The synthetic progestin norethindrone causes thyroid endocrine disruption in adult zebrafish.

Synthetic progestin norethindrone (NET) widely used in oral contraceptives, hormonal therapy and livestock farming has been detected in various aquatic ecosystems. Recent studies have shown that NET can cause thyroid endocrine disruption in amphibians. However, studies are still lacking on thyroid axis of fish. In the present study, we investigated thyroid hormone levels (T3 and T4) and transcriptional patterns of 15 genes of the hypothalamic-pituitary-thyroid axis (HPT axis) in adult zebrafish that were exposed to solvent control and three measured concentrations of NET (7, 84 and 810 ng/L) for 90 days. The results indicated that NET significantly lowered T3 and T4 levels in both female and male zebrafish. Transcriptional expression profiles of some of the HPT-axis related genes were disrupted. Specifically, the expression levels of tshb and pax8 have increased significantly while dio2 and ugt1ab have decreased in females. In male, however, tshb expression levels were increased while ttr, ugt1ab, thra and thrb were decreased. The overall results demonstrate that NET disrupts thyroid endocrine system by interfering at multiple sites along HPT axis in adult zebrafish.

Influence of endogenous and synthetic female sex hormones on human blood cells in vitro studied with comet assay.

The comet assay has been conducted with numerous cell lines to assess in vitro genotoxicity. In order to use the comet assay as part of an in vitro test for evaluating genotoxicity, however, there are cell-specific factors that need to be better understood. In this present study we have evaluated some factors that may impact upon the DNA damage detected in whole blood (WB) cells and lymphocytes (ILs). Experiments were conducted comparing responses of both cells, and investigating the effects of the female hormonal cycle, and oral contraceptive (OC) use on DNA damage detection in the in vitro comet assay, at three sampling time. No significant differences were detected in the basal levels of DNA damage detected in ILs and WB cells from women OC users and non-users and from men. Basal DNA damage in ILs was unaffected by gender and stage of the menstrual cycle or the stage of the treatment schedule. Our results also indicated that the H2O2 induces DNA damage in human lymphocytes independently of gender, low-dose OC use and hormonal fluctuation. However, data showed that in 3rd sampling of menstrual cycle, lymphocytes were more resistant to H2O2-induced DNA damage than those from OC users and men.

Genotoxic potential of medroxyprogesterone acetate in cultured human peripheral blood lymphocytes.

Medroxyprogesterone acetate was studied at three different concentrations (1, 5 and 10 microM), for its genotoxic effects in human peripheral blood lymphocyte culture using chromosomal aberrations and sister chromatid exchanges as parameters. Duplicate peripheral blood cultures were treated with three different concentrations (1, 5 and 10 microM) of medroxyprogesterone acetate. The study was carried out both in the absence as well as in the presence of metabolic activation (S9 mix) with and without NADP. Medroxyprogesterone acetate was found genotoxic at 5 and 10 microM in the presence of S9 mix with NADP. To study the possible mechanism of the genotoxicity of medroxyprogesterone acetate, superoxide dismutase and catalase at different doses were used separately and in combination with 10 microM of medroxyprogesterone at different doses in the presence of S9 mix with NADP. Superoxide dismutase treatment results in an increase of the genotoxic damage but catalase treatment reduce the genotoxic damage of medroxyprogesterone acetate. Catalase treatment in combination with superoxide dismutase also results in the further reduction of the genotoxic damage. The results of the present study reveal that medroxyprogesterone acetate is genotoxic only in the presence of metabolic activation (S9 mix) with NADP. Treatments with superoxide dismutase and catalase suggests the possible generation of reactive oxygen species by redox cycling of various forms of quinones, similar to estrogens, that are the results of aromatic hydroxylation by cytochrome P450s.

17-alpha-ethinylestradiol and norgestrel in combination induce micronucleus increases and aneuploidy in human lymphocyte and fibroblast cultures.

