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.

Effects of the synthetic progestin levonorgestrel on some aspects of thyroid physiology in common carp (Cyprinus carpio).

The objective of the present study was to assess the effects of levonorgestrel (LNG), a synthetic progestin, on early development and the thyroid system of carp using morphological, histological, immunohistochemical, and gene expression analysis. Fish were exposed to LNG at three levels (3, 31, and 310 ng L-1) from eggs to the onset of juvenile stage (47 days). LNG had no significant effect on early development in common carp or on the occurrence of morphological anomalies. No pathological alterations of the thyroid follicles were found. Immunohistochemical examination of the thyroid follicles using antibodies against thyroxin did not show any differences in fish exposed to 310 ng L-1 LNG compared to the controls. mRNA expression of iodothyronine deiodinases (dio1, 2, 3) was differentially affected by LNG treatment during carp development. Most importantly, dio3 was markedly downregulated in fish exposed to all three LNG levels compared to the controls at the conclusion of the experiment (47 days post-fertilization). A decrease in dio1 or dio3 or an increase in dio2 transcription observed at different time points of the study may be a sign of hypothyroidism. mRNA expression of genes npr, esr1, and esr2b in the body and npr and esr2b in the head of fish exposed to 310 ng L-1 LNG was significantly upregulated compared to the solvent control group at the end of the test. Together, these results show that levonorgestrel caused parallel changes in the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-gonad axes.

Exposure to levonorgestrel-based birth control pill in early life and its persistent effects in zebrafish.

The consumption of progestins has increased considerably in recent decades, as has their disposal into the environment. These substances can negatively affect the reproduction, physiology, and behavior of non-target organisms, such as fish. We aimed to evaluate the effects of exposure to environmentally relevant concentrations of levonorgestrel-control birth based (1.3, 13.3, 133, and 1330 ng/L) on the development and behavior of zebrafish (Danio rerio) in terms of mortality, hatching, spontaneous movement, and larval and adult behavioral tests. Exposure caused anxiogenic-like behavior in larvae, which persisted in adults, as demonstrated by the light-dark test. In contrast, it caused anxiolytic-like behavior in the novel tank test. There was a high mortality rate at all tested concentrations and increases in the hormone cortisol at 13.3 ng/L that affected the sex ratio. These changes may lead to an ecological imbalance, emphasizing the risk of early exposure to progestins in the environment.

Dydrogesterone and levonorgestrel at environmentally relevant concentrations have antagonist effects with rhythmic oscillation in brain and eyes of zebrafish.

Synthetic progestins levonorgestrel (LNG) and dydrogesterone (DDG) are frequency detected in surface water. Combined effects of LNG and DDG on gonad differentiation are similar to LNG single exposure in juvenile zebrafish. However, LNG and DDG mixtures have stronger effects on spermatogenesis in testes of adult zebrafish, which show variable at different life stage. Effects of LNG and DDG mixtures on eyes and brain remain unknown. Here we investigated effects of LNG, DDG and their mixtures on eyes and brain. Zebrafish were exposed to LNG, DDG and their mixtures from 2 hpf to 144 dpf. Rhythm and vision related biological processes were enriched in eyes and brain in LNG and DDG treatments, which indicated rhythmic oscillation in eyes and brain. The qPCR data revealed that both LNG and DDG decreased transcription of arntl2 and clocka, while increased transcription of per1a, per1b, rpe65a and tefa in eyes and brain. However, DDG and LNG mixtures had slight effect on transcription of genes related to rhythm and vision. In addition, LNG and DDG reduced the thickness of inner nuclear layer in the eyes. Bliss independent model revealed that LNG and DDG had antagonist effects on transcription and histology in eyes and brain. Moreover, LNG and DDG formed the same hydrogen bonds with green-sensitive opsin-4 and rhodopsin kinase GRK7a. Taken together, LNG and DDG competed with each other for the same binding residues resulting in antagonist effect in their mixtures treatments, and have significant ecological implications to assess combined effects of progestins mixtures on fish in different organs.

Endocrine disrupting effects induced by levonorgestrel linked to altered DNA methylation in rare minnow (Gobiocypris rarus).

