Levonorgestrel causes feminization and dose-dependent masculinization in medaka fish (Oryzias latipes): Endocrine-disruption activity and its correlation with sex reversal.

The effect of a synthetic progestin, levonorgestrel (LNG), on the sex of exposed embryos was examined in medaka fish (Oryzias latipes). The aims of this study are to clarify the dual effect of LNG on sex and the correlation with its androgenic/estrogenic potential in medaka. LNG exposure causes significant dose-dependent masculinization (0.1-100 µg/L), whereas a decrease in the masculinization ratio is observed at 100 µg/L. LNG also causes significant feminization at 1-100 µg/L, but not in a dose-dependent manner. Exposure of estrogen-responsive gene (choriogeninH-EGFP) transgenic embryos to 100 µg/L LNG produced significant fluorescent signals in hatched fry. In vitro transcriptional assays indicated that LNG at 10-7-10-5 M induced significant activity for estrogen receptor (ESR)2a and ESR2b, but not for ESR1. In pre-self-feeding fry at 5 days post hatching (dph), 1-100 µg/L LNG caused a significant increase in the mRNA of choriogeninH, irrespective of genetic sex. Moreover, LNG (10-10-10-5 M) also caused a significant increase in the transcriptional activity of androgen receptor (AR) α and ARß in vitro, and 0.1 µg/L LNG significantly increased the mRNA levels of a testis-differentiation initiation factor, gonadal soma-derived factor (gsdf), as an androgen-upregulated and estrogen-downregulated gene, in 5 dph XX fry to levels similar to those in the control XY fry. However, 100 and 10 µg/L LNG suppressed or did not induce gsdf mRNA expression in XY and XX fry, respectively. Together, these findings show that LNG exerts estrogenic and androgenic activities in different concentration ranges, which correlate with the ratio of LNG-induced sex reversal. These results suggest for the first time, that medaka exposure to LNG can induce masculinization and feminization, based on the balance between androgenic and estrogenic activities, and the protocol applied in this study represents an alternative to the traditional animal model used to screen for endocrine-disrupting potential.

Estrogen-progestin oral contraceptive and nicotine exposure synergistically confers cardio-renoprotection in female Wistar rats.

Approximately fifty percent of premenopausal women who smoke cigarettes or on nicotine replacement therapy are also on hormonal contraceptives, especially oral estrogen-progestin. Oral estrogen-progestin therapy has been reported to promote insulin resistance (IR) which causes lipid influx into non-adipose tissue and impairs Na+/K+ -ATPase activity, especially in the heart and kidney. However, the effects of nicotine on excess lipid and altered Na+/K+ -ATPase activity associated with the use of estrogen-progestin therapy have not been fully elucidated. This study therefore aimed at investigating the effect of nicotine on cardiac and renal lipid influx and Na+/K+ -ATPase activity during estrogen-progestin therapy. Twenty-four female Wistar rats grouped into 4 (n = 6/group) received (p.o.) vehicle, nicotine (1.0 mg/kg) with or without estrogen-progestin steroids (1.0 µg ethinyl estradiol and 5.0 µg levonorgestrel) and estrogen-progestin only daily for 6 weeks. Data showed that estrogen-progestin treatment or nicotine exposure caused IR, hyperinsulinemia, increased cardiac and renal uric acid, malondialdehyde, triglyceride, glycogen synthase kinase-3, plasminogen activator inhibitor-1, reduced bilirubin and circulating estradiol. Estrogen-progestin treatment led to decreased cardiac Na+/K+-ATPase activity while nicotine did not alter Na+/K+-ATPase activity but increased plasma and tissue cotinine. Renal Na+/K+-ATPase activity was not altered by the treatments. However, all these alterations were reversed following combined administration of oral estrogen-progestin therapy and nicotine. The present study therefore demonstrates that oral estrogen-progestin therapy and nicotine exposure synergistically prevents IR-linked cardio-renotoxicity with corresponding improvement in cardiac and renal lipid accumulation, oxidative stress, inflammation and Na+/K+-ATPase activity.

