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.

Norethindrone alters mating behaviors, ovary histology, hormone production and transcriptional expression of steroidogenic genes in zebrafish (Danio rerio).

The impact of progestins (i.e. synthetic forms of progesterone) on aquatic organisms has drawn increasing attention due to their widespread occurrence in the aquatic environments and potential effects on the endocrine system of fish. In this study, the effects of norethindrone (NET, a progestin) on the reproductive behavior, sex hormone production and transcriptional expressions were evaluated by exposing female zebrafish to NET at 0, 3.1, 36.2 and 398.6 ng L-1 for 60 days. Results showed that NET impaired the mating behaviors of female at 36.2 and 398.6 ng L-1 exhibited by males and increased the frequency of atretic follicular cells in the ovary exposed to NET at 398.6 ng L-1. As for sex hormones, plasma testosterone concentration in zebrafish increased, while estradiol concentration decreased. Up-regulation of genes (Npr, Mpra, Mprß, Fshß, Lß, Tshb, Nis and Dio2) was detected in the brain of fish exposed to NET at 398.6 ng L-1. The transcriptional levels of genes (Esr1, Vtg1, Ar, Cyp19a, Cyp11b and Ptgs2) were generally inhibited in the ovary of zebrafish by NET at 398.6 ng L-1. Moreover, the transcripts of genes (Vtg1, Esr1, Ar and Pgr) in the liver were reduced by NET at 36.2 and 398.6 ng L-1. Our findings suggest that NET can potentially diminish the of fish populations not only by damaging their reproductive organs, but also by altering their mating behavior through the changes in the expressions of genes responsible for the production of sex hormones.

The progestin norethindrone alters growth, reproductive histology and gene expression in zebrafish (Danio rerio).

The aim of this study was to assess the long-term effects of synthetic progestin norethindrone (NET) on the growth, reproductive histology, and transcriptional expression profiles of genes associated with the hypothalamic-pituitary-gonadal (HPG) axis and germ cells in adult zebrafish. Adult zebrafish were exposed to 7, 84 and 810 ng/L NET for 90 days. The results showed that exposure to 810 ng/L NET caused a significant decrease in growth of females and males. The ovary weight and GSI was significantly reduced by NET at concentrations of 84 or 810 ng/L, which came along with the delay of ovary maturation in females. However, NET at all treatments resulted in acceleration of sperm maturation in males. In the ovaries of females, a strong inhibition of cyp19a1a gene was observed following exposure to NET at 810 ng/L. Similarly, NET at the highest treatment led to a significant down-regulation of cyp17, cyp19a1a, vasa, nanos1, dazl and dmc1 genes in the testes of males. Taken together, the overall results demonstrated that NET could impact growth and gonadal maturation, with significant alterations of transcriptional expression genes along HPG axis and germ cells.

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.

Resveratrol ameliorates prenatal progestin exposure-induced autism-like behavior through ERß activation.

Background: Recent literatures indicate that maternal hormone exposure is a risk factor for autism spectrum disorder (ASD). We hypothesize that prenatal progestin exposure may counteract the neuroprotective effect of estrogen and contribute to ASD development, and we aim to develop a method to ameliorate prenatal progestin exposure-induced autism-like behavior. Methods: Experiment 1: Prenatal progestin exposure-induced offspring are treated with resveratrol (RSV) through either prenatal or postnatal exposure and then used for autism-like behavior testing and other biomedical analyses. Experiment 2: Prenatal norethindrone (NET) exposure-induced offspring are treated with ERß knockdown lentivirus together with RSV for further testing. Experiment 3: Pregnant dams are treated with prenatal NET exposure together with RSV, and the offspring are used for further testing. Results: Eight kinds of clinically relevant progestins were used for prenatal exposure in pregnant dams, and the offspring showed decreased ERß expression in the amygdala with autism-like behavior. Oral administration of either postnatal or prenatal RSV treatment significantly reversed this effect with ERß activation and ameliorated autism-like behavior. Further investigation showed that RSV activates ERß and its target genes by demethylation of DNA and histone on the ERß promoter, and then minimizes progestin-induced oxidative stress as well as the dysfunction of mitochondria and lipid metabolism in the brain, subsequently ameliorating autism-like behavior. Conclusions: We conclude that resveratrol ameliorates prenatal progestin exposure-induced autism-like behavior through ERß activation. Our data suggest that prenatal progestin exposure is a strong risk factor for autism-like behavior. Many potential clinical progestin applications, including oral contraceptive pills, preterm birth drugs, and progestin-contaminated drinking water or seafood, may be risk factors for ASD. In addition, RSV may be a good candidate for clinically rescuing or preventing ASD symptoms in humans, while high doses of resveratrol used in the animals may be a potential limitation for human application.

