Alterations to the vision-associated transcriptome of zebrafish (Danio rerio) following developmental norethindrone exposure.

Synthetic sex steroids, like the synthetic progestin norethindrone (NET), can affect a wide variety of biological processes via highly conserved mechanisms. NET is prevalent in surface waters, yet the sub-lethal effects of NET exposure are not are net yet well characterized in aquatic biota. A few targeted gene expression and behavioral studies have concluded that NET affects the vision of adult fish; however, early life stage (ELS) fish are often more sensitive to contaminants. Furthermore, many species of fish rely heavily on visual perception for survival during development. The goal of the present study was to characterize the effects of developmental exposure to environmentally relevant concentrations of NET on the visual system of ELS zebrafish, using transcriptomics and histological methods. Results indicate that exposure to relatively low levels of NET in aquatic systems may be sufficient to affect the visual function of developing fish.

Histopathologic Characterization of Mifepristone-induced Ovarian Toxicity in Cynomolgus Monkeys.

Mifepristone, which is an orally active synthetic steroid with antiprogesterone activity, is known as an ovarian toxicant. Because the available data regarding the histopathologic characteristics of ovarian toxicity in nonhuman primates are limited, the present study was undertaken in order to investigate detailed histopathologic changes accompanying mifepristone-induced ovarian toxicity and its relationship to changes in menstrual cycle and circulating sex steroid hormone. Twenty mg/kg of mifepristone was orally administered daily to 4 cynomolgus monkeys for 2 months. Mifepristone inhibited the cyclic increases in circulating estradiol-17ß and progesterone levels with associated absence of menstruation. Histopathologically, the ovary in the treated animals showed follicular phase without changes in the percentage of atretic antral follicles, and reduced endometrial thickness was noted in the uterus. These changes indicated that a certain degree of antral follicle development had been retained in spite of the menstrual cycle having been arrested in mifepristone-treated animals. Our investigation suggested that it is important to perform detailed histopathologic examination of reproductive organs with precise knowledge of the characteristics of each menstrual stage to detect ovarian toxicity in nonhuman primates. Monitoring menstrual signs and circulating sex steroid hormone levels provides additional evidence for the investigation of the mechanism of ovarian toxicity.

Exposure effects of levonorgestrel on oogenesis in the fathead minnow (Pimephales promelas).

The synthetic progestin levonorgestrel is commonly utilized in human oral contraceptives. It enters the environment as a component of wastewater treatment plant effluent, and has been measured at low ng/L concentrations in surface waters. It has been shown to activate fish androgen receptors, causing the physical masculinization of females, changes in reproductive behavior, and decreases in fecundity. In the present study, the effects of levonorgestrel exposure on early-stage oogenesis in the fathead minnow (Pimephales promelas) was examined. Adult females were exposed to 0, 10, or 100 ng/L levonorgestrel for 14 d using a flow-through exposure system. The ovaries from each female were then removed via dissection and weighed for gonadosomatic index (GSI) calculations, and oocytes from one lobe preserved in Serra's fixative. Total numbers of late-stage vitellogenic oocytes exhibiting a germinal vesicle were then quantified. In a second exposure, blood plasma samples were collected from adult females and analyzed for vitellogenin concentrations using enzyme-linked immunosorbent assay. Females exposed to both concentrations of levonorgestrel developed male secondary sexual characteristics in a dose-dependent manner, and ovaries contained significantly fewer late stage oocytes. Exposure to 100 ng/L of levonorgestrel resulted in decreased GSI and blood plasma vitellogenin concentrations. The results suggest that female exposure to levonorgestrel alone may have profound effects on reproduction in progestin-contaminated environments. Environ Toxicol Chem 2017;36:3299-3304. © 2017 SETAC.

The progestin levonorgestrel disrupts gonadotropin expression and sex steroid levels in pubertal roach (Rutilus rutilus).