Ethinylestradiol and Levonorgestrel as Active Agents in Normal Skin, and Pathological Conditions Induced by UVB Exposure: In Vitro and In Ovo Assessments.

The link between melanoma development and the use of oral combined contraceptives is not fully elucidated, and the data concerning this issue are scarce and controversial. In the present study, we show that the components of oral contraceptives, ethinylestradiol (EE), levonorgestrel (LNG), and their combination (EE + LNG) ± UVB (ultraviolet B radiation) induced differential effects on healthy (human keratinocytes, fibroblasts, and primary epidermal melanocytes, and murine epidermis cells) and melanoma cells (human-A375 and murine-B164A5), as follows: (i) at low doses (1 µM), the hormones were devoid of significant toxicity on healthy cells, but in melanoma cells, they triggered cell death via apoptosis; (ii) higher doses (10 µM) were associated with cytotoxicity in all cells, the most affected being the melanoma cells; (iii) UVB irradiation proved to be toxic for all types of cells; (iv) UVB irradiation + hormonal stimulation led to a synergistic cytotoxicity in the case of human melanoma cells-A375 and improved viability rates of healthy and B164A5 cells. A weak irritant potential exerted by EE and EE + LNG (10 µM) was assessed by the means of a chick chorioallantoic membrane assay. Further studies are required to elucidate the hormones' cell type-dependent antimelanoma effect and the role played by melanin in this context.

Anti-inflammatory and antithrombotic effects of nicotine exposure in oral contraceptive-induced insulin resistance are glucocorticoid-independent.

BACKGROUND: Reports showed that estrogen-progestin oral contraceptive (COC) or tobacco smoking causes increased risk of cardiovascular diseases (CVD) in premenopausal women. Studies also suggest that nicotine, a major tobacco alkaloid, may worsen or improve atherothrombotic CVD. Altered hemorheology, prothrombotic and pro-inflammatory biomarkers, have been implicated in the development of atherothrombotic CVD events. However, the effect of non-smoking nicotine exposure on these biomarkers during COC treatment is not yet established. We therefore sought to determine the effects of nicotine exposure during COC treatment on these biomarkers, and also tested the hypothesis that the nicotine effects would be glucocorticoid-dependent. METHODS: Female Sprague-Dawley rats aged 10 weeks were given (po) vehicle, low-dose nicotine (0.1mg/kg) or high-dose nicotine (1.0mg/kg) with or without COC steroids (5.0µg/kg ethinylestradiol and 25.0µg/kg levonorgestrel) daily for 6 weeks. RESULTS: COC treatment or nicotine exposure led to increased insulin resistance (IR), hemorheological (blood viscosity, hematocrit and plasma viscosity), prothrombotic (plasminogen activator inhibitor-1), pro-inflammatory (uric acid, C-reactive protein, neutrophil/lymphocyte and platelet/lymphocyte ratios) biomarkers and corticosterone. However, these effects except that on corticosterone were abrogated by nicotine exposure during COC treatment. CONCLUSIONS: Our study indicates that nicotine- or COC-induced IR may be mediated via inflammatory/thrombotic pathway. The results imply that nicotine exposure could impact negatively on atherothrombotic biomarkers in COC non-users, whereas the impact in COC users could be positive. The results also suggest that the anti-inflammatory, antithrombotic and blood viscosity-lowering effects of nicotine exposure during COC use is circulating glucocorticoid-independent.

Developmental exposure to progestins causes male bias and precocious puberty in zebrafish (Danio rerio).