The progestin norethisterone affects thyroid hormone-dependent metamorphosis of Xenopus laevis tadpoles at environmentally relevant concentrations.

Previously, levonorgestrel (LNG) has been shown to be an endocrine disruptor of the amphibian thyroid system. In the present study, we investigated whether anti-thyroidal effects are a common property of progestins other than LNG. Premetamorphic Xenopus laevis tadpoles were exposed to norethisterone (NET) and dienogest DIE (each at 0.1-10nM) and LNG (10nM) until completion of metamorphosis. LNG and NET at all concentrations caused a significant developmental retardation whereas DIE did not impair time to metamorphosis. In LNG and 10nM NET exposed animals, tsh mRNA levels increased considerably later than the developmental delay occurred and thyroid histopathology showed no signs of TSH-hyperstimulation. Instead, thyroid glands from these treatments appeared inactive in producing thyroid hormones. Thyroidal transcript levels of dio2 and dio3 were increased by treatments with LNG and NET at 1nM and 10nM, whereas iyd mRNA was reduced by LNG and 10nM NET. Expression of slc5α5 was not changed by any treatment. Effects of DIE differed from those induced by LNG and NET. No developmental delay was measurable; however, tshß and dio2 mRNAs were increased in pituitary glands of tadpoles exposed to 1.0nM and 10nM DIE. Thyroid histopathology displayed no abnormalities and thyroidal mRNA expression of the genes analyzed (slc5α5, iyd, dio2, dio3) was not changed by DIE. Overall, our results provide evidence that the anti-thyroidal effects already known from LNG are also present in another progestin, namely NET, even at environmentally relevant concentrations. In conclusion we suggest that progestins do not only pose an environmental risk in terms of their impact on reproductive success of aquatic vertebrates, but also with respect to their anti-thyroidal properties affecting amphibian metamorphosis.

Chemical analysis of fish bile extracts for monitoring endocrine disrupting chemical exposure in water: Bisphenol A, alkylphenols, and norethindrone.

The present study determined concentrations of estrogenic bisphenol A (BPA), nonylphenol, octylphenol (4-tert-octylphenol), butylphenol (4-tert-butylphenol), and progestogenic norethindrone by liquid chromatography-tandem mass spectrometry in bile extracts from field fish from the Xin'an River and market fish in Shanghai, China. Compared with the field fish, endocrine disrupting chemical (EDC) concentrations in market fish bile were at relatively high levels with high detectable rates. The average concentrations of BPA, nonylphenol, 4-tert-octylphenol, 4-tert-butylphenol, and norethindrone in field fish bile were 30.1 µg/L, 203 µg/L, 4.69 µg/L, 7.84 µg/L, and 0.514 µg/L, respectively; in market fish bile they were 240 µg/L, 528 µg/L, 76.5 µg/L, 12.8 µg/L, and 5.26 µg/L, respectively; and in the surface water of Xin'an River they were 38.8 ng/L, 7.91 ng/L, 1.98 ng/L, 2.66 ng/L, and 0.116 ng/L, respectively. The average of total estrogenic activity of river water was 3.32 ng/L estradiol equivalents. High bioconcentration factors (BCFs) were discovered for all 5 EDCs (≧998-fold) in field fish bile. Furthermore, the authors analyzed the BCF value of BPA in fish bile after 30-d exposure to environmentally relevant concentrations of BPA in the laboratory, and the analysis revealed that BCF in fish bile (BCF(Fish bile)) changed in an inverse concentration-dependent manner based on the log10-transformed BPA concentration in water. Strikingly, the data from the field study were well fitted within this trend. The data together suggested that analysis of fish bile extracts could be an efficient method for assessing waterborne EDCs exposure for aquatic biota.

