Transcriptomic and physiological changes in western mosquitofish (Gambusia affinis) after exposure to norgestrel.

Norgestrel (NGT) is a synthetic progestin used in human and veterinary medicine. Adult female mosquitofish were exposed to NGT for 42 d at 377 ng L-1. The fin morphology and the liver transcriptome were assessed. NGT exposure increased ray 4:6 length ratio. As compared to the control, NGT treatment affected the expression of 11,772 annotated transcripts in female mosquitofish. Specifically, we found 5780 were repressed while 5992 were significantly induced. Gene ontology (GO) analysis showed that 53 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and 158 GO terms were significantly over expressed. Genes showing the largest magnitude of expression changes were related to fin development, androgen biosynthesis, and lipid and fatty acid metabolisms, suggesting the involvement of these biological processes in response to NGT exposure in G. affinis. This first comprehensive study on the transcriptomic alterations by NGT in G. affinis not only provides valuable information on the development of molecular markers but also opens new avenues for studies on the molecular mechanisms of effects of NGT in particular and possibly other progestins in G. affinis.

Alterations of secondary sex characteristics, reproductive histology and behaviors by norgestrel in the western mosquitofish (Gambusia affinis).

Synthetic hormones in wastewater effluents released into the aquatic environments may interfere with the normal endocrine systems of fish in receiving streams. Norgestrel (NGT) is a synthetic progestin widely used in oral contraceptives and frequently detected in wastewater effluents. In this study, adult female mosquitofish (Gambusia affinis) were exposed to three environmentally relevant concentrations of norgestrel (NGT) (i.e., 3.6, 35.8, and 368.0 ng L-1) for 42 d, fin morphology, histology of the ovary, and reproductive behaviors were evaluated. The results showed that NGT at all three concentrations caused an increased frequency of atretic follicular cells in ovaries and impaired mating behaviors exhibited by males toward the NGT-exposed females. In mosquitofish exposed to NGT at 35.8 and 368 ng L-1, the anal fin of females had an increased length ratio of ray4/ray 6, an increased width of ray 3, and increased number of segments in ray 3. The histopathological analysis showed that exposure to NGT increased the incidence of spermatogenesis in ovaries. Mating behavior was impaired 58.4%, 65.7%, and 76.4% (P < 0.01 in all cases) when mosquitofish were exposed to NGT at 3.6, 35.6 and 368.0 ng L-1, respectively. The rapid masculinization, the increased frequency of atretic follicles, the incidence of spermatogenesis in the ovary of female fish, and the altered reproductive behaviors suggest that wild populations of mosquitofish could be similarly affected inhabiting in NGT contaminated environments.

Transcriptional and histological alterations in gonad of adult zebrafish after exposure to the synthetic progestin norgestrel.

The aim of the present study was to investigate the effects of norgestrel (NGT) on gonadal development in adult zebrafish. Adult zebrafish were exposed to NGT for 14 d at 871 ng L-1 for microarray analysis, and a follow-up experiment was conducted to further study the targeted pathway in adult zebrafish after exposure to NGT at 6.7, 83, and 912 ng L-1 by quantitative polymerase chain reaction (qPCR) and histological analysis. The microarray analysis revealed that 11 545 transcripts were identified. Gene ontology analysis showed organ development, system development, multicellular organismal development, single-organism developmental process, and developmental process were significantly enriched. A Venn diagram displayed 434 target genes involved in organ development, and these genes were common in these 5 development-related processes. Kyoto Encyclopedia of Genes and Genomes analysis showed that the notch signaling pathway was the top toxicity pathway, and it was selected as the target pathway for further qPCR analysis. The qPCR analysis revealed significant and dose-dependent alterations of most target genes involved in the notch signaling pathway in the gonads, even at an environmentally relevant concentration of 6.7 ng L-1 . The transcriptional patterns were consistent with the notch signaling cascade. In addition, NGT significantly increased the frequency of mature sperm and decreased the frequency of immature sperm at all concentrations. Meanwhile, NGT treatment increased the percentage of mature vitellogenic oocytes and atretic follicles at 912 ng L-1 but decreased the percentage of immature vitellogenic oocytes. Thus, the present study demonstrated significant developmental toxicity in the gonad of adult zebrafish even at environmentally relevant NGT concentrations. Environ Toxicol Chem 2017;36:3267-3276. © 2017 SETAC.