Progestins are worldwide environmental contaminants, however, their ecotoxicological risks and underlying molecular mechanisms of effects are not fully understood. In this study, newly hatched rare minnow (Gobiocypris rarus) larvae were exposed to environmentally realistic concentrations (1 and 10 ng/L) of levonorgestrel (LNG) for 6 months. The sex ratios were not affected by LNG at both concentrations, but the growth was significantly inhibited at 10 ng/L while promoted at 1 ng/L. Histological analysis revealed impaired gonadal development. Plasma concentrations of estradiol in females and testosterone in both sexes were significantly induced after exposure to 1 ng/L LNG; plasma concentrations of 11-ketotestosterone were markedly increased in females exposed to 10 ng/L LNG and in males exposed to both concentrations of LNG. The transcription of cyp19a1a was significantly up-regulated in ovaries exposed to LNG at both concentrations, while cyp17a1 was down-regulated in testes exposed to 10 ng/L LNG. The global DNA methylation level was significantly decreased in testes exposed to 10 ng/L LNG, which might be associated with inhibited spermatogenesis. Gender-specific changes in CpG methylation patterns were induced by LNG in the 5' flanking region of cyp19a1a, with hypomethylation in ovaries but hypermethylation in testes, which was linked to the regulation of cyp19a1a transcription. The results suggest that LNG could induce endocrine disrupting effects in fish at environmentally realistic concentrations, which may be linked to altered DNA methylation. This study indicates potentially high ecological risk of LNG to fish populations, and warrants researches on regulatory mechanisms of epigenetic modifications in progestin-induced effects.

Effects of the Gestagen Levonorgestrel in a Life Cycle Test with Zebrafish (Danio rerio).

The amount of pharmaceuticals transferred to the aquatic environment via municipal and hospital waste water is steadily increasing. The progress in medical research has resulted in the manufacture of active substances of increased stability, specificity, and potency, which can trigger adverse effects in aquatic organisms. Moreover, advanced analytical methods allow the detection of pharmaceuticals in environmental matrices at very low concentrations, which increases the number of substances to be assessed. Levonorgestrel is a synthetic gestagen commonly used in medicinal products for contraception. Because progestogenic compounds could have an impact on fish maturation processes, a life cycle test was performed to assess the effects of levonorgestrel exposure of the embryonic to the adult stages of zebrafish (Danio rerio) at mean measured concentrations of 0.06, 0.16, 0.47, 1.64, and 5.45 ng/L. Apical endpoints were survival, growth, reproduction, and sex ratio. Determination of endocrine modulation was completed by measurement of vitellogenin and 11-keto testosterone in blood plasma, as well as by histopathological analysis of gonads. For all parameters, control values were within the recommended quality range. The most prominent levonorgestrel effect was a shift toward an increased number of male fish at 1.64 and especially 5.45 ng/L, at which point all fish were histologically determined to be males and no spawning occurred; 11-keto testosterone was significantly decreased. A no-observed-effect concentration (NOEC) of 0.47 ng levonorgestrel/L was confirmed by the fertilization capability of adult fish, the male maturation stages, and female gonad histopathology. Whereas hatch and juvenile growth were not affected, posthatch survival was significantly impeded at ≥0.47 ng levonorgestrel/L, although it was not clearly related to the test concentration. For male length and weight, the same NOEC of 0.16 ng/L was obtained at study termination. Environ Toxicol Chem 2022;41:580-591. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Levonorgestrel and dydrogesterone affect sex determination via different pathways in zebrafish.

Levonorgestrel (LNG) and dydrogesterone (DDG) are two commonly used synthetic progestins that have been detected in aquatic environments. They could affect fish sex differentiation, but the underlying mechanisms remain unknown. Here we investigated the effects of LNG (5 ng L-1 and 50 ng L-1), DDG (100 ng L-1) and their mixtures on gonadal differentiation and sex determination in zebrafish at transcriptomic and histological levels from 2 hours post-fertilization (eleutheroembryos) to 144 days post-fertilization (sexual maturity). Germ cell development and oogenesis pathways were significantly enriched in LNG and the mixture of LNG and DDG treatments, while insulin and apoptosis pathways in the DDG treatment. LNG and the mixture of LNG and DDG strongly decreased transcripts of germ cell development and oogenesis related genes, while DDG increased the transcripts of insulin and apoptosis related genes at 28 days post fertilization (dpf) and 35 dpf. Furthermore, DDG caused ∼ 90% males, and LNG and the mixture of LNG and DDG resulted in 100% males on all sampling dates. Specifically, most males in LNG and the mixture of LNG and DDG treatments were "Type I" males without juvenile oocytes at 28 dpf and 35 dpf, while those in DDG treatment were "Type II" and "Type III" males with a few juvenile oocytes. These results indicated that LNG and DDG promoted testicular differentiation via different pathways to cause male bias. LNG and DDG mixtures have similar effect on testicular differentiation to LNG alone. The findings from this study could have significant ecological implications to fish populations.