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.

Reproductive responses in fathead minnow and Japanese medaka following exposure to a synthetic progestin, Norethindrone.

Synthetic progestins, such as Norethindrone (NET), are common ingredients in oral contraceptives and in treatment for post-menopausal problems. Given the widespread use of oral contraceptives and post-menopausal treatments, several reports have targeted and identified progestins in aquatic environments. In fish, progestins play an important role in the stimulation of oocyte final maturation and ovulation in females, stimulation of spermiation and sperm motility in males, and the initiation of meiosis in both sexes. They also have a role as pheromones in some species. Given the pivotal role that progestins play in reproduction, their appreciable daily dose (i.e. microg to mg range in contraceptives and hormone replacement therapies) and continuous use pattern, it is important to understand the potential risk these compounds pose once discharged into the aquatic environment. Since little published data are available on this class of compounds, our research focused on the reproductive effects of NET on the fathead minnow and Japanese medaka. A 28 day static-renewal reproduction study with Japanese medaka indicated that NET produces a significant decrease in fecundity at aqueous concentrations >or=25 ng/L. A 21 day flow-though fathead minnow reproduction study also demonstrated that NET causes a significant decrease in fecundity in the low ng/L range. Fathead minnow morphological changes (i.e. female fin spots) suggest that NET exposure may have a potent androgenic effect on fish; however, plasma 11-Ketotestosterone (11-KT) concentrations were reduced in males at the highest exposure concentration. Collectively, these data indicate that further investigation of reproductive responses associated with synthetic progestins is warranted.

Novel genotoxicity assays identify norethindrone to activate p53 and phosphorylate H2AX.

Norethindrone is a commonly used drug for contraception and hormone replacement therapy, whose carcinogenic potential is still controversial. We applied a novel and particularly sensitive method to screen for DNA damage with special attention to double-strand breaks (DSBs) and identified norethindrone to be likely genotoxic and therefore potentially mutagenic: a p53-reporter assay served as a first, high-throughput screening method and was followed by the immunofluorescent detection of phosphorylated H2AX as a sensitive assay for the presence of DSBs. Norethindrone at concentrations of 2-100 microg/ml activated p53 and phosphorylated H2AX specifically and in a dose-dependent manner. No p53 activation or H2AX phosphorylation was detected using a panel of structurally/functionally related drugs. The overall amount of DNA damage induced by norethindrone was low as compared with etoposide and ionizing radiation. Consistently, norethindrone treatment did not cause a cell cycle arrest. DSBs were not detected with the neutral comet assay, a less sensitive method for DSB assessment than H2AX phosphorylation. Our findings in the p53-reporter and gamma-H2AX assays could not be ascribed to common DSB-causing artifacts in standard genotoxicity screening, including drug precipitation, high cytotoxicity levels and increased apoptosis. Therefore, our study suggests that norethindrone induces DSBs in our experimental setting, both complementing and adding a new aspect to the existing literature on the genotoxic potential of norethindrone. As the effective concentrations of norethindrone used in our assays were approximately 100- to 1000-fold higher than therapeutical doses, the significance of these findings with regard to human exposure still remains to be determined.

Toxicity of 17alpha-ethynylestradiol and norethindrone, constituents of an oral contraceptive pill to the swimming and reproduction of cladoceran Daphnia magna, with special reference to their synergetic effect.

