DNA damage, alterations in the expression of antioxidant enzyme genes and in the histoarchitecture of gill cells of zebrafish exposed to 17-α-ethinylestradiol.

Endocrine disruptors, such as estrogen, are chemical substances with the potential to alter the hormonal balance of organisms. Their origin can be natural or artificial, and they can act at very low doses. The estrogen 17α-ethinylestradiol (EE2) is used worldwide as an oral contraceptive and is a potential contaminant in aquatic ecosystems. It is well documented that these environmental pollutants can act directly or indirectly on the reproductive system, impairing development and fertility. However, little is known about the alteration of the cell oxidative status induced by EE2. The main objective of this study was to evaluate the effect on the gill cells of adult zebrafish exposed in vivo to EE2, analyzing cell histology, DNA damage and the expression levels of genes encoding the main enzymes involved in oxidative stress pathways. The histological study showed that EE2 produces moderate to high damage to the gill tissue, an increase in gill cell DNA damage and the mRNA levels of the genes corresponding to the manganese superoxide dismutase (Mn-sod) and catalase (cat) after exposure to 5 ng/L EE2. The results indicate that EE2 causes tissue alterations, DNA damage and oxidative stress. EE2 produced important alterations in the gills, a fundamental organ for the survival of fish. There is a clear need for further research on the ecological consequences of EDCs on non-target organisms.

Histological investigation in aging male and female gerbil prostates after prenatal exposure to pequi (Caryocar brasiliense Cambess) oil and 17α-ethinylestradiol.

The female prostate, also known as Skene's gland, is present in both humans and rodents. Prenatal exposure to ethinylestradiol (EE2), a synthetic estrogen found in oral contraceptives, induces pormotes neoplasic prostate lesions in gerbils (Meriones unguiculatus). Conversely, pequi oil (Pe), extracted from the Brazilian Cerrado fruit, has antioxidant, anti-inflammatory, and anticancer properties, mitigates risks associated with chronic diseases related to lifestyle and aging. This study evaluates the impact of prenatal exposure to Pe (300 mg/kg) on senile gerbil offspring's male and female prostates under normal conditions and EE2 exposure (15 µg/kg/day). Histological and morphometric analyses revealed that Pe reduced male body weight and prostate epithelial height, along with a thinner muscle layer. In females, EE2 exposure reduced prostatic weight, while Pe exposure lowered epithelial height and the relative stromal compartment volume, increasing the muscle layer. Pequi oil holds potential in mitigating alterations induced by exposure to the endocrine disruptor EE2.

Physicochemical and bioinformatic characterization of Oreochromis niloticus vitellogenin as an endocrine disruption biomarker.

Aquatic biota is increasingly being exposed to chemical pollutants due to human activities and the relationship between the level of environmental pollution and fish reproduction is a continuously ongoing issue. The vitellogenin (Vtg) protein synthesis can be induced in the liver of juvenile and male fish after stimulation of the estrogen receptor and therefore, Vtg has been used as a biomarker of xenoestrogen exposure in several fish species. The current study reported the first physicochemical characterization of Vtg from Oreochromis niloticus. Adult male fish were exposed to 17α-ethinylestradiol for Vtg induction. Purified vitellogenin from plasma showed low stability at 25 and 4 °C in saline conditions, and good stability in acidic (low pH) or in heated conditions. The 3D modeling provided useful information on the structure of O. niloticus Vtg showing conserved structural features. According to bioinformatics and experimental results, there are important structural differences between the two chemical forms of Vtg (VtgAb and VtgC) in a phylogenetic context. The present results add information about the development of ecotoxicological immunoassays to study the endocrine disruption in O. niloticus improving the Vtg performance as a biomarker of reproduction in fish.

Acute exposure to 17-α-ethinylestradiol disrupt the embryonic development and oxidative status of Danio rerio.