Occurrence of Levonorgestrel in Water Systems and Its Effects on Aquatic Organisms: A Review.

Levonorgestrel is one of the active ingredients of oral contraceptives detected in the aquatic environment at concentrations in the order of ng/L. During the past decade, a wealth of new information about levonorgestrel has been produced, with several studies having reported negative effects in the reproduction and growth of aquatic organisms after exposure to this emerging contaminant of concern. In the present study, the data about its levels in water and its effects on aquatic organisms were integrated and used to perform an updated preliminary aquatic risk assessment for levonorgestrel based on the guideline for Environmental Risk Assessment of Medicinal Products for Human Use from the European Medicines Agency. The aim was to investigate if this pharmaceutical has a risk for adverse effects on aquatic organisms (i.e. for organisms residing in surface water and groundwater). The results evidenced that levonorgestrel is likely to pose an environmental risk to surface water (risk quotient >1). Based on these results, a more refined risk assessment for this pharmaceutical is needed. Besides, our findings highlight the need for investigation under the adverse outcome pathway (AOP) framework, as well as for further studies about toxicological interactions between levonorgestrel and other synthetic steroids.

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.

Combined effects of increased temperature and levonorgestrel exposure on zebrafish female liver, using stereology and immunohistochemistry against catalase, CYP1A, HSP90 and vitellogenin.

Climate change and pharmaceuticals contamination constitute two of the most relevant stressors on the aquatic ecosystems, however, there is a huge lack of information regarding the interactive effects of both stressors. For that, a mesocosm experiment was implemented where adult zebrafish were exposed to combined temperature and the progestin levonorgestrel (LNG) for 21 days. Considering that the liver is one of the organs where there is a greater metabolization and accumulation of toxicants, the main objective of this work was to assess the effects of both stressors on the female zebrafish hepatocytes morphology and functioning, through stereological and immunohistochemical techniques. Our results revealed an increase of coefficient of variation of the number distribution of hepatocytes volume (CVN(υ)) for individuals exposed to LNG, which denotes an increase of the hepatocytes size variability and is suggestive of functional impacts. This was corroborated by the signs of increased glycogen content with the exposure to increased LNG concentrations and temperature, indicating modified hepatocyte glycogen metabolism. Such disturbances can be considered indicators that the fish had to deal with impacts caused by the stress factors. Regarding the immunoreactivity, from the four proteins selected (catalase, CYP1A, HSP90 and Vtg), just in two of them (catalase and Vtg) were observed some responses to both stressors. For catalase there was a hormetic response, in which exposure to lower LNG concentrations caused a significant higher positive immunostaining than under higher LNG concentrations. While, for Vtg, significant effects of temperature and LNG existed, in which a decline in Vtg immunostaining was observed with exposure to higher temperature and lower LNG concentrations. These results should be seen as a warning sign about fine impacts of multiple stressors, such as temperature and progestogens, on the structure and functioning of zebrafish liver and potentially in other aquatic organisms, and on their health implications.

Assessment of endocrine disruptor effects of levonorgestrel and its photoproducts: Environmental implications of released fractions after their photocatalytic removal.

The presence of levonorgestrel (LNG) in water bodies via direct discharge and human excretion has been reported worldwide, but its effects on the reproduction of aquatic species and humans are still unknown. Owing to its recalcitrant properties, LNG is not completely removed during wastewater treatment plants, and many species may be exposed to low traces of this compound from discharged effluents. Thus, in this study, a photocatalytic process for removing LNG along with screening of endocrine disruptor effects for risk assessment was applied. Although the removal rate of LNG by ultraviolet C (UV-C) radiation was >90%, reproductive toxicity testing using the BeWo cell line exposed to LNG and its degraded fraction showed the reduced production of basal human chorionic gonadotropin hormone (ß-hCG) by more than 73%, from 8.90 mIU mL-1 to <2.39 mIU mL-1, with both LNG and the degraded fraction. ß-hCG hormone has been implicated in the viability of trophoblastic cells during the first trimester of pregnancy; therefore, degraded fractions and waterborne LNG may affect reproduction in some aquatic species and humans with low level of exposure.

Genotoxic effects of combined multiple stressors on Gammarus locusta haemocytes: Interactions between temperature, pCO2 and the synthetic progestin levonorgestrel.