Toxicity of 17alpha-ethynylestradiol (EE2) and norethindrone (NOR), constituents of low dosage oral contraceptives, was assessed for the freshwater cladoceran Daphnia magna. Acute toxicity tests showed that 5 ppm of EE2, the highest concentration in this study, never inhibited swimming, whereas NOR inhibited swimming at >3 ppm: 48 h EC(50) for NOR was 6.41 ppm. Chronic toxicity tests were carried out for 25 days by measuring the number of offspring, moltings and sex ratios of neonates at 20, 100 and 500 ppb. EE2>100 ppb significantly decreased the number of offspring to 75% of the control; however, no effect was observed in molting and sex ratios at <500 ppb. NOR did not affect reproduction and sex ratios at <500 ppb. Mixture of EE2 (5.88 ppb) and NOR (94.12 ppb) also significantly decreased the number of offspring to 57% of the control. This result indicates the importance of examining synergetic effects of chemicals in the context of natural environments which face exposure to myriad chemicals.

Oral contraceptives worsen endotoxin-induced liver injury in rats.

BACKGROUND: Oral contraceptives are widely used; however, these drugs occasionally cause liver injury. Recently, it was reported that estriol worsens alcoholic liver injury by the mechanism involving activation of Kupffer cells as a result of gut-derived endotoxin. However, the relationship between oral contraceptives and endotoxin-induced liver injury has not been elucidated. Here we show that oral contraceptives sensitize Kupffer cells via a mechanism dependent on increased gut permeability to endotoxin. METHODS: Female Wistar rats (200-250 g) were given intraperitoneally a combination of estradiol (35 ng/kg of 17 alpha-Ethynylestradiol) and progesterone (2 microg/kg of Norethindrone), each dose being similar to that contained in oral contraceptives (EP treatment). After 24 hr, a sublethal dose of lipopolysaccharide (LPS; 5 mg/kg) was injected via the tail vein. In some experiments, antibiotics (150 mg/kg/day of polymyxin B and 450 mg/kg/day of neomycin) were administered orally for 4 days before EP treatment. Gut permeability was measured in isolated segments of ileum by translocation of horseradish peroxidase. Kupffer cells were isolated and cultured in RPMI 1640 + 10% fetal bovine serum for 24 hr. After addition of LPS (100 ng/ml) to the culture medium, intracellular calcium concentration ([Ca2+](i) ) was measured with fura-2. RESULTS: Liver histology in rats given EP treatment intraperitoneally followed by an injection of LPS (5 mg/kg) 24 hr later revealed pronounced liver damage with massive necrosis. Whereas mean values of alanine aminotransferase (ALT) in the control, nontreated rats were 30 +/- 6 IU/liter, ALT increased to 75 +/- 21 IU/liter 24 hr after LPS injection. This increase was aggravated 6-fold (483 +/- 118 IU/liter; p< 0.05) by EP treatment. The EP treatment-induced increase in ALT was completely blocked by antibiotics (82 +/- 26 IU/liter; p< 0.05). Gut permeability was increased approximately 10-fold with EP treatment. This increase in gut permeability was not altered by treatment with nonabsorbable antibiotics. In isolated Kupffer cells, LPS increased [Ca2+](i) of Kupffer cells in control rats from basal levels (36 +/- 8 nmol/liter) to 100 +/- 8 nmol/liter. In contrast, the LPS-induced [Ca2+](i) elevation was approximately 3-fold greater in the group given EP treatment before 24 hr (305 +/- 17 nmol/liter; p< 0.05). This increase was also blocked completely by treatment with antibiotics (128 +/- 13 nmol/liter). Similar results were obtained for LPS-induced tumor necrosis factor-alpha production by Kupffer cells from either control or EP treatment group. The increased tumor necrosis factor-alpha production as a result of EP treatment was blocked completely by antibiotics. CONCLUSIONS: These results indicate that EP treatment in vivo sensitizes Kupffer cells to LPS via a mechanism dependent on the portal increase of gut-derived endotoxin. This event suggests that oral contraceptives exacerbate alcoholic liver injury.

Pharmacology and toxicology of ethinyl estradiol and norethindrone acetate in experimental animals.