Long-term exposure to environmentally relevant concentrations of progesterone and norgestrel affects sex differentiation in zebrafish (Danio rerio).

The aim of this study was to investigate the effects of progestins on the sex differentiation of zebrafish by measuring the sex ratio and transcriptions of genes related to sex differentiation (Amh, Dmrt1, Figa, Sox9a and Sox9b genes) as well as sex hormone levels and transcriptional expression profiles along the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-adrenal (HPA) axes in juvenile zebrafish. Exposure of zebrafish to 4, 33, 63ngL(-1) progesterone (P4) or 4, 34, 77ngL(-1) norgestrel (NGT) started at 20 days post fertilization (dpf) and ended at 60 dpf. The results showed that exposure to P4 caused a significant increase in proportion of females as well as significant down-regulation of Amh gene and up-regulation of Figa at a concentration of 63ngL(-1). However, the shift in the sex ratio toward males was observed following exposure to 34 and 77ngL(-1) NGT, which came along with the significant induction of Dmrt1 gene and inhibition of Figa gene. The sex hormones in exposed fish were measured with estrone being detected only in the fish exposed to the highest P4 concentration; whereas estradiol and androstenedione were detected only in the fish of the control and lowest NGT concentration. Furthermore, the increase in females was associated with the significant up-regulation of several key genes controlling the synthesis of sex hormones (i.e., Cyp17, Cyp19a1a and Hsd3b) following exposure to 63ngL(-1) P4 whereas the significant down-regulation of Cyp11a1, Cyp17, Cyp19a1a and Hsd3b genes was observed in the male-biased populations caused by 34 and 77ngL(-1) NGT. The overall results imply that both P4 and NGT could significantly affect sex differentiation in zebrafish, and that changes may be reflected by altered sex hormone levels and transcriptional expression profiles of genes related to synthesis of sex hormones.

Oral contraceptives and nicotine synergistically exacerbate cerebral ischemic injury in the female brain.

Oral contraceptives (OC) and smoking-derived nicotine (N) are known to synergistically increase the risk and severity of cerebral ischemia in women. Although it has been known for some time that long-term use of OC and nicotine will have an increased risk of peripheral thrombus formation, little is known about how the combination of OC and nicotine increases severity of brain ischemia. Recent laboratory studies simulating the conditions of nicotine exposure produced by cigarette smoking and OC regimen of women in female rats confirms that the severity of ischemic hippocampal damage is far greater in female rats simultaneously exposed to OC than to nicotine alone. These studies also demonstrated that the concurrent exposure of OC and nicotine reduces endogenous 17ß-estradiol levels and inhibits estrogen signaling in the brain of female rats. The endogenous 17ß-estradiol plays a key role in cerebrovascular protection in women during their pre-menopausal life and loss of circulating estrogen at reproductive senescence increases both the incidence and severity of cerebrovascular diseases. Therefore, OC and nicotine induced severe post-ischemic damage might be a consequence of lack of estrogen signaling in the brain. In the present review we highlight possible mechanisms by which OC and nicotine inhibits estrogen signaling that could be responsible for severe ischemic damage in females.

Protective effect of nordihydroguaiaretic acid (NDGA) against norgestrel induced genotoxic damage.

Nordihydroguaiaretic acid (NDGA) is a phenolic lignan and possesses antioxidant and number of properties potentially useful to man. The effect of NDGA was studied against norgestrel induced genotoxic damage, using sister chromatid exchanges (SCEs), chromosomal aberrations (CAs), mitotic index (MI) and replication index (RI) as parameters. Amounts of 5, 10 and 20 microM of norgestrel was tested for its genotoxic effect in the absence as well as presence of S9 mix, and was found to be genotoxic at 10 and 20 microM in the presence of S9 mix. Again, 10 microM of norgestrel was treated with 0.5 and 1 microM of NDGA, separately, in the presence of S9 mix. Similar treatment was given with 20 microM of norgestrel. Treatments given with NDGA result in the reduction of SCE, CA and increase of MI as well as RI, suggesting its protective action on human lymphocytes in vitro against the norgestrel induced genotoxic damage.