Progestins are aquatic contaminants that in low concentrations can impair fish reproduction. The mechanisms are likely multiple since different progestins interact with other steroid receptors in addition to progesterone receptors. Puberty is the process when animals first acquire the capability to reproduce and it comprises maturation of sperm and eggs. In zebrafish, puberty is initiated around 45days post fertilization (dpf) in females and around 53-55 dpf in males, and is marked by increased production of pituitary gonadotropins. We exposed juvenile zebrafish from 20 to 80 dpf to the androgenic progestin levonorgestrel at concentrations of 5.5, 79 and 834ngL(-1) and to the non-androgenic progestin progesterone at concentrations of 3.7, 77 and 1122ngL(-1), during sexual differentiation and puberty. Levonorgestrel exposure caused 100% males even at the lowest concentration tested whereas progesterone did not affect the sex ratio. Transcript levels of the gonadal genes amh, CYP11B and CYP19a1a indicated that the masculinizing effect of levonorgestrel occurred very rapidly. Transcript concentrations of gonadotropins in pituitaries were low in control fish at 44 dpf, but high at 55 dpf and onward. In fish exposed to levonorgestrel or progesterone gonadotropin transcript concentrations were high already at 44 dpf, indicating that both progestins caused precocious puberty. Gonad histology at 50 dpf confirmed a well advanced sexual maturation, but only in males. Our results show that progestins can affect sexual development in fish and that the androgenic progestin levonorgestrel induces a male phenotype at concentrations similar to those detected in aquatic environments.

Molecular and histological endpoints for developmental reproductive toxicity in Xenopus tropicalis: Levonorgestrel perturbs anti-Müllerian hormone and progesterone receptor expression.

There is an increasing concern regarding the risks associated with developmental exposure to endocrine disrupting chemicals and the consequences for reproductive capability. The present study aimed to refine the Xenopus (Silurana) tropicalis test system for developmental reproductive toxicity by characterising molecular and histological features of sexual development, and to explore effects of exposure to the progestagen levonorgestrel (LNG). Larvae were exposed to LNG (0, 3, 30, 300 ng/L) over the first three weeks of development, encompassing the beginning of gonadal differentiation. mRNA levels of amh (anti-Müllerian hormone), amhr2 (amh receptor 2), ipgr (intracellular progesterone receptor), mpgr beta (membrane progesterone receptor beta), and cyp19a1 (cytochrome p450 19a1) were quantified in larvae and juveniles (4 weeks post-metamorphosis). Relative cyp19a1 and amh expression was used as a molecular marker for phenotypic sex of larvae. Gonadal and Müllerian duct development were characterised histologically in juveniles. Compared to controls, LNG exposure increased the expression of amh and ipgr in male larvae. In juveniles, mpgr beta expression was increased in both sexes and amhr2 expression was decreased in males, implying persistent effects of developmental progestagen exposure on amh and pgr expression signalling. No effects of LNG on the gonadal or Müllerian duct development were found, implying that the exposure window was not critical with regard to these endpoints. In juveniles, folliculogenesis had initiated and the Müllerian ducts were larger in females than in males. This new knowledge on sexual development in X. tropicalis is useful in the development of early life-stage endpoints for developmental reproductive toxicity.

Mixture effects of levonorgestrel and ethinylestradiol: estrogenic biomarkers and hormone receptor mRNA expression during sexual programming.

Synthetic progesterone (progestins) and estrogens are widely used pharmaceuticals. Given that their simultaneous unintentional exposure occurs in wildlife and also in human infants, data on mixture effects of combined exposures to these hormones during development is needed. Using the Xenopus (Silurana) tropicalis test system we investigated mixture effects of levonorgestrel (LNG) and ethinylestradiol (EE2) on hormone sensitive endpoints. After larval exposure to LNG (0.1nM), or EE2 (0.1nM) singly, or in combination with LNG (0.01, 0.1, 1.0nM), the gonadal sex ratio was determined histologically and hepatic mRNA levels of genes encoding vitellogenin (vtg beta1) and the estrogen (esr1, esr2), progesterone (ipgr) and androgen (ar) receptors were quantified using quantitative PCR. All EE2-exposed groups showed female-biased sex ratios and increased vtg beta1 mRNA levels compared with the controls. Compared with the EE2-alone group (positive control) there were no significant alterations in vtg beta1 levels or in sex ratios in the co-exposure groups. Exposure to LNG-alone caused an increase in ar mRNA levels in females, but not in males, compared to the controls and the co-exposed groups, indicating that co-exposure to EE2 counteracted the LNG-induced ar levels. No treatment related impacts on the mRNA expression of esr1, esr2, and ipgr in female tadpoles were found, suggesting that these endpoints are insensitive to long-term exposure to estrogen or progestin. Due to the EE2-induced female-biased sex ratios, the mRNA expression data for the low number of males in the EE2-exposed groups were not statistically analyzed. In conclusion, our results suggest that induced vtg expression is a robust biomarker for estrogenic activity in exposure scenarios involving both estrogens and progestins. Developmental exposure to LNG caused an induction of hepatic ar mRNA expression that was antagonized by combined exposure to EE2 and LNG. To our knowledge this is the first study to report effects of combined exposures to EE2 and LNG during the period of sexual programming.