Climate change and pharmaceutical contamination are two priority research topics due to their impacts in the aquatic ecosystems and in the food chain structure. In the bottom of many food chains are the invertebrates, like the amphipods, which are important environmental and ecotoxicological models. In this study, we combined the increase of temperature [ambient and warming temperature], pCO2 [normocapnia and hypercapnia] and the synthetic progestin levonorgestrel (LNG) [environmentally relevant concentration (10 ng L-1) and 100-fold higher (1000 ng L-1)] to evaluate the genotoxic effects on the amphipod Gammarus locusta haemocytes, using the comet assay technique. Additionally, the study examined protective/potentiating effects of the three tested factors against hydrogen peroxide (H2O2)-induced DNA damage in haemocytes after ex vivo exposure. Our data revealed no significant effects of any of the three stressors on DNA damage of G. locusta haemocytes or protection against H2O2-induced DNA damage after twenty-one days exposure. Only a significant effect of the solvent was visible, since it was able to induce higher DNA damage (i.e. strand breaks) on exposed individuals. On the other hand, LNG exposure seemed to induce a slight increase of DNA damage after H2O2 exposure. Our findings suggest that more short-term studies to conclude about the genotoxicity and/or protective effects of the stress factors in G. locusta should be made, attending to the fast turnover rate of repairing cells that could have masked impacts seen only after the end of the experiment.

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.

The fate of prazosin and levonorgestrel after electrochemical degradation process: Monitoring by-products using LC-TOF/MS.

Prazosin (PRZ) and levonorgestrel (LNG) are widely used as an anti-disease drugs due to their biological activity in the human body. The frequent detection of these compounds in water samples requires alternative technologies for the removal of both compounds. After electrochemical degradation of PRZ and LNG, the parent compounds could be completely removed after treatment, but the identification and characterization of by-products are necessary as well. In this study, the effects of NaCl concentration and applied voltage were investigated during the electrochemical degradation process. The results revealed that the increase of NaCl concentration and applied voltage could promote the generation of hypochlorite OCl- and then enhance the degradation of PRZ and LNG. After initial study, 6V and 0.2g NaCl were selected for further experiments (96% and 99% removal of PRZ and LNG after 40min, respectively). Energy consumption was also evaluated and calculated for PRZ and LNG at 3, 6 and 8V. Solid phase extraction (SPE) method plays an important role in enhancing the detection limit of by-products. Furthermore, characterization and identification of chlorinated and non-chlorinated by-products were conducted using an accurate liquid chromatography-time of flight/mass spectrometry LC-TOF/MS instrument. The monitoring of products during the electrochemical degradation process was performed at 6V and 0.2g NaCl in a 50mL solution. The results indicated that two chlorinated products were formed during the electrochemical process. The toxicity of by-products toward E. coli bacteria was investigated at 37°C and 20hr incubation time.

Histopathological Evaluation of Combined Impacts of the Synthetic Progestin Levonorgestrel and Temperature on the Female Zebrafish Maturation Using a Semi-quantitative Grading Analysis-Is it Enough?

Pharmaceuticals contamination (e.g., synthetic progestins), and global climate change, represent two of the most stressful factors affecting aquatic species. To our knowledge, there is huge gap of data regarding the combined effects of both stressors on vertebrates' reproduction. Thus, it is crucial to implement rapid screenings of measurable histopathological alterations in fish gonads. For that, we propose: (1) an evaluation of the combined effects of progestin (levonorgestrel) and temperature on maturation of zebrafish female gonads, using a semi-quantitative method (i.e., grading) and (2) testing the robustness of the grading analysis comparatively to a quantitative method (i.e., stereology). Grading analysis showed a decrease on maturation stage of ovaries exposed to both stressors. Although grading is less robust than stereological analysis, it is recommended for a preliminary approach, since it gives a correct idea on trends and it is fast and cost-effective. For a detailed histological assessment, we recommend a stereological study.

Exposure to levonorgestrel increases nest acquisition success and decreases sperm motility in the male fathead minnow (Pimephales promelas).