Evaluation of genotoxic potential of synthetic progestins-norethindrone and norgestrel in human lymphocytes in vitro.

The genotoxicity study of two widely used contraceptive synthetic progestins, i.e. norgestrel and norethindrone was carried out on human lymphocyte chromosomes using chromosomal aberrations (CA), sister chromatid exchanges (SCE) and cell growth kinetics as parameters. The study was carried out both in the presence as well as in the absence of metabolic activation (S(9) mix). The lymphocytes were exposed to three different concentrations of the drugs (20, 40 and 75 microg/ml for norethindrone and 10, 25 and 50 microg/ml for norgestrel) for three different durations (24, 48 and 72 h). The drug norethindrone was found to be non-genotoxic at any concentration and at any exposure duration either in the presence or in the absence of S(9) mix. But another drug norgestrel was found to affect the genetic material. It induces CA, SCE at significant level, and inhibits lymphocyte proliferation at 25 and 50 microg/ml of concentrations only. In the presence of S(9) mix the values obtained for CA, SCE and mitotic index (MI) were more significant. A time and dose relationship was also observed. It was concluded that norgestrel itself and possibly its metabolites are potent mutagens beyond a particular dose in human lymphocytes.

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.

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.

Transdermal dual-controlled delivery of contraceptive drugs: formulation development, in vitro and in vivo evaluations, and clinical performance.

Several transdermal contraceptive device (TCD) formulations were developed to provide a dual-controlled transdermal delivery of levonorgestrel (LN), a potent progestin, and 17 beta-estradiol (E2), a natural estrogen. Using a sensitive HPLC method, the in vitro release and skin permeation profiles of LN and E2 from various TCD formulations were simultaneously characterized in the hydrodynamically well-calibrated Valia-Chien skin permeation cells and both were found to follow zero-order kinetics. The rates of drug release and skin permeation were observed to vary significantly depending upon some formulation parameters. Six-month stability studies were performed on seven formulations at room and elevated temperatures (37 and 45 degrees C), and two (Formulations 4 and 5) were found to be acceptable, based on drug recovery, release rate, and skin permeation rate data. Judging from the 6-month accelerated stability studies, it is projected these two formulations will have shelf-life of at least 2 years. As a result of development of an efficient manufacturing process, Formulation 4 was selected for further evaluation. One-week primary skin irritation evaluation in 6 rabbits indicated that Formulation 4 is nonirritating, and it was thus selected for Phase I clinical bioavailability/dose proportionality studies in 12 healthy female volunteers of child-bearing age. Results of pharmacokinetic and pharmacodynamic analyses demonstrated that it is capable of achieving and maintaining a steady-state serum level of LN throughout the 3-week treatment period by weekly applications of one or two TCD patches (10 or 20 cm2). A dose proportionality was obtained in the serum drug levels, daily dose delivered, and contraception efficacy. An excellent correlation was obtained for the rates of transdermal delivery determined by the in vitro studies using human cadaver skin, the in vivo studies in rabbits, and the clinical studies in living subjects.

From menarche to menopause: coronary artery atherosclerosis and protection in cynomolgus monkeys.

The effects on atherogenesis of stress, pregnancy, and oral contraceptive therapy were studied in a nonhuman primate model. The stress of social subordination was associated with ovarian dysfunction, unfavorable lipoprotein changes, and increased coronary artery atherosclerosis compared with nonstressed (socially dominant) or normal monkeys. Although pregnant animals exhibited lower high-density lipoprotein cholesterol concentrations, they had only one half as much diet-induced coronary artery atherosclerosis as their nonpregnant counterparts. Monkeys treated with an Ovral-like regimen also exhibited adverse lipoprotein changes. Nevertheless, prevalence and extent of coronary artery plaques decreased. We conclude that estrogen is an important factor in the animals' "female protection" against diet-induced atherosclerosis. We also suggest that the lowering of high-density lipoproteins by the progestin component of higher-dose contraceptives is not necessarily atherogenic if a sufficiently potent exogenous estrogen is administered concomitantly.