For over 30 years various combinations of synthetic estrogens and progestins have been used in oral contraceptive formulations. Ethinyl estradiol (EE) and norethindrone acetate (NA) are common synthetic hormones used in oral contraceptives such as Loestrin, Brevicon, Ortho-Novum, Norlestrin, and Norinyl. In recent years these oral contraceptives have been considered for development in other therapeutic indications. Given the use of these agents for other clinical indications with different and larger target populations, an updated comprehensive review of the toxicology literature of estrogens and progestins is warranted. This review will summarize available data on the pharmacology and toxicology of estrogens and progestins with an emphasis on the specific synthetic hormones EE and NA. Ethinyl estradiol and norethindrone acetate alone or in combination, possess low acute and chronic toxicity. In some studies, EE and/or NA increased the incidence of specific tumors in susceptible strains of rodents and dogs, but not monkeys. These agents are not teratogenic when given in combination. Alone EE and NA have clastogenic properties. Overall, the animal data demonstrates that long-term exposure to EE and NA formulations pose very little health risks to humans.

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.

Induction of micronuclei and of enzyme-altered foci in the liver of female rats exposed to progesterone and three synthetic progestins.

Progesterone (PG) and three structurally similar synthetic progestins-norethisterone (NE), allylestrenol (AE), and dydrogesterone (DG)-have been compared for their ability to induce the formation of micronuclei and of enzyme-altered foci in the liver of female rats. In the micronucleus assay, carried out in rats given a single p.o. dose of 100 mg kg-1 3 days before partial hepatectomy and sacrificed for cell sampling 2 days later, the frequency of micronucleated hepatocytes was 3.5-fold higher than in controls with PG, 2.8-fold with DG, 2.2-fold with NE and 2.1-fold with AE, but the increase was statistically significant only for PG. In the liver foci assay, performed to evaluate the tumor initiating activity of p. o. dosing with 100 mg kg-1 once a week for 6 successive weeks, the values of the number and area of gamma-glutamyltranspeptidase-positive foci were, as compared to controls, 15.9- and 100-fold higher with NE, and 13.9- and 52-fold higher with AE, but only the increase of area produced by NE was statistically significant; PG and DG did not display in this test any activities. Considered together with previous findings, these results suggest that NE might be biotransformed in the liver into reactive species and thus behave as a weak genotoxic agent.

In vitro cytotoxicity and teratogenicity of norethisterone and levonorgestrel released from hollow nylon monofilaments.

Direct delivery of progestogens to the uterus may be of use in the treatment of dysfunctional uterine bleeding and the sequelae of the menopause as it has the potential to overcome the problems of systemic administration. This study characterised the release of norethisterone and levonorgestrel into an aqueous medium from hollow nylon fibres with dimensions suitable for easy insertion through the post-menopausal cervix. Cell culture techniques were used to assess potential cytotoxic and teratogenic effects. The results demonstrated that the hollow fibres released norethisterone and levonorgestrel at mean rates of 0.5 and 0.6 micrograms/day over 14 days, respectively. There were indications, however, that both steroids were toxic to endometrial cells at concentrations of approximately 5 micrograms/ml, and both drugs showed signs of potential teratogenicity at 10 micrograms/ml. Delivery of the same doses of norethisterone using the hollow fibres reduced the effects on the endometrial and fetal cells. Delivery of levonorgestrel from the hollow fibres had no effect on the endometrial cell toxicity but potentiated the effects on the fetal cells. These results suggest that the hollow nylon fibres may be of use for the delivery of norethisterone to the uterus but that they are inappropriate for the delivery of levonorgestrel.

Effects of ethinylestradiol-norethisterone acetate combinations on the ultrastructure of liver, kidney, ovary and endometrium cells.

Sexually mature female Wistar rats were given 0.05 mg ethinylestradiol (EE) + 0.5 mg norethisterone acetate (NET), or 0.1 mg EE + 1.0 mg NET for 6 or 12 sexual cycles, i.e., for 30 or 60 days. Rat hepatocytes, renal proximal tubule cells, ovarian granulosa cells and endometrial gland cells showed ultrastructural changes that correlated with the dose and duration of EE/NET treatment. The most common ultrastructural features were as follows: reduced RER, mitochondrial damage, and an increased number of lysosomes. The study has shown that EE/NET combinations used in oral contraception produce ultrastructural lesions, which may impair protein biosynthesis and energy production processes, and may simultaneously enhance cellular catabolism.