Progestins are utilized as a component of human contraceptives, and commonly enter the environment via wastewater treatment plant effluent. Certain progestins activate fish androgen receptors and cause decreases in fecundity and masculinization of females. We used a nest acquisition assay and computer-assisted sperm analysis to examine the effects of levonorgestrel on male fathead minnow (Pimephales promelas) reproductive fitness. Males were exposed to 0, 10, or 100 ng/L levonorgestrel for 14 d. Combinations of a control male and a male from one of the treatments were placed into a competitive nesting assay, and the time each male spent holding the nest and time spent exhibiting aggressive behaviors were analyzed at 48 h postexposure. Semen samples were analyzed for total motility, straight-line velocity, curvilinear velocity, average path velocity, linearity, beat cross frequency, and wobble at 0, 30, 60, 90, and 120 s postactivation. Males exposed to either 10 or 100 ng/L of levonorgestrel exhibited increased nest acquisition success and lower levels of aggression compared with control-control pairings, as well as decreases in multiple sperm motion characteristics. Our results suggest that further research is required to ascertain the effects of levonorgestrel on male gamete quality and reproductive behaviors. Environ Toxicol Chem 2018;37:1131-1137. © 2017 SETAC.

Interactive effects of increased temperature, pCO2 and the synthetic progestin levonorgestrel on the fitness and breeding of the amphipod Gammarus locusta.

Given the lack of knowledge regarding climate change-chemical exposure interactions, it is vital to evaluate how these two drivers jointly impact aquatic species. Thus, for the first time, we aimed at investigating the combined effects of increased temperature, pCO2 and the synthetic progestin levonorgestrel on survival, growth, consumption rate and reproduction of the amphipod Gammarus locusta. For that, a full factorial design manipulating temperature [ambient temperature and warming (+4 °C)], pCO2 [normocapnia and hypercapnia (Δ pH 0.5 units)] and the progestin levonorgestrel (LNG: L1 - 10 ngLL-1 and L2 - 1000 ngLL-1, control - no progestin and solvent control - vehicle ethanol (0.01%)) was implemented for 21 days. G. locusta was strongly negatively affected by warming, experiencing higher mortality rates (50-80%) than in any other treatments. Instead, growth rates were significantly affected by interactions of LNG with temperature and pCO2. It was observed, in the short-term (7d) that under ambient temperature (18 °C) and hypercapnic conditions (pH 7.6), the LNG presence promoted the amphipod's growth, while in the medium-term (21d) this response was not observed. Relative consumption rates (RCRs), during the first week were higher than in the third week. Furthermore, in the first week, RCRs were negatively affected by higher temperature while in the third week, RCRs were negatively affected by acidification. Furthermore, it was observed a negative effect of higher temperature and acidification on G. locusta fecundity, contrarily to LNG. Concluding, the impact of increased temperature and pCO2 was clearly more adverse for the species than exposure to the synthetic progestin, however, some interactions between the progestin and the climate factors were observed. Thus, in a future scenario of global change, the presence of LNG (and other progestins alike) may modulate to a certain level the effects of climate drivers (and vice-versa) on the gammarids fitness and reproduction.

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.

Warming modulates the effects of the endocrine disruptor progestin levonorgestrel on the zebrafish fitness, ovary maturation kinetics and reproduction success.

Interactive effects between multiple stressors, namely climate drivers (e.g., temperature) and chemical pollution (e.g., endocrine disruptors) are poorly studied. Here, it was for the first time evaluated the combinatory effects of temperature and a synthetic progestin, levonorgestrel (LNG), on the fitness and reproductive-related endpoints of zebrafish (Danio rerio). A multi-factorial design was implemented by manipulating both temperature [setting as baseline an ambient temperature of 27 °C, against warming (+3 °C)] and LNG levels (10 ngL-1 and 1000 ngL-1). Groups of males and females were exposed sub-acutely, for 21-days. Increased temperature caused an overall decrease in the females' gonadosomatic index (GSI), during the pre-reproduction phase, LNG did not affect GSI. In addition, fecundity (number of ovulated eggs) was negatively affected by both temperature and LNG, being the effect of the latter more intense. Fish exposed to the highest LNG concentration (at both temperatures) did not reproduce, but also in those exposed to the lowest dose of progestin at a higher temperature, a complete reproductive failure occurred. These results reflect what was observed in the stereological analysis of the ovary maturation stages prior to reproduction. Accordingly, the higher the LNG concentration, the lower the degree of maturation of the ovary. This was exacerbated by the higher temperature. As to embryonated eggs, they hatched significantly faster at higher temperatures, but exposure to 10 ngL-1 of LNG (at 27 °C) reduced significantly the hatching rate, comparing to control. Further, the recrudescence of the ovary 48 h after spawning seems to be not affected by both stressors. Our data suggest that in a future scenario of global warming and synthetic hormones exposure, the reproduction of fish species, such as the zebrafish, can be endangered, which can put at risk their success, and consequently affect the structure and functioning of associated aquatic ecosystems.

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.