The morphologic effects of synthetic reproductive steroids on the mammary gland of rhesus monkeys. Mestranol, ethynerone, mestranol-ethynerone, chloroethynyl norgestrel-mestranol, and anagestone acetate-mestranol combinations.

A total of 213 treated and 16 control monkeys comprising 12 experimental groups was evaluated for determination of the long-term (10 years) effects of various dosages of a variety of synthetic oral contraceptive steroids on the mammary glands of rhesus monkeys. The steroid hormones included mestranol, ethynerone, a combination of mestranol and ethynerone, chlorethynyl norgestrel plus mestranol, and anagestone acetate plus mestranol. Various degrees of physiologic lobular hyperplasia and lactational changes were observed in association with all of these steroid hormones; these changes appeared dose-dependent. Mestranol caused a proliferative atypia ranging from a minimal to a moderate degree in 8 of 34 (23%) animals, but it was not dose-related. Eleven of 15 monkeys (73%) administered ethynerone developed proliferative atypia, ranging in degree from minimal to severe, including one invasive carcinoma and 2 lesions resembling intraductal carcinoma in the human. The mestranol and ethynerone combination produced a proliferative atypia in 22 of 52 animals (42%), including five identical to intraductal carcinoma in the human and one identical to lobular neoplasia. Of the 40 monkeys administered anagestone acetate and mestranol, 20 (50%) developed proliferative atypias; the atypias ranged from mild to severe and included five resembling intraductal carcinoma in human breast. The chloroethynyl norgestrel and mestranol combination induced proliferative atypia in 25 of 52 monkeys (49%); six of these atypias were severe and indistinguishable from intraductal carcinoma of the human breast; and one, if in the human breast, would reflect a solid variant of an invasive carcinoma. Only 2 of the 16 control monkeys (12%) developed proliferative atypias, and these were of minimal to mild degree. The occurrence of severe degrees of atypia identical to intraductal carcinoma in the human breast and invasive carcinoma associated with hormone administration suggests a carcinogenic effect.

Malignant neoplasms of decidual origin (deciduosarcomas) induced by estrogen-progestin-releasing intravaginal devices in rabbits.

A combination of estrogen and levonorgestrel was continuously delivered to 23 adult rabbits for up to 2 years via a Silastic ring device sutured into the vagina. Twenty-one control rabbits were given similar rings devoid of drugs. A marked decidual reaction of the endometrium occurred in 16 of 23 test rabbits. In 14 test rabbits (61%) malignant tumors developed of decidual type cells not heretofore described. The deciduosarcomas were composed of anaplastic cells that invaded the uterine walls, uterine lymphatics, and in 4 of 13 (31%) rabbits that survived 2 years of treatment, the tumors metastasized to the lungs. Several deciduosarcomas appeared to arise within the spleen or other abdominal organs. Other drug-related lesions included uterine or vaginal polyps, endometrial atrophy, and focal necrosis and mineralization of the uterine wall. Cells from several deciduosarcomas failed to produce tumors in nude mice or to colonize on soft agar. No decidualization or decidual neoplasms were seen in the controls.

Chronic steroid contraceptive treatment decreases renal alpha-adrenoceptor levels in the rat.

Female Sprague-Dawley rats were injected s.c. with ethynyloestradiol (EE2, 0.2 microgram/day) and levonorgestrel (NG, 2.0 micrograms/day) separately and in combination (EE2/NG). Binding of [3H]rauwolscine (alpha 2-adrenoceptor specific) and [3H]prazosin (alpha 1-adrenoceptor specific) was examined in crude membrane suspensions prepared from whole rat kidney after 3, 6 and 12 weeks of steroid administration. Receptor affinity was high for both ligands (KD, equilibrium dissociation constant [3H]rauwolscine, congruent to 2.0 nM; [3H]prazosin, congruent to 0.2 nM) and was not altered in rats chronically treated with steroid contraceptives. The Bmax (maximum density of binding sites) for [3H]prazosin binding was not altered, indicating no change in the number of renal alpha 1-adrenoceptors. NG administered alone did not affect the numbers of alpha 1- or alpha 2-adrenoceptors. Catechol metabolites of endogenous oestrogens did not displace the binding of either radioligand, suggesting that these metabolites do not directly interact with renal alpha-adrenoceptors. However, after 12 weeks treatment, the number of [3H]rauwolscine binding sites was reduced in both EE2 (Bmax, 133 +/- 7 fmol/mg protein)- and combined EE2/NG (135 +/- 11 fmol/mg protein)-treated rats, compared to controls (162 +/- 9 fmol/mg protein). Since renal alpha 2-adrenoceptors inhibit renin release, this reduction in alpha 2-adrenoceptor number may contribute to increased renin levels associated with oestrogen-induced hypertension.