17-Alpha-ethinylestradiol (EE2) is an estrogen derived from estradiol (E2). This compound and is one of the most widely used drugs both in humans and animals. Numerous studies have reported the ability of EE2 to alter sex determination and delay sexual maturity, but there are toxic effects that need to be explored. In this work, we analyzed the effect of EE2 on embryonic development and oxidative stress biomarkers in Danio rerio. For this effect, zebrafish embryos in the blastula period (2.5 h post fecundation) were exposed to different concentrations of EE2 (36-106 ng L-1) until 96 hpf. Survival, alterations to embryonic development, and teratogenic effects were evaluated using a stereomicroscope. Furthermore, oxidative stress biomarkers: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) activities, lipid peroxidation (LPX), hydroperoxide content (HPX), and protein carbonyl content (POX) were evaluated at 72 and 96 hpf using spectrophotometric methods. LC50 and EC50 of malformations got values of 82 ng L-1 and 57.7 ng L-1, respectively. The main teratogenic effects found were: chorda malformation, body malformation, and developmental delay. These alterations occurred at 86, 96, and 106 ng L-1. Integrated biomarker index showed that the oxidative stress biomarkers that had the most influence on embryos were SOD, CAT, GPX, and LPX. Overall, our results allow us to conclude that low concentrations of EE2 may potentially alter the development and oxidative status in the early life stages of zebrafish. Therefore, this bio-active estrogen can be considered a hazardous substance for fish.

Prenatal and pubertal exposure to 17α-ethinylestradiol cause morphological changes in the prostate of old gerbils.

This study evaluated such as exposure to ethinylestradiol during the prenatal (18th-22nd day) and pubertal (42nd-49th day) periods acts on the male ventral prostate and female prostate of 12-month old gerbils. We performed the analysis to serum hormone levels for estradiol and testosterone. The prostates were submitted to morphometric and immunohistochemical analyses. Exposure to ethinylestradiol during these developmental periods decreased the testosterone serum levels in males and increased the estradiol serum levels in females. Morphologically, prostate intraepithelial neoplasia and disorders in the arrangement of the fibrous components were observed in the prostate glands of both sexes of gerbil exposed to ethinylestradiol during development periods. In the male prostate, the ethinylestradiol promoted decreased in the frequency of positive epithelial cell for androgen receptor (AR) and increased the frequency of positive stromal cell for estrogen receptor α. However, in the female prostate, this synthetic estrogen caused AR upregulation and increased cell proliferation. This study shows that the exposure to ethinylestradiol during development phases alters the morphology and the hormonal signaling in the male and female prostates of old gerbils, confirming the action of ethinylestradiol as endocrine disruptor.

Estrogen induces shift in abundances of specific groups of the coral microbiome.

Synthetic estrogens such as ethinylestradiol (EE2) are persistent micropollutants that are not effectively removed from wastewater by conventional treatments. These contaminants are released into waterbodies, where they disrupt endocrine systems of organisms and cause harmful effects such as feminization, infertility, reproduction problems and genital malformations. The consequences of this pollution for key marine ecosystems such as coral reefs and their associated microbiomes are underexplored. We evaluated the effects of EE2 concentrations of 100 ng L-1 and 100 µg L-1 on the coral metaorganism Mussismilia harttii. The results indicated no effects on visible bleaching or Fv/Fm ratios in the corals during a 17-day microcosm experiment. However, next-generation sequencing of 16S rDNA revealed a statistically significant effect of high EE2 concentrations on OTU richness, and shifts in specific microbial groups after treatments with or without EE2. These groups might be bioindicators of early shifts in the metaorganism composition caused by EE2 contamination.

Effects of 17α-ethinylestradiol on sex ratio, gonadal histology and perianal hyperpigmentation of Cnesterodon decemmaculatus (Pisces, Poeciliidae) during a full-lifecycle exposure.