The aim of the present study was to investigate the effects of the synthetic progestin levonorgestrel (LNG) on the reproductive endocrine system of a teleost fish, the roach (Rutilus rutilus). Pubertal roach were exposed for 28 days in a flow-through system to four concentrations of LNG (3, 31, 312, and 3124 ng/l). Both males and females treated with 3124 ng/l LNG exhibited the upregulated levels of vitellogenin and oestrogen receptor 1 mRNA in the liver. At the same concentration, LNG caused a significant upregulation of the mRNA expression of the gene encoding luteinising hormone ß-subunit (lhß) and the suppression of the mRNA expression of the gene encoding follicle-stimulating hormone ß-subunit (fshß) in the pituitary of both male and female roach. A lower LNG concentration (312 ng/l) suppressed mRNA expression of fshß in males only. Females treated with 3124 ng/l LNG exhibited significantly lower plasma 11-ketotestosterone (11-KT) and oestradiol (E2) concentrations, whereas their testosterone (T) level was higher compared with the control. Females exposed to 312 ng/l LNG presented significantly lower plasma E2 concentrations. Males exposed to ≥31 ng/l LNG exhibited significantly reduced 11-KT levels. As determined through a histological analysis, the ovaries of females were not affected by LNG exposure, whereas the testes of males exposed to 31 and 312 ng/l LNG exhibited a significantly higher percentage of spermatogonia B compared with the control. The results of the present study demonstrate that LNG disrupts the reproductive system of pubertal roach by affecting the pituitary gonadotropin expression and the sex steroid levels. This disruption was determined to occur in males after exposure to an environmentally relevant concentration (31 ng/l). Moreover, the highest tested concentration of LNG (3124 ng/l) exerted an oestrogenic effect on fish of both sexes.

Occurrence, fate and removal of synthetic oral contraceptives (SOCs) in the natural environment: a review.

Synthetic oral contraceptives (SOCs) are a group of compounds with progestagenic and/or androgenic activities, with some also possessing estrogenic activities. Recent research has documented that some of these emerging contaminants have adverse effects on aquatic organisms at very low concentrations. To facilitate the evaluation of their latent risks, published works on their occurrence and fate in the environment are reviewed. Androgenic/progestagenic relative potencies or relative binding affinity of these SOCs as well as their physicochemical properties and toxicity are summarized. Appropriate analytical methods are outlined for various environmental sample types, including methods of sample preparation and limit of detection/quantification (LOD/LOQ). Finally results on their occurrence and fate in wastewater treatment plants (WWTPs) and other environments are critically examined.

Deletion of cyclooxygenase 2 in mouse mammary epithelial cells delays breast cancer onset through augmentation of type 1 immune responses in tumors.

Inhibition of cyclooxygenase (COX) 2, which is associated with >40% of breast cancers, decreases the risk of tumorigenesis and breast cancer recurrence. To study the role of COX-2 in breast cancer, we engineered mice that lack selectively mammary epithelial cell (MEC) COX-2 (COX-2 KO(MEC)). Compared with wild type (WT), MEC from COX-2 KO(MEC) mice expressed >90% less COX-2 messenger RNA (mRNA) and protein and produced 90% less of the dominant pro-oncogenic COX-2 product, prostaglandin (PG) E(2). We confirmed COX-2 as the principle source of PGE(2) in MEC treated with selective COX-2 and COX-1 inhibitors. Tumors were induced in mice using medroxyprogesterone acetate and 7,12-dimethylbenz[a]anthracene. Breast cancer onset was significantly delayed in COX-2 KO(MEC) compared with WT (P = 0.03), equivalent to the delay following systemic COX-2 inhibition with rofecoxib. Compared with WT, COX-2 KO(MEC) tumors showed increased mRNA for Caspase-3, Ki-67 and common markers for leukocytes (CD45) and macrophages (F4/80). Analysis of multiple markers/cytokines, namely CD86, inducible nitric oxide synthase (iNOS), interleukin-6, tumor necrosis factor α (TNFα) and Tim-3 indicated a shift toward antitumorigenic type 1 immune responses in COX-2 KO(MEC) tumors. Immunohistochemical analysis confirmed elevated expression of CD45, F4/80 and CD86 in COX-2 KO(MEC) tumors. Concordant with a role for COX-2 in restraining M1 macrophage polarization, CD86 and TNFα expression were offset by exogenous PGE(2) in bone marrow-derived macrophages polarized in vitro to the M1 phenotype. Our data reveal the importance of epithelial COX-2 in tumor promotion and indicate that deletion of epithelial COX-2 may skew tumor immunity toward type 1 responses, coincident with delayed tumor development.