Facts about an implantable contraceptive: memorandum from a WHO meeting.

This Memorandum reviews the results of research undertaken in animals and human subjects on the implantable contraceptive, Norplant, and where relevant, its components, levonorgestrel and Silastic. Results from clinical trials, including effectiveness and side-effects, are evaluated and service delivery aspects commented on. The Memorandum concludes with a statement regarding the use of Norplant as an option for long-term reversible contraception.

The influence of cigarette smoke and treatment with contraceptive hormones on the fibrinolytic activity in the rat.

The fibrinolytic activity in the vessel wall (FAV), in plasma and in euglobulin solution (FAE) was determined in rats exposed to cigarette smoke in an inhalation chamber or treated with contraceptive hormones. In the smoke-exposed rats increased FAE and significantly decreased FAV were demonstrated. No effect on fibrinolysis was recorded in the hormone-treated rats, in which mean plasma levels of ethinylestradiol and d-norgestrel were 74 pg/ml and 0.17 ng/ml, respectively, six hours after the last subcutaneous administration.

Further investigations on the cytological effects of some contraceptives.

Anovlar induces a mitodepressive effect, producing abnormal prophases and a considerable number of micronuclei, i.e. Anovlar can produce major cytological abnormalities. On the other hand, Lyndiol induces minor abnormalities leaving the process of mitosis to proceed almost normally. Microgynon 30 did not show any effect on any of the mitotic stages. Our conclusion may offer an explanation for the contradictory results found in the literature upon the cytological effect of oral contraceptives, although the major chemical constitution of all contraceptives used is the same, still there are minor differences in their chemical structure. These minor differences in chemical structure may be the deciding cause whether or not a contraceptive is harmful.

PIP: Anovlar induces a mitodepressive effect, producing abnormal prophases and a considerable number of micronuclei, i.e., Anovlar can produce major cytological abnormalities. Conversely, Lyndiol induces minor abnormalities leaving the process of mitosis to proceed almost normally. Microgynon 30 did not show any effect on any of the mitotic stages. The conclusion may offer an explanation for the contradictory results found in the literature of the cytological effect of oral contraceptives, although the major chemical constitution of all contraceptives used is the same, still there are minor differences in their chemical structure. These minor differences may be the deciding factor as to whether or not a contraceptive is harmful.

A comparative study of two estrogen dosages in combined oral contraceptives among Sudanese women.

A prospective study of two combined oral contraceptives was conducted in the Sudan. No pregnancies occurred. Overall incidence of side effects was low. Headache was most frequently reported. Elevations were observed for weight, systolic and diastolic blood pressures, and SGOT and SGPT values while a decrease was seen for hemoglobin levels. Menstrual irregularities were not a problem for the users. Total 6-month use discontinuation rates were low for both pill groups.

Toxicity of the progestagen STS 557 compared to levonorgestrel in beagles after oral administration for 6 months.

Female and male beagle dogs were administered orally STS 557 (17 alpha-cyanomethyl-17 beta-hydroxy-13 beta-methyl-gon-4,9(10)-dien-3-one) for 6 months at dose levels of 0.01, 0.1, or 1.0 mg/kg/day, and levonorgestrel at a dose of 1.0 mg/kg/day, respectively. The results mainly confirmed the gestagenic efficacy on the reproductive organs of both compounds acting directly or via the anterior pituitary gland. In contrast to levonorgestrel, STS 557 did not show any androgenic activity, but had slightly estrogenic effects. Neither clinical, functional nor morphological investigations revealed toxic side effects of the drugs on the liver, the kidneys, the bone marrow, or on blood clotting function.