The effects of 17α-ethinylestradiol (EE2) on sex ratio, gonopodium morphology, and gonadal histology of C. decemmaculatus were assessed by a full-lifecycle exposure experiment. Newborn fish were waterborne exposed to 30, 100, and 300 ng EE2/L for 90 d, using 50 fish per treatment. Additionally, in December of 2016, a field survey was conducted on a C. decemmaculatus population inhabiting the Girado Creek downstream of the Chascomus city wastewater effluent discharge. After 90 d of exposure, EE2 was able to histologically skew the sex ratio toward females and inhibit the full gonopodium development since the lowest tested concentration (LOEC = 30 ng/L). At higher concentrations, EE2 was toxic, inducing mortality in a concentration-dependent fashion (90 d-LC50 = 109.9 ng/L) and altering the gonadal histoarchitecture, causing neither testes nor ovaries discernible histologically (LOEC = 100 ng/L). In addition, a novel response, perianal hyperpigmentation, was discovered been induced by the EE2 exposure in a concentration-dependent fashion (90 d-EC50 = 39.3 ng/L). A higher proportion of females and perianal hyperpigmentation were observed in wild fish collected from the Girado Creek. The major reached conclusions are: i) EE2 induce different effects on the sexual traits of C. decemmaculatus when exposed from early-life or adult stages. ii) The most sensitive effects observed in the laboratory occur in a creek receiving wastewater effluent. iii) The perianal hyperpigmentation comes-up as a promising biomarker of exposure to estrogenic compounds.

Toxicity of three emerging contaminants to non-target marine organisms.

Coastal areas are continually impacted by anthropic activities because they shelter large urban conglomerates. Urban effluents directly or indirectly end up reaching the marine environment, releasing a large number of pollutants which include the so-called contaminants of emerging concern (CECs), since the conventional treatment plants are not effective in removing these compounds from the effluents. These substances include hormones, pharmaceuticals and personal care products, nanoparticles, biocides, among others. The aim of this study was to evaluate the toxicity of the 17α-ethinylestradiol (EE2), acetylsalicylic acid (ASA), and bisphenol-A (BPA) to two marine crustaceans and one echinoderm, evaluating the following parameters: survival (Artemia sp. and Mysidopsis juniae), embryo-larval development (Echinometra lucunter). The LC50 values calculated in the acute toxicity tests showed that the compounds were more toxic to M. juniae than to the Artemia sp. Among the three contaminants, EE2 was the most toxic (LC50-48h = 18.4 ± 2.7 mg L-1 to Artemia sp.; LC50-96h = 0.36 ± 0.07 mg L-1 to M. juniae). The three tested compounds affected significantly the embryonic development of the sea urchin in all tested concentrations, including ecologically relevant concentrations, indicating the potential risk that these contaminants may present to the marine biota.

Exposure to E2 and EE2 environmental concentrations affect different components of the Brain-Pituitary-Gonadal axis in pejerrey fish (Odontesthes bonariensis).

The present study focuses on the effects of E2 and EE2 environmental concentrations on different components of the reproductive axis of pejerrey (Odontesthes bonariensis), a native fish species from Pampas lakes of Argentina. The results obtained demonstrated that E2 and EE2 separate or mixed, could disrupt key pathways of the pejerrey Brain-Pituitary-Gonadal axis. First, it was observed that at the brain level, gnrh-III and cyp19a1b mRNA expression increased significantly in the exposed fish. Secondly, in the pituitary fshb and lhb mRNA expression levels, the study did not show any differences between treated and control groups. Thirdly, fshr and lhcgr transcript levels showed a significant decrease at testicular level. Nevertheless, testosterone plasmatic levels remained unchanged in exposed fish. In addition, in a histological analysis, it was possible to find pyknotic nuclei in estrogen only on treated fish testis linked to a reduction in the GSI index and a decrease in the length of spermatogenic lobules. All these findings highlighted the fact that environmental concentrations of E2, EE2 and their mixture disrupted the endocrine-reproductive axis of pejerrey, being the testis the main direct target.

Pubertal exposure to ethinylestradiol promotes different effects on the morphology of the prostate of the male and female gerbil during aging.