The synthetic gestagen levonorgestrel impairs metamorphosis in Xenopus laevis by disruption of the thyroid system.

Synthetic gestagens, including levonorgestrel (LNG), are active compounds in contraceptives, and several studies report their occurrence in surface waters. However, information about endocrine-disrupting effects in nontarget organisms is scarce. The present study investigated effects of LNG exposure on thyroid hormone-dependent metamorphosis of Xenopus laevis. Premetamorphic X. laevis tadpoles at Nieuwkoop and Faber (NF) stage 48 were exposed in a flow-through culture system to four LNG concentrations (10(-11), 10(-10), 10(-9), and 10(-8)M) over the period of metamorphosis. At NF 58 and 66, tadpoles were examined sex specifically. Developmental time and organismal responses were recorded and correlated with molecular and histopathological endpoints. Exposure to 10(-8)M LNG caused an inhibition of metamorphosis resulting in developmental arrest at early climax stages as giant tadpoles or tailed frogs. In brain-pituitary tissue of NF 58 tadpoles, gene expression of thyroid-stimulating hormone (ß-subunit; TSHß), TH receptor ß (TRß), and deiodinase type 3 (D3) was not changed. Instead, prolactin (PRL) messenger RNA (mRNA) was significantly increased by 10(-9)M LNG in females and by 10(-8)M LNG in both sexes. In NF 66 tadpoles, mRNA levels of TSHß mRNA were significantly increased in the 10(-9) and 10(-8)M LNG treatment groups indicating a hypothyroid state. No changes of TRß, D3, and PRL gene expression were detected. Histopathological evaluation of thyroid gland sections revealed no typical sign of hypothyroidism but rather an inactivated appearance of the thyroid. In conclusion, our data demonstrate for the first time a completely new aspect of thyroid system disruption caused by synthetic gestagens in developing amphibians.

Effects of some endocrine disruptors on the proliferation and viability of human endometrial endothelial cells in vitro.

Endocrine disrupting chemicals (EDCs) pose a potential threat to human reproductive health. We studied the proliferation and viability of human endometrial endothelial cells (HEECs) in vitro after exposure to 2,2-bis(o,p-chlorophenyl)-1,1,1-trichloroethane (o,p'-DDT), 3,3',4,4'-tetrachlorobiphenyl (CB 77), 3,3',4,4',5-pentachlorobiphenyl (CB 126), di-n-butyl phthalate (DBP), bisphenol A (BPA), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and 17beta-oestradiol, progesterone, 17alpha-ethynyl oestradiol and levonorgestrel. Cell proliferation was studied using immunocytochemistry for PCNA expression and a 5-bromo-2'-deoxyuridine assay. Cell viability was studied by vital staining with propidium iodide and Hoechst 33258. HEECs in primary culture responded with increased proliferation to oestradiol and with decreased proliferation to levonorgestrel and the EDCs. Some EDCs also affected cell viability and increased the proportion of necrotic cells. However, the decrease in proliferation in response to DBP and TCDD cannot be explained by cell death. In light of these results, it is possible that the EDCs could have effects in vivo as well as in vitro, and influence processes involving for example endometrial angiogenesis.

Stearoyl-CoA desaturase is involved in the control of proliferation, anchorage-independent growth, and survival in human transformed cells.