Oral contraceptives are highly efficient and easily administered drugs; however, it must not be forgotten that they are composed of chemical substances which can be classified as potential carcinogens. Testing of a substance for genotoxicity represents a reliable approach both to evaluate the genetic hazard and to obtain information on its possible tumorigenic (cancerogenic) properties. The present study was undertaken to evaluate through carefully planned and controlled investigations the in vitro cytogenetic effects of oral contraceptives (ethynilestradiol and norgestrel mixed in the proportion 1:5) using three different concentrations, with two different durations of treatment (48 and 72 h), on two types of human cells (lymphocytes and fibroblasts) and a series of short-term test procedures: sister chromatid exchange (SCE), micronucleus test (MN), and chromosome aberrations (CA). In addition, the FISH procedure and in vitro anaphase and metaphase preparation analyses were performed. In contrast to CA and SCE frequencies, the frequency of MN in treated blood lymphocytes showed higher values by comparison with the controls, although the difference was statistically significant only for the lowest concentration (P = 0. 016). When using pancentromeric alphoid probes, the FISH procedure gave positive signals in more than 85% of micronuclei, clearly indicating that MN may contain whole chromosomes rather than acentric fragments. Unlike the lymphocytes, the fibroblasts showed dose-dependent effects, although those treated with the highest hormone concentrations showed an increased number of highly damaged cells (cytoplasmatic vacuolization, nuclear fragmentation, etc.), a decreased number of anaphase cells, a large number of which were abnormal, and a reduction of mitotic index. In conclusion, our data confirm that hormones do not induce structural chromosome aberrations in lymphocytes and indicate that ethynilestradiol and norgestrel have an aneugenic effect on fibroblast and lymphocyte cultures; FISH analysis on micronuclei from lymphocyte cultures and anaphase preparations from fibroblast cultures support this hypothesis. Teratogenesis Carcinog. Mutagen. 20:147-159, 2000.

Effect of oral contraceptive steroid hormones on metabolic parameters of streptozotocin-induced diabetic rat.

This study examined the effect of 0.05 mg norgestrel + 0.01 ethinyl estradiol (NEE) Kg x body wt(-1) on body weight, random blood glucose, glycosylated hemoglobin, and plasma insulin levels in streptozotocin-induced diabetic rats. Weight loss, blood glucose, glycosylated hemoglobin, and plasma insulin values of rats treated with NEE before and after the onset of diabetes were not significantly different from that of untreated diabetic rats. In conclusion, oral administration of these contraceptive steroid hormones does not significantly alter the metabolic parameters of diabetic rats.

[Increased serum alkaline phosphatase induced by DT-5061, an oral contraceptive, in rats].

DT-5061, a steroid oral contraceptive which contains norethisterone (NET) and ethinyl estradiol (EE) in a ratio of 100:7, was administered orally to rats for 14 days in order to investigate a possible origin of the increased serum alkaline phosphatase (ALP). Increased serum total ALP was noted in rats receiving DT-5061 at 5.35 mg/kg/day or EE at 0.35 mg/kg/day. Electrophoresis of serum ALP revealed that both liver and bone-type ALP isozymes were increased in the DT-5061 5.35 mg/kg and EE 0.35 mg/kg groups, and the ratio of increase in the liver-type was greater than that in the bone-type although the increase in concentration of the bone-type was greater as compared to the liver-type. ALP level in the liver was elevated together with an increase in liver weight, consistently with the increased serum liver-type isozyme. However, neither histological changes indicative of cholestasis nor increase in serum leucine aminopeptidase, bilirubin, GOT or GPT were seen. No changes were observed in bone ALP activity; hence inconsistent with the increased serum bone-type isozyme. From these results, it is considered that the increased serum ALP induced by this drug was due to the increased liver-type isozyme induced in the liver and to the increased bone-type isozyme, and among the ingredients of this oral contraceptive, EE was mainly involved in the increased serum ALP induced by this drug.

[Effects of adrenalectomy on the increased serum alkaline phosphatase activity induced by DT-5061, an oral contraceptive, in rats].

DT-5061, a steroid oral contraceptive which contains norethisterone (NET) and ethinyl estradiol (EE) was administered orally to adrenalectomized rats for 3 days to investigate involvement of the adrenals in the increased serum alkaline phosphatase (ALP). In addition, corticosterone or aldosterone were administered to the adrenalectomized rats to examine their effects on serum ALP. Increases in serum total ALP, liver-type and bone-type ALP isozymes were observed in rats with intact adrenals following administration of DT-5061, but these responses were not noted in adrenalectomized rats. Increases in liver weight and ALP activity in the liver after administration of the drug were reduced but not abolished in the adrenalectomized rats. The adrenalectomized rats receiving corticosterone showed increases in serum total ALP, liver-type ALP isozyme and liver weight but did not exhibit any increase in bone-type ALP isozyme. On the other hand, aldosterone did not increase and even reduced serum ALP although ALP activity in the liver was increased in the adrenalectomized rats.