In rodents, the final growth and maturation of the prostate occur at puberty, a crucial period for prostate development. The present study is a serological, morphological, morphometric, and immunohistochemical analysis of the effects of exposure to ethinylestradiol (EE) (15 µg/kg/day) during puberty (EE/PUB group) on the male ventral and female prostate in senile gerbils. In the study, male and female gerbils (Meriones unguiculatus) (42 days) received by gavage 15 µg/kg/day of EE (a component of the contraceptive pill), diluted in 100 µL of Nujol® for 1 week (EE/PUB group). In the control group, males and females were not treated. Animals were killed (n = 5) after 12 months in the experimental groups. In the senile male in the EE/PUB group, we observed a reduction in testosterone levels and a decrease in the prostatic epithelial thickness, as well as in the thickness of the muscle layer. In addition, an increase in PIN multiplicity and prostatic inflammation was observed. In the senile female in the EE/PUB group, we observed increased testosterone and estradiol levels, an enhanced prostatic epithelial thickness and an increase in the thickness of the muscle layer. Immunohistochemical analysis revealed an increase in positive cells (%) for AR and PCNA in the male prostate and an increase in positive basal cells for p63 in the female prostate of the EE/PUB group. Exposure to EE during puberty resulted in an inhibitory action on the male ventral prostate and an anabolic effect on the female prostate in senile gerbils. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 477-489, 2017.

Intersex and liver alterations induced by long-term sublethal exposure to 17α-ethinylestradiol in adult male Cnesterodon decemmaculatus (Pisces: Poeciliidae).

The aim of the present study was to assess the responses of the gonopodium morphology and the gonadal and liver histology of adult male Cnesterodon decemmaculatus to sublethal long-term exposure concentrations of 17α-ethinylestradiol (EE2). Two experiments were conducted exposing the fish to waterborne concentrations of EE2 ranging from 20 ng/L to 200 ng/L for 8 wk, 12 wk, and 16 wk. Intersex gonads were observed after 8 wk and 16 wk in fish exposed to 200 ng EE2/L and 100 ng EE2/L, respectively. Oocytes' development from testis germ cells and replacement of the efferent duct periodic acid-Schiff-positive secretion surrounding spermatozeugmata by parenchymal tissue and duct structure alterations were the major observed changes in the gonads. In contrast, no response was observed in the gonopodium morphology. Liver histology was also altered, showing increasing steatosis, single-cell necrosis to generalized necrosis, and disruption of acinar organization from 100 ng EE2/L to 200 ng EE2/L. In summary, the present results showed that although EE2 was not able to alter the morphology of a developed gonopodium, it was capable of inducing development of testicular oocytes in adult male C. decemmaculatus at environmentally relevant concentrations. Thus, externally normal but intersex C. decemmaculatus males would be expected in the wastewater-receiving streams that the species inhabits. According to the literature, the present study would be the first indicating estrogen-induced intersex in adult male poeciliid. Environ Toxicol Chem 2017;36:1738-1745. © 2016 SETAC.

Neonatal exposure to ethinylestradiol increases ventral prostate growth and promotes epithelial hyperplasia and inflammation in adult male gerbils.

The aim of this study was to analyse morphologically the ventral prostate of adult Mongolian gerbils exposed to ethinylestradiol (EE) during the first week of postnatal development. Lactating females received daily, by gavage, doses of 10 µg/kg of EE diluted in 100 µl of mineral oil from the 1st to 10th postnatal day of the pups (EE group). In the control group (C), the lactating females received only the vehicle. Upon completing 120 days of age, the male offspring were euthanized and the prostates collected for analyses. We employed morphological, stereological-morphometrical, immunohistochemical and ultrastructural methods. The results showed that the postnatal exposure to EE doubled the prostatic complex weight, increasing the epithelial and stromal compartments, in addition to the secretory activity of the ventral lobe of the prostate. All glands exposed to EE showed strong stromal remodelling, and some foci of epithelial hyperplasia and inflammatory infiltrate in both luminal and epithelial or stromal compartments. Cells positive for anti-AR and anti-PCNA reactions increased into the epithelial and stromal tissues. ERα-positive cells, which are normally found in the stromal compartment of intact prostates, were frequently observed in the prostatic epithelium of treated animals. This study demonstrated that the exposure to EE during postnatal development causes histophysiological alterations in this gland, predisposing to the development of prostatic lesions during life. These results are important for public health, considering that women worldwide have commonly used EE. Moreover, the bioaccumulation of this chemical has increased in different ecosystems.