The influence of gill and liver metabolism on the predicted bioconcentration of three pharmaceuticals in fish.

The potential for xenobiotic compounds to bioconcentrate is typically expressed through the bioconcentration factor (BCF), which has gained increased regulatory significance over the past decade. Due to the expense of in vivo bioconcentration studies and the growing regulatory need to assess bioconcentration potential, BCF is often calculated via single-compartment models, using K(OW) as the primary input. Recent efforts to refine BCF models have focused on physiological factors, including the ability of the organism to eliminate the compound through metabolic transformation. This study looks at the ability of in vitro biotransformation assays using S9 fractions to provide an indication of metabolic potential. Given the importance of the fish gill and liver in metabolic transformation, the metabolic loss of ibuprofen, norethindrone and propranolol was measured using rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) gill and liver S9 fractions. Metabolic transformation rates (k(M)) were calculated and integrated into a refined BCF model. A significant difference was noted between BCF solely based on K(OW) and BCF including k(M). These studies indicate that the inclusion of k(M) in BCF models can bring predicted bioconcentration estimates closer to in vivo values.

Protective role of nordihydroguaiaretic acid (NDGA) against the genotoxic damage induced by ethynodiol diacetate in human lymphocytes in vitro.

Antioxidants and plant products are reported to reduce the genotoxic damage of steroids. In our present study we have tested different dosages of nordihydroguaiaretic acid (NDGA) against the genotoxic damage induced by ethynodiol diacetate in the presence of S9 mix. Treatments with nordihydroguaiaretic acid (NDGA) results in the reduction of the genotoxic damage. A significant decrease was observed at all the tested doses of NDGA in sister chromatic exchanges of number of abnormal cells. The results suggest a protective role of NDGA against the genotoxic damage.

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.

Oxidation of bisphenol A, 17beta-estradiol, and 17alpha-ethynyl estradiol and byproduct estrogenicity.

A human breast cancer cell line (MCF-7) was used to investigate the cumulative estrogenicity profiles elicited during the oxidation of three estrogenic compounds [bisphenol A (BPA), 17beta-estradiol (E2), and 17alpha-ethynyl estradiol (EE2)]. High-performance liquid chromatography (HPLC) with a method detection limit (MDL) of approximately 1 nM was used to measure the initial and final concentrations of test compounds during oxidation. Both chlorination and ozonation removed from 75% to >99% of the test compounds in distilled water. Increasing contact time and chlorination dose improved compound removal. Chlorination byproducts of BPA, E2, and EE2 elicited low levels of estrogenicity over an extended period of time. For equivalent molar oxidant dosages, ozone and chlorine had comparable residual proliferative effect values and >99% loss of the parent compounds. For oxidation studies of estrogenic chemicals, ammonium chloride was found to adequately quench residual chlorine without interfering with cell culture assay. Oxidation of test compounds with chlorine and ozone resulted in a similar estrogenicity trend, with a relative higher level of estrogenicity elicited during the early phases of oxidation, which gradually dissipated over the extended exposure time to a stable point. Oxidation with ozone resulted in the rapid transformation of test compounds, reaching a stabilized estrogenic level in 10 min, whereas for chlorination it took more than 120 min for elicited estrogenicity to stabilize.

[Estrogenic and contraceptive activity of 17-R-hydroximino derivatives of 9alpha-hydroxy-11beta-nitroxyestrones]. / Estrogennaia i kontratseptivnaia aktivnost' 17-R-oksiminoproizvodnykh 9alpha-oksi-11beta-nitroksiéstronov.

