and NA are presented separately for the rat, dog, and monkey. Since oral contraceptives are taken for extended periods of time and estrogenic and progestogenic agents act in concert with each other, emphasis was placed on studies that investigated the effects of co-administration
Of EE and NA.
3.5.2.1 Rat
Chronic administration of EE and NA to rats alone or in combination is associated with predominantly exaggerated endocrine stimulation. Albino rats fed a diet containing Norlestrin (a combination of EE/NA, 1: 50) at doses between 0.3 and 4 mg/kg-day for 2 years resulted in dose-related growth retardation, transient alopecia, mastopathy, liver hyperplasia, and gonadal atrophy [40]. A similar spectrum of effects was observed in rats administered the same doses of NA alone or EE at doses 50-fold lower [40-41]. However, both of these agents whether given alone or in combination appeared to have a positive influence on the health of the rats with longer survival, longer time to tumor formation, and decreased incidence and severity of degenerative aging diseases [41].
The low chronic toxicity of EE and NA is consistent with the lack of changes in organ weight, function, or a combination NA and the methylether form of EE at 0.2 mg/kg-day [43]. However, higher doses (1 and 2 mg/kg-day) led to increased organ/body weight ratios (liver, testes, thymus, ovaries, uterus) in the absence of hematologic or histopathological correlates. The ability of certain estrogenic and progestogenic agents to induce tumors in rodents has long been known [44]. High-dose administration of various estrogenic and progestogenic agents alone or in combination to susceptible strains of rodents has been shown to increase the incidence of specific tumors in the pituitary, uterus, breast, ovary, and liver [46-47].
3.5.2.2 Dog
Chronic studies with estrogen/progestin combinations (0.8–1.0 mg/kg-day) elicited various effects including pyometra, uterine perforation, alopecia, thickened and wrinkled skin, decreased hemoglobin/hematocrit, and mammary tumors [48]. Induction of mammary tumors in dogs by various combinations of estrogenic and progestogenic compounds has not been seen in all studies [49]. High doses of potent estrogens or progestins with estrogenic properties also appear to promote tumors in the smooth muscle of the uterus and vagina and occasionally in the transitional epithelium of the bladder.
Like with other estrogenic/progestogenic formulations, the most prominent effects of long-term administration of EE and NA (1: 20) to dogs are alopecia, cystic endometrial hyperplasia, and severe pyometra [50]. These effects occurred at doses ranging from 21 to 525 ¹g/kg-day. However, treated dogs were in good health throughout the study, had lower cholesterol levels than controls, and were without evidence of mammary or hepatic nodules or tumors. There were, however, effects on the hematological system with a dose-related decrease in erythrocyte counts, packed cell volume, and hemoglobin [50].
3.5.2.3 Monkey
The results of long-term monkey studies indicate that overall, long-term treatment with high doses of progestins, estrogens, or their combinations fails to produce significant signs of systemic toxicity or tumor development [51-52]. Female rhesus monkeys treated with Norlestrin (EE/NA, 1: 50) up to 2.55 mg/kg-day for 10 years displayed no significant signs of toxicity or tumorigenicity [53]. Treatmentrelated effects were confounded by the effects of aging and consisted of exaggerated pharmacological symptoms of oral contraceptive exposure, such as uterine and ovarian atrophy and mammary dilation. All doses were well tolerated and survival was unaffected. No hematologic or target organ pathologic findings attributable to steroid treatment were noted. These results are in agreement with an overall lack of toxicity and only an occasional mammary nodule or local mammary hyperplasia observed in monkeys given high doses of Norlestrin for a period of 5 to 10 years [54]. Together, the data in monkeys demonstrate that long-term exposure to a combination of EE/NA well above therapeutic exposures is well tolerated and does not increase tumor incidence in any tissue.
3.6 REPRODUCTIVE/DEVELOPMENTAL TOXICITY
As an extension of the pharmacology and clinical use of synthetic estrogens and progestins alone or in combination, these agents can prevent reproduction when given at the appropriate time and dose. The mechanism for their contraceptive effects is complex, but involves interruption of tubular transport of the ova, as well as hormone-induced changes to the uterine lining which prevents suitable implantation and subsequent development of the fertilized ovum (Yanagimachi and Sato, 1968). Following cessation of oral contraceptive treatment, normal fertility in animals is fully restored within 6 months (Peterson and Edgren, 1965). The antifertility effects of these agents have been discussed in greater detail elsewhere (Goldzieher and Fotherby, 1994).
Data collected from rodents and nonhuman primates show that combination oral contraceptives are embryolethal, but are not teratogenic. Developmental effects from administration of synthetic sex hormones to pregnant rodents and rabbits are inconsistent and are dependent on the compound, the dose, the species, and the timing of administration. Combined synthetic estrogen and progestin formulations, including Norlestrin, do not appear to be teratogenic in variousanimal species including mice (Takano et al., 1966), rats (Saunders and Elton, 1967; Edgren and Clancy, 1968), and rabbits (Saunders and Elton, 1967; Chang, 1974). Progestins alone are teratogenic in several species if given at an appropriate dose and time (Johnstone and Franklin, 1964; Warton and Scott, 1964; Gidley et al., 1970). The primary developmental effect of NA administration to pregnant mammals is female virilization, although skeletal abnormalities, cryptorchidism, hydrocephalus, and club feet have been reported (Warton and Scott, 1964; Andrew et al., 1972; Schardein, 1993). Pregnant rhesus monkeys given Norlestrin (EE/NA, 1 : 50) at doses ranging from 5 to 25 mg/kg-day during early (gestational days 21 to 35) or late gestation (gestational days 33 to 46) or throughout organogenesis (gestational days 21 to 46) experienced a higher rate of fetal mortality than controls (Prahalada and Hendrickx, 1983). Surviving offspring were without teratogenic or histopathologic findings. There were also no teratogenic effects in fetuses exposed in utero at 50 mg/kg during early organogenesis (days 21–35) and examined on gestational day 50. No abnormalities were detected in a subset of these animals at 1/2 years of age. A similar lack of teratogenic effects has been reported in other nonhuman primate species treated with high doses of EE/NA, although virilization of females was occasionally seen (Hendrickx et al., 1987). In addition, monkeys exposed in utero to Norlestrin up to 10 mg/kg from gestational day 21 to 45 did not show abnormalities in neurobehavior, activity level, or motor function during the first year of life (Golub et al., 1983).