Efeitos de hormônios esteroides de contraceptivos orais combinados sobre os parâmetros comportamentais de Betta splendens (Regan, 1909) / Effects of steroid hormones from combined oral contraceptives on the behavioral and physiological parameters of Betta splendens (Regan, 1909)

Os hormônios esteroides presentes em várias gerações de contraceptivos orais combinados (COC) podem se apresentar como disruptores endócrinos, produzindo alterações no comportamento e na fisiologia de peixes. Diante disso, o objetivo deste trabalho foi avaliar os efeitos de hormônios esteroides presentes em COC sobre os parâmetros comportamentais de Betta splendens, um peixe ornamental usado na aquariofilia e bastante agressivo. Machos adultos foram observados pelo método ad libitum para confecção do etograma e divididos em cinco grupos, controle e expostos aos hormônios: 17β-estradiol (E2); levonorgestrel e etinilestradiol (LEA - segunda geração de COC); gestodeno e etinilestradiol (GEA - terceira geração de COC); e drospirenona e etinilestradiol (DEA - quarta geração de COC). Os peixes foram expostos por 30 dias à concentração final de 10ng/L. Foram avaliados os comportamentos pelos métodos de varredura instantânea e animal-focal, bem como o consumo de ração. No método varredura, o comportamento descansar apresentou o maior valor (54,4±10,1%) no grupo E2 (P<0,05). Os comportamentos agressivos de carga (16,1±3,6%) e recuar e carga (16,4±5,1%) apresentaram os maiores valores no grupo controle em relação aos demais grupos (P<0,05). Os animais do grupo E2 apresentaram maior frequência de comportamentos inativos (76,1%) comparados aos de outros grupos. O comportamento atípico natação errática não foi observado no grupo controle, mas foi observado nos grupos experimentais. Os grupos E2, LEA, GEA e DEA apresentaram redução nos comportamentos agressivos (10%) quando comparados ao grupo controle pelo método animal-focal. Não foram observadas diferenças na exibição desses comportamentos ao se compararem os animais expostos às diferentes gerações de contraceptivos e no consumo de ração. Pode-se concluir que 17β-estradiol causou mais efeitos aos peixes e que diferentes gerações de COC apresentaram efeitos tóxicos semelhantes em relação aos comportamentos observados.(AU)

The steroid hormones present in many generations of combined oral contraceptives (COC) can act as endocrine disruptors inducing changes in the behavior and physiology of fish. In this context, the aim of this work was to evaluate the effects of steroid hormones present in COC on behavioral parameters of Betta splendes, an aggressive ornamental fish used in the aquariophily. Adult males were observed with the ad libitum method to develop an ethogram and were divided into five groups, Control and exposed to hormones: 17β-estradiol (E2), levonorgestrel and ethinylestradiol (LEA - 2nd COC generation), gestodene and ethinylestradiol (GEA - 3rd COC generation) and drospirenone and ethinylestradiol (DEA - 4th COC Generation). Fish were exposed for 30 days to a final concentration of 10ng/L. The behavior was evaluated by scan sampling and animal-focal methods, and feed intake. In the scan sampling method, the Resting behavior showed the highest value (54.4±10.1%) in E2 group (P<0.05). The aggressive behavior Rush (16.1±3.6%) and Back and Rush (16.4±5.1%) showed the highest values in the control group, compared to the other groups (P<0.05). Animals in the E2 group showed higher frequency of inactive behaviors (76.1%) compared to other groups. Furthermore, the atypical behavior Erratic swimming was not observed in the control group, but it was observed in the experimental groups. The E2, LEA, GEA and DEA groups showed reduction in aggressive behavior (10%) compared to the control group by the animal-focal method. Moreover, no difference was observed in the exhibition of these behaviors and feed intake comparing animals exposed to the different generations of contraceptives. It can be concluded that 17β-estradiol has caused more effects on fish and different generations of COC showed similar toxic effects in the observed behaviors.(AU)