FDA requirements for nonclinical testing of contraceptive steroids.

Written guidelines for the preclinical testing of contraceptive steroids have not been revised since 1968 despite the fact that many important changes have been implemented by the FDA's Division of Metabolism and Endocrine Drug Products. This paper describes the new preclinical testing requirements and the rationale for their implementation.

PIP: The Division of Metabolism and Endocrine Drug Products of the US Food and Drug Administration (FDA) has effected many significant changes since it last revised its written guidelines for preclinical testing of contraceptive steroids in 1968. Yet it did not update the guidelines, which pertain to both male and female contraceptives, until 1992. Type and duration of preclinical studies for Clinical Phase I are single-dose studies in rats and mice using an amount that produces overt toxicity and which is administered through the same route as intended for humans and repeat dose studies in rats and monkeys (up to 200 times the human dose for rats and 50 times the human dose for monkeys) for at least 1 month. The FDA is flexible about the duration of preclinical studies for Phase II as long as the duration is at least as long as the duration of the proposed clinical trial (6-month maximum for rats and 1-year maximum for monkeys). Preclinical trials for Phase II are the same studies as for Phase II as well as genotoxicity tests. For genotoxicity tests, the FDA recommends in vitro mammalian cell gene mutation assay with and without metabolic activation, in vitro chromosome aberration test in mammalian cells with and without metabolic activation, and the mouse micronucleus test for chromosome damage. Preclinical studies for FDA are a 6-month toxicology study in rats, a 12-month toxicology study in monkeys, and a 2-year carcinogenicity study in rats or mice. The FDA can consider other species for toxicology testing. Pharmacology tests, generally done in rodents, determine drug effects on neurological, cardiovascular, and immunological parameters. Researchers should use steady state conditions to study pharmacokinetic parameters including, Tmax, Cmax, T1/2, and area under the curve. Toxicologic tests on contraceptives with estrogen/progestogen combinations should use the ratio to be marketed or, if they have more than 1 ratio, the lowest ratio. FDA remains flexible to any deviations for which there is a sound scientific rationale.

Effects of prolonged administration of the 19-nor-testosterone derivatives norethindrone and norgestrel to female NZB/W mice: comparison with medroxyprogesterone and ethinyl estradiol.

To evaluate effects of commonly used progestational estrogenic contraceptive steroids in a hormone-responsive model of lupus, we treated female NZB/W mice before clinical disease (6 wks of age) and after onset of lupus (24 wks of age) with doses of hormones titered to suppress reproduction. We report efficacy of norethindrone (NE) and norgestrel (NG), progestins derived from 19-nor-testosterone, in delaying expression of anti-DNA antibodies. Mice implanted with NG at 24 wks of age had prolonged lifespans. In contrast, the hydroxyprogesterone derivative, medroxyprogesterone acetate (MP), did not affect autoimmune disease. These observations suggest that prolonged administration of 19-nor-testosterone derivatives, in small doses adequate to suppress reproduction, may have ameliorative effects in systemic lupus erythematosus. Mice receiving ethinyl estradiol (EE) plus courses of tetracycline to suppress cystitis had active anti-DNA responses. In 60% of EE-treated mice, however, early deaths resulted from malignant lymphomas and complications of obstructive uropathy. Estrogen toxicity, rather than accelerated lupus, was the major cause of death in NZB/W mice treated with EE.

Globulines anormalment precipitables (GAP) in Nigerian users of progestogen-only pill.

The globulines anormalment precipitables (GAP), which have been reported to be raised in current and former oral contraceptive users, were measured in Nigerian subjects which included male volunteers. The results showed that GAP were present in males who had never used contraceptives and that the mean values were lowest in intrauterine contraceptive device (IUD) users and highest in females who had never used any contraceptives. However, current contraceptive users had lower mean GAP values than either former users or never users. It was therefore concluded that GAP levels alone cannot be used to predict the development of thromboembolic complications in pill users, and that ethinyl estradiol cannot be the main physiological stimulus for GAP synthesis.