The estrogenic or uterotropic (UA) and contraceptive (CA) activity of two modified steroids representing 17-R-oximino-9 alpha-oxy-11 beta-nitroxyestrone derivatives (compounds I and II) was experimentally studied by oral administration in female rats in comparison to ethynylestradiol (EED) and mestranol. Compounds I and II showed significant UA (about half that of EED and mestranol) and a pronounced contraceptive effect (exceeding that of mestranol). The dissociation of the drug effects for CA is also indicated by the index of contraception (IC) or the CA/UA ratio. The administration of compounds I and II did not lead to a loss of animals in the acute toxicity tests on female mice.

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.

Behaviour and occurrence of estrogens in municipal sewage treatment plants--II. Aerobic batch experiments with activated sludge.

Aerobic batch experiments containing a diluted slurry of activated sludge from a real sewage treatment plant (STP) near Frankfurt/Main were undertaken, in order to investigate the persistence of natural estrogens and contraceptives under aerobic conditions. The batch experiments showed that while in contact with activated sludge the natural estrogen 17 beta-estradiol was oxidized to estrone, which was further eliminated in the batch experiments in an approximate linear time dependence. Further degradation products of estrone were not observed. 16 alpha-hydroxyestrone was rapidly eliminated, again without detection of further degradation products. The contraceptive 17 alpha-ethinylestradiol was principally persistent under the selected aerobic conditions, whereas mestranol was rapidly eliminated and small portions of 17 alpha-ethinylestradiol were formed by demethylation. Additionally, two glucuronides of 17 beta-estradiol (17 beta-estradiol-17-glucuronide and 17 beta-estradiol-3-glucuronide) were cleaved in contact with the diluted activated sludge solution and thus 17 beta-estradiol was released. The glucuronidase activity of the activated sludge was further confirmed by the cleavage of 4-methylumbelliferyl-beta-D-glucuronide (MUF-beta-glucuronide) in a solution of a activated sludge slurry and Milli-Q-water (1:100, v/v). The turnover rate obtained was approximately steady state, with a turnover rate of 0.1 mumol/l for the released MUF. Hence, it is very likely that the glucuronic acid moiety of 17 beta-estradiol glucuronides and other estrogen glucuronides become cleaved in a real municipal STP, so that the concentrations of the free estrogens increase.

Behavior and occurrence of estrogens in municipal sewage treatment plants--I. Investigations in Germany, Canada and Brazil.

The developed method enables the quantification of estrogens in sewage samples down to 1 ng/l and in river water down to 0.5 ng/l. Mean recoveries of the analytes in ground water after SPE extraction, clean-up and derivatization generally exceeded 75%. The determined R.S.D. varied from 0 to 14% at a spiking level of 0.05 microgram/l. Even in the raw influent and the final effluent from municipal STPs the mean recoveries of estrogens were mostly above 70%. Using this method the behavior and occurrence of natural estrogens and synthetic contraceptives in municipal sewage treatment plants (STP) were investigated in German and Canadian facilities. In the sewage of a German municipal STP close to Frankfurt/Main 17 beta-estradiol and estrone were determined, with mean concentrations of 0.015 microgram/l and 0.027 microgram/l, respectively. In two investigated municipal STPs, 17 beta-estradiol and 16 alpha-hydroxyestrone were eliminated with a higher efficiency than 17 alpha-ethinylestradiol and estrone. In Canadian and German STP discharges estrone, 17 beta-estradiol, 17 alpha-ethinylestradiol and 16 alpha-hydroxyestrone were frequently detected within the lower ng/l-range. A maximum concentration was found for estrone with 70 ng/l. In 15 investigated German rivers and streams only estrone was present with a maximum concentration of 1.6 ng/l.

Effects on hemostasis after two-year use of low dose combined oral contraceptives with gestodene or levonorgestrel.