Efeitos de hormônios esteroides de contraceptivos orais combinados sobre os parâmetros comportamentais de Betta splendens (Regan, 1909) / Effects of steroid hormones from combined oral contraceptives on the behavioral and physiological parameters of Betta splendens (Regan, 1909)

Os hormônios esteroides presentes em várias gerações de contraceptivos orais combinados (COC) podem se apresentar como disruptores endócrinos, produzindo alterações no comportamento e na fisiologia de peixes. Diante disso, o objetivo deste trabalho foi avaliar os efeitos de hormônios esteroides presentes em COC sobre os parâmetros comportamentais de Betta splendens, um peixe ornamental usado na aquariofilia e bastante agressivo. Machos adultos foram observados pelo método ad libitum para confecção do etograma e divididos em cinco grupos, controle e expostos aos hormônios: 17β-estradiol (E2); levonorgestrel e etinilestradiol (LEA - segunda geração de COC); gestodeno e etinilestradiol (GEA - terceira geração de COC); e drospirenona e etinilestradiol (DEA - quarta geração de COC). Os peixes foram expostos por 30 dias à concentração final de 10ng/L. Foram avaliados os comportamentos pelos métodos de varredura instantânea e animal-focal, bem como o consumo de ração. No método varredura, o comportamento descansar apresentou o maior valor (54,4±10,1%) no grupo E2 (P<0,05). Os comportamentos agressivos de carga (16,1±3,6%) e recuar e carga (16,4±5,1%) apresentaram os maiores valores no grupo controle em relação aos demais grupos (P<0,05). Os animais do grupo E2 apresentaram maior frequência de comportamentos inativos (76,1%) comparados aos de outros grupos. O comportamento atípico natação errática não foi observado no grupo controle, mas foi observado nos grupos experimentais. Os grupos E2, LEA, GEA e DEA apresentaram redução nos comportamentos agressivos (10%) quando comparados ao grupo controle pelo método animal-focal. Não foram observadas diferenças na exibição desses comportamentos ao se compararem os animais expostos às diferentes gerações de contraceptivos e no consumo de ração. Pode-se concluir que 17β-estradiol causou mais efeitos aos peixes e que diferentes gerações de COC apresentaram efeitos tóxicos semelhantes em relação aos comportamentos observados.

The steroid hormones present in many generations of combined oral contraceptives (COC) can act as endocrine disruptors inducing changes in the behavior and physiology of fish. In this context, the aim of this work was to evaluate the effects of steroid hormones present in COC on behavioral parameters of Betta splendes, an aggressive ornamental fish used in the aquariophily. Adult males were observed with the ad libitum method to develop an ethogram and were divided into five groups, Control and exposed to hormones: 17β-estradiol (E2), levonorgestrel and ethinylestradiol (LEA - 2nd COC generation), gestodene and ethinylestradiol (GEA - 3rd COC generation) and drospirenone and ethinylestradiol (DEA - 4th COC Generation). Fish were exposed for 30 days to a final concentration of 10ng/L. The behavior was evaluated by scan sampling and animal-focal methods, and feed intake. In the scan sampling method, the Resting behavior showed the highest value (54.4±10.1%) in E2 group (P<0.05). The aggressive behavior Rush (16.1±3.6%) and Back and Rush (16.4±5.1%) showed the highest values in the control group, compared to the other groups (P<0.05). Animals in the E2 group showed higher frequency of inactive behaviors (76.1%) compared to other groups. Furthermore, the atypical behavior Erratic swimming was not observed in the control group, but it was observed in the experimental groups. The E2, LEA, GEA and DEA groups showed reduction in aggressive behavior (10%) compared to the control group by the animal-focal method. Moreover, no difference was observed in the exhibition of these behaviors and feed intake comparing animals exposed to the different generations of contraceptives. It can be concluded that 17β-estradiol has caused more effects on fish and different generations of COC showed similar toxic effects in the observed behaviors.

Impact of an environmental relevant concentration of 17α-ethinylestradiol on the cardiac function of bullfrog tadpoles.