Saturated and monounsaturated fatty acids are the most abundant fatty acid species in mammalian organisms, and their distribution is regulated by stearoyl-CoA desaturase, the enzyme that converts saturated into monounsaturated fatty acids. A positive correlation between high monounsaturated fatty acid levels and neoplastic transformation has been reported, but little is still known about the regulation of stearoyl-CoA desaturase in cell proliferation and apoptosis, as well as in cancer development. Here we report that simian virus 40-transformed human lung fibroblasts bearing a knockdown of human stearoyl-CoA desaturase by stable antisense cDNA transfection (hSCDas cells) showed a considerable reduction in monounsaturated fatty acids, cholesterol, and phospholipid synthesis, compared with empty vector transfected-simian virus 40 cell line (control cells). hSCDas cells also exhibited high cellular levels of saturated free fatty acids and triacylglycerol. Interestingly, stearoyl-CoA desaturase-depleted cells exhibited a dramatic decrease in proliferation rate and abolition of anchorage-independent growth. Prolonged exposure to exogenous oleic acid did not reverse either the slower proliferation or loss of anchorage-independent growth of hSCDas cells, suggesting that endogenous synthesis of monounsaturated fatty acids is essential for rapid cell replication and invasiveness, two hallmarks of neoplastic transformation. Moreover, apoptosis was increased in hSCDas cells in a ceramide-independent manner. Finally, stearoyl-CoA desaturase-deficient cells were more sensitive to palmitic acid-induced apoptosis compared with control cells. Our data suggest that, by globally regulating lipid metabolism, stearoyl-CoA desaturase activity modulates cell proliferation and survival and emphasize the important role of endogenously synthesized monounsaturated fatty acids in sustaining the neoplastic phenotype of transformed cells.

The development and regression of deciduosarcomas and other lesions caused by estrogens and progestins in rabbits.

A series of experiments were conducted to study the histopathological effects of a combination of exogenous estrogens and progestins in mature rabbits. Estradiol (14-45 microg/day) and levonorgestrel (30-233 microg/day) were administered by intravaginal or subdermal Silastic devices for various time intervals to study the development of lesions with time and to determine if lesions regressed following withdrawal of the steroids. The origin of splenic decidual tumors (primary or metastasis from the uterus) was determined by administering the same steroid combination to castrated male rabbits. It was determined that uterine decidualization is present after 7 days of steroid treatment and that neoplasms of decidual cells may appear in the uterus after only 30 days of steroid administration. Decidual changes were observed frequently in uterine arteries, often concurrent with infarct-like areas of necrosis of the uterine wall. Withdrawal of contraceptive steroids for 14-120 days after 60 days' administration resulted in atrophy and disappearance of decidual cells and decidual tumors. Decidual neoplasms developed in the spleen of all castrated male rabbits given subdermal steroids, demonstrating that these tumors can arise as primary neoplasms of the spleen. The foregoing lesions appear to be peculiar to the rabbit and, together with previous data, suggest the rabbit to be a poor model for evaluating the effects of contraceptive steroids in other species.

The roles of estrogen and progestin in producing deciduosarcoma and other lesions in the rabbit.

The interactions of estrogens and progestins in producing decidualization, deciduosarcoma. and other lesions in the rabbit were explored. Steroids were delivered by silicone elastomer implants placed subdermally except for oral dosing in 1 experiment. Varying doses of levonorgestrel (LNG) were given with and without estradiol (E2) and varying doses of E2 with and without LNG. LNG alone delivered at an estimated mean dose of 233 microg/day did not result in endometrial decidualization or deciduosarcoma. Both conditions occurred when E2 was added to the regimen and increased as the dose of E2 was increased. Sixty microg of E2 per day produced endometrial decidualization in all test animals in a 2-month exposure, but deciduosarcoma occurred only when LNG was also supplied and increased as the LNG dose was increased. Progesterone given with E2 resulted in deciduosarcoma in most rabbits. Ethynylestradiol alone at 30 microg/day delivered by implants produced splenic and ovarian deciduosarcomas in 1 of 5 test animals. Adding LNG resulted in more numerous and widespread deciduosarcomas. These experiments indicate that exogenous estrogen is necessary for decidualization of the endometrium and to production of deciduosarcoma in the nonpregnant rabbit. Exogenous progestin promotes the process. Necrosis of the uterine wall tended to increase with increasing dose of estrogens.