Comparative aspects of contraceptive steroids: effects observed in the monkey.

The use of the non-human primate in long-term studies of contraceptive steroids has been questioned because of time, expense and apparent lack of results predictive for humans. Controversies have arisen primarily over the occurrence of mammary nodules in studies of different contraceptive steroids and the occurrence of uterine tumors in 2 high-dose group monkeys in the Depo-Provera study. The long-term studies have been criticized because of the experimental design and the small number of monkeys per dose group. Individual studies by themselves did not reveal lesions other than those expected from an exaggerated pharmacologic response of target tissues; however, a pattern may emerge from reviewing and combining results of different studies that indicate the results of these studies are in agreement with the clinical findings in man. Effects of contraceptive steroids on the mammary gland and genital organs will be discussed. Data from 17 contraceptive steroid studies involving 264 untreated control and 733 treated non-human primates were available.

Comparative aspects of contraceptive steroids: effects observed in rats.

Current regulatory guidelines for testing contraceptive drugs in long-term rodent studies have established dosages based on multiples of the proposed human usage level. These multiples in rodents are 1-2, 10, and 50. The estrogen/progestogen ratio for most human contraceptive drugs ranges from 1/5 to 1/80. One of the biological endpoints in arriving at the human estrogen/progestogen ratio is the development of an endometrial decidualization response. The ratio necessary to achieve a similar uterine response in the rat is 1:10,000 to 1:20,000. Thus, dosages in the rodent, when based only on a multiple of the proposed human usage level, result in a highly estrogenic combination with estrogen being completely dominant. Continuously elevated estrogen in the rat is toxic to dopaminergic neurons in the hypothalamus which secrete prolactin inhibiting factor (PIF). Hyperplasia of pituitary lactotrophs occurs from both the direct stimulatory effect of estrogen and the uninhibited secretory activity of lactotrophs related to depressed PIF secretions. Prolactinomas result. Increased levels of prolactin lead to mammary gland stimulation and tumor development. Dosage levels for future rodent studies of contraceptive drugs should be based on pharmacokinetics, endocrine profiles, and biological endpoints rather than on multiples of the human usage level.

Guidelines and requirements for the evaluation of contraceptive steroids.

Since their introduction in the early 1960s, the oral contraceptive (OCs) steroids have been subjected to preclinical and clinical investigations unprecedented in medical history. As a result of such extensive studies, it is now possible to make a comprehensive review of preclinical and clinical data on oral contraceptives. The OCs were introduced at a time when the Food and Drug Administration (FDA) was undergoing drastic changes as a result of the thalidomide tragedy, the introduction of the Kefauver-Harris Amendment, and the desire for greater control over the pharmaceutical industry. The initial requirements for the safety evaluation of OCs were identical to those of other drugs. There were no explicit requirements for OCs although it was generally felt that the requirements should be more stringent because the OCs were being used in otherwise healthy women for long periods of time and with minimal medical supervision. In the 1960s when it became apparent from ongoing studies that there was an increased incidence of mammary tumors in dogs treated with some progestins, the FDA made the decision to terminate clinical studies and established the requirement for 7- and 10-yr studies in dogs and monkeys, respectively. The primary purpose of this paper is to present an historical perspective of the evolution of the preclinical requirements for the evaluation of the safety of OCs prior to their use in the various phases (I, II, III) of clinical trials. Some proposed changes in the requirements are discussed. This information will form the basis for other presentations dealing with the safety assessment of OCs in rats, dogs, and monkeys.

PIP: Sufficient preclinical and clinical data are now available to formulate guidelines on the use of oral contraceptives (OCs). The 1961 introduction of OCs coincided with the implemented of detailed study testing requirements of new drugs, including 3 phases of clinical trials each preceded by animal toxicity studies. Phase I human safety studies involved 10-20 subjects and focus on drug tolerance and clinical pharmacology. Phase II trials seek to demonstrate optimal dosage, efficacy, and relative safety in 50-100 subjects. Phase III trails are conducted under field conditions and involve up to 1000 human subjects. Before Phase III trials are initiated, 2-year studies in rats, dogs, and monkeys must be completed and 7- and 10-year studies in dogs and monkeys initiated. Over the past 3 decades, it has become apparent that there are significant differences in the responsiveness of target organs to OCs between humans and rats, dogs, and monkeys--the recommended animal models. The impossibility of accurately and completely predicting all the effects of OCs on humans on the basis of animal research has led to proposals to update the official requirements for the preclinical and clinical assessment of new fertility regulating drugs. A 1987 meeting on Methods for Improving the Safety Requirements for Contraceptive Steroids has developed guidelines aimed at increasing the reliability of OC safety data. The US Food and Drug Administration has accepted many of these proposals and added others, including acute toxicity studies in 3-4 species and multidose studies of toxicity in a rodent and monkey. Final approval requires 1-year toxicity studies in rodent and nonrodent species and a 3-year dog study.