We studied 67 healthy women who were randomly allocated to receive third generation gestodene (Gynera) or second generation levonorgestrel (Microgynon 30) combination of low-dose estrogen oral contraceptives (OCs) for their hemostatic effects over 2 years. Hemostatic changes were apparent within 3 months of OC use. Hematocrit (Hct) was not affected, but hemoglobin (Hb) concentration decreased by 18 months. Shortened prothrombin time (PT) and activated plasma thromboplastin time (APTT) were associated with elevated fibrinogen within the 12-month use of both OCs. Factor VII was reduced only in Micro 30 during the 18 months of use. Enhanced thrombin-antithrombin (TAT)-complex level was seen at 18 months of Gynera use. Prothrombin fragment1+2 (F1+2) rise was seen at 3 months with Micro 30. Reduced antithrombin III (ATIII) activity was seen at 18 months with Gynera and at 24 months with Micro 30. Increased protein C activity was seen at 3 months and reduced protein S occurred at 18 months of Gynera use. Tissue plasminogen activator (t-PA) activity was enhanced for 6 months in both OCs with raised D-dimer levels for 12 months with Gynera and 6 months with Micro 30. Decreased t-PA antigen was seen at 18 months and decreased urokinaselike plasminogen activator (u-PA) antigen occurred throughout the 24 months of both OCs use. Enhanced u-PA activity was only seen in Gynera users. Elevated plasminogen levels were apparent throughout both OCs use. PAI-1 levels were significantly decreased with Micro 30. With Gynera, the decreased PAI-1 activity was seen only at 18 months and PAI-1 antigen at 12 months. No change in platelets and von Willebrand factor (vWF) were seen in long-term OC use except that beta-thromboglobulin (beta-TG) showed decreased trends reaching statistical significance by 18 and 24 months of Micro 30 use and by 24 months of Gynera use. A further significant decrease in beta-TG, u-PA antigen, ATIII, and protein S levels were seen 3 months after pill stoppage compared with pretreatment levels. Activated protein C resistance (APCR) was negative in all subjects before and during OC use. The study indicated dynamic balance between coagulation and fibrinolysis with no endothelial activation. However, because some hemostatic markers showed wide fluctuations during OC use, a longer term study is warranted to investigate any adverse hemostatic changes that might enhance the risks of venous thromboembolism in Asian subjects known to be less prone to thrombosis.

Role of oral contraceptive in atherosclerosis.

The possibility that risk of a atherosclerosis complication increases with oral contraceptive use was examined by studying the effect of oral pill containing 0.067 mg menstranol and 0.667 mg ehtynodiol diacetate/kg body weight on the metabolism of lipids in female rats fed a hypercholesterolemic diet for three months. Experimental group clearly exhibited higher levels of triglycerides and cholesterol in plasma and tissues, increase in aorta observed to be two folds. Increased hepatic cholesterogenesis was noted with treatment of oral contraceptive as indicated by higher activity of HMG-CoA reductase. Activity of lipoprotein lipase of extrahepatic tissue was depressed in experimental group. Activity of plasma LCAT, an enzyme involved in the transport of cholesterol from tissues, was also lower with treatment of oral contraceptive. However, activity of malic enzyme and glucose-6-phosphate dehydrogenase enhanced considerably with administration of oral pill. The increase in plasma and aortic cholesterol levels, increase in LDL+VLDL cholesterol and considerable decrease in HDL cholesterol in animals treated with oral contraceptives and fed with atherogenic diet, indicates that prolonged administration of oral pill may predispose towards atherosclerosis.

The toxic pathophysiology of retinol hypervitaminosis and norethisterone enantate in rats.

The use of the long acting contraceptive was accompanied by clinically overt toxic manifestations on the liver, glucose tolerance, in addition to the danger of carcinogenicity. Also chronic hypervitaminosis "A" leads to a variety of toxic manifestations of obscure mechanism. The objective of this experimental work was directed to study the toxicity of the isolated and combined augmented doses of the two therapeutic agents on the female albino rat. The results of the present study evidenced that the long acting contraceptive norethisterone enantate is potentially hepato- and nephro-toxic. More damage to the liver; and luteinization of theca and stroma cells of the ovaries occurred as a result of retinol hypervitaminosis. The brunt of toxicity and damage on the test organs after coadministration of both chemicals proved to be synergetic except on the ovarian tissues.