Comparative aspects of contraceptive steroids--effects observed in beagle dogs.

The effects of oral contraceptives have been studied in the beagle bitch for periods up to 7 yr. High doses of these potent estrogen: progestogen (E:P) combinations have been shown to promote tumors in the mammary glands, smooth muscle of the tubular genitalia, and occasionally in the transitional epithelium of the neck/trigone area of the urinary bladder. The contraceptive formulations used in humans are balanced with an E:P ratio of about 1:5 to 1:80 to produce a desired decidual response in the uterus. The corresponding ratio for producing the decidual reaction in the dog is 1:1,000 to 1:3,000 with the result that the dog is grossly overdosed with estrogens when given the human formulation at the usual multiples of up to 25 times the human dose. Smooth muscle tumors of the tubular reproductive tract are common sequelae to estrogen overstimulation in the dog and are known to occur in other species, including the humans. The dog also has major differences in hormonal control and sensitivity when compared to humans. Progestogens stimulate synthesis and release of growth hormone (GH) in dogs which in turn is the major stimulant (with progestogens) of mammary growth and tumors. Evidence is accumulating which indicates that most if not all progestogens can produce mammary tumors in the dog if given by the correct route and at high enough dosage. In contrast, GH in humans is not increased nor does it have any significant mammotrophic role. Mammary tumors in dogs related to oral contraceptives are now widely considered to be irrelevant as a model or predictor for human tumors. Transitional cell tumors in the urinary bladder seem to be a species specific phenomenon seen on occasion in the dog, but not in the rat, monkey, or human. The usual location in the neck/trigone area may be related to the embryologic origin of this portion of the bladder, which derives from tissues more closely related to the genital organs than does the rest of the bladder.

Oral contraceptives: significance of their effects in man and relationship to findings in animal models.

Combination oral contraceptives have been available since 1960. They contain both an estrogen and a progestogen and have been studied extensively in both lower animals and humans and have been the subject of special regulatory requirements for toxicological and clinical studies. The initial oral contraceptives, by today's standards, contained very high levels of both hormones. There has been a continuous decrease in the dose of both the estrogen and the progestogen during the past quarter century, with continued maintenance of high degree of effectiveness. This decrease of dosage has been stimulated by findings from prospective clinical trials and retrospective case control trials. As additional information has been gained with oral contraceptives, new benefits beyond their effectiveness as contraceptives have been realized. Today's oral contraceptives provide a high degree of effectiveness, low incidence of nuisance side effects, and low incidence of major adverse effects.

PIP: A historical review of the 28-year history of oral contraceptives from the viewpoint of correlation or lack thereof between drug toxic and pathologic effects seen in laboratory animals and those seen clinically is presented. Early high dose pills were expected to cause growth of uterine fibroids, but instead they had the unexpected effect of an estrogen dose-related venous thrombosis risk. Work on rats predicted that pills would cause liver cancers, but instead to slightly increase the incidence of being liver adenomas in women. Similarly, rat research predicted pituitary microadenomas. Pituitary effects in women, while rare, are thought to be due to prescription of pills to women with irregular cycles of pituitary origin. Progestins of the 17-acetoxy series were considered likely to produce breast cancers, as they had in beagle dogs. They apparently have not done so in women. They were reports in the mid-1970s that sequential pills containing 100 mcg ethinyl estradiol cause endometrial carcinoma. These pills have been discontinued. Recent evidence has been accumulating that low-dose pills containing levonorgestrel increase blood pressure and possible LDL-cholesterol. Risk of death from vascular disease, however, seems to be concentrated in women who smoke, especially those over 35.

Evaluating steroidal contraceptives: pre-clinical and clinical approaches.

PIP: The Special Program of Research, Development and Research Training in Human Reproduction of the World Health Organization held a meeting in Geneva in February of 1987 to review the results of animal and human studies analyzing contraceptive steroids. In reviewing this data, assessment of pre-clinical toxicology and human risk would be possible. The studies reviewed included: studies on steroid exposure and breast disease, the protective factor of combined oral contraceptives (OC) (against ovarian cancer), hormonal contraceptives and cervical cancer, the relationship of gall bladder disease to OC use, congenital malformations and steroid hormone, OC use and its effect on breast milk and other topics. Basic principles on pre-clinical testing were adopted. These included: 1) the establishment of safety is a joint responsibility of experimental toxicology and clinical pharmacology. 2) Testing should include detailed analysis of the hormonal effect of new types in several animal species. 3) Species specific data should be collected. 4) Studies to detect cumulative toxicity should be performed. 5) Reproductive toxicity should concentrate on exposure throughout the embryonic period. 6) In vivo and in vitro tests should be conducted to investigate the mutagenic potential of new contraceptive steroids. 7) Long-term carcinogenicity study should be performed, preferably on the rat. 8) When new delivery systems are used to administer established steroidal contraceptives, experimental safety studies should assess potential interactions with the system and on the possibility of adverse effects of the delivery system. 9) Post-registration surveillance is on integral part of the risk assessment process.^ieng

Effects of female sex steroids on concanavalin A-mediated agglutination of hepatocytes from nonregenerating and regenerating rat liver and hepatic tumor marker enzymes.

Effect of treatment of female rats with an oral contraceptive agent (OCA), Ovulen-50, for 7 weeks on agglutination of hepatocytes with concanavalin A (con A) and activities of certain tumor marker enzymes were examined to find out if OCA treatment is related to preneoplastic or neoplastic processes. Hepatocytes from regenerating and nonregenerating livers of control female rats showed negligible agglutination with Con A, whereas hepatocytes from non regenerating but not from the regenerating livers of female rats treated with a combination of 5 micrograms ethinyl estradiol and 100 micrograms ethynodiol diacetate showed agglutination. Of the tumor marker enzymes such as hepatic glucose 6-phosphatase, gamma-glutamyl transpeptidase (gamma-GT), and arginase examined in the liver, only gamma-glutamyl transpeptidase showed a significant increase in activity in the steroid-treated rats. Plasma alkaline phosphatase activity was also higher in the treated animals. However, the magnitude of the changes observed was relatively small and perhaps unrelated to the neoplastic process.

Contraceptive steroids and coronary artery atherosclerosis in cynomolgus macaques.

The influence of two types of steroidal contraception on the extent of coronary, aortic, carotid, and iliaco-femoral atherosclerosis was assessed in 57 cynomolgus macaques with moderate diet-induced hyperlipoproteinemia. Thirteen animals were treated with an intravaginal ring that released 17 beta-estradiol and levonorgestrel. Fifteen females were treated with an oral contraceptive (OC) composed of ethinyl estradiol and norgestrel. Fifteen females received placebo vaginal rings, and 14 males were untreated. The contraceptive treatments resulted in similar large reductions in plasma high-density lipoprotein (HDL) cholesterol concentrations. Neither treatment influenced the prevalence of coronary artery atherosclerosis. However, treatment with the contraceptive vaginal ring was associated with increased extent of coronary artery atherosclerosis (plaque size) relative to untreated females, whereas treatment with the OC was not. The contrasting effects of the two treatments could not be explained by differences in total plasma cholesterol, HDL cholesterol, or blood pressure. The results suggest that the greater estrogenic influence associated with the ethinyl estradiol-containing OC resulted in inhibition of coronary artery atherosclerosis despite a pronounced progestin-induced lowering of plasma HDL cholesterol concentration and, further, that hormonal balance may have a marked influence on the relationship between plasma lipids and atherogenesis.

Pre-clinical evaluation of contraceptive steroids: regulatory requirements and scientific expectations.

The development of new contraceptive steroids placed great pressures on regulatory agencies. There was insufficient time to develop a novel pre-clinical safety evaluation, hence their toxicity in animals was assessed as with other drugs. The approach of regulatory agencies to toxicity and carcinogenicity testing of these steroids are discussed and evaluated.