This study evaluated if a concentration of 17α-ethinylestradiol (EE2 - 10 ng L(-1) for 96 h) normally found in Brazilian surface waters exerts any impact on cardiac function of bullfrog tadpoles (25 Gosner stage), Lithobates catesbeianus. During exposure, the animals' activity level (AL -% of active individuals) was monitored twice a day. Then, the in loco heart rate (f(H) - bpm) was determined, as well as the relative ventricular mass (RVM - % of body mass). Afterwards, cardiac ventricles were mounted for isometric force recordings (CS - mN mm(-2)), and determination of the cardiac pumping capacity (CPC - mN mm(-2) min(-1)). EE2 did not affect tadpoles' AL, although it resulted in a tachycardia in animals exposed to EE2 (f(H) = 66 bpm) when compared to controls (f(H) = 52 bpm), suggesting that EE2 acts directly on the cardiac muscle of tadpoles, rather than being a result of an increased cardiac demand due to a higher activity level (i.e., avoidance response). Additionally, EE2 exerted a positive inotropic response, which resulted in a higher CPC, which occurred independently of an increase in the number of myofibrils of EE2-exposed animals, since RVM remained similar between experimental groups. Thus, the increase on cardiac demand induced by the exposure to EE2 elevates considerably the animal energy expenditure, diverting a large amount of energy that tadpoles could use for their growth and development. These alterations can make amphibians more susceptible to predators and reduce the likelihood to reach reproductive stage.

Effects of estradiol and ethinylestradiol on sperm quality, fertilization, and embryo-larval survival of pejerrey fish (Odontesthes bonariensis).

17ß-Estradiol (E2) and synthetic 17α-Ethinylestradiol (EE2) are estrogenic compounds present in surface waters as a consequence of municipal sewage discharges. The aim of this study was to evaluate the effects of E2, EE2 and its mixtures on different reproductive parameters and embryo-larval survival in pejerrey fish (Odontesthes bonariensis). In order to analyze the effect of these compounds on sperm quality, fertilization%, embryo-larval survival (%), and the point of no return (PNR), different assays were performed using concentrations 175, 350, 700 and 1400 ng/L of E2; 22.5, 45, 90 and 180 ng/L of EE2 and mixtures M1 (175 E2+22.5 EE2, ng/L), M2 (350 E2+45 EE2, ng/L), M3 (700 E2+90 EE2, ng/L) and M4 (1400 E2+180 EE2 ng/L). No significant differences in motility parameters were observed between E2 and EE2 treatments and the control group. However, a significant decrease in motility% was recorded for all mixtures tested compared with the control samples. For fertilization%, only sperm activated with M4 showed a significant decrease compared with the control group. In the case of embryo survival, there was only a significant decrease in the highest concentration of EE2 compared with the control group. For the mixtures, M3 is the one that had the most adverse effect on embryo survival. In larval survival, there was a significant decrease in concentration 175 and 700 ng/L of E2 compared with the control group. In EE2 treatments, the ones with a significant reduction in larval survival were concentration 45 and 90 ng/L. And for the mixture treatments, M1, M3 and M4 had a significantly lower larval survival than the control group. In comparison to other treatments, M1 demonstrated a significant difference in PNR when compared with the control group. The results obtained demonstrated that the exposure to mixtures of E2 and EE2 affected fish sperm motility, fertilization% and, embryo and larval survival even at relevant environmental concentrations highlighting the necessity of considering the effects of pollutants mixtures in ecotoxicological studies.

Effects of the synthetic estrogen 17α-ethinylestradiol on aromatase expression, reproductive behavior and sperm quality in the fish Jenynsia multidentata.

The synthetic estrogen 17α-ethynylestradiol (EE2) has been increasingly detected in sewage effluents in the last two decades. The aim of the present study was determined if EE2 exposure adversely affected reproduction in internally fertilizing fish species Jenynsia multidentata. Sexual behavior, brain and gonadal aromatase expression as well as sperm quality were evaluated. The brain aromatase expression, reproductive behavior, spermatozoa viability and gonadosomatic index were sensitive biomarkers of EE2 effects on this species. The condition factor, hepatosomatic index, gonadal aromatase expression, sperm count and sperm velocities were unaltered after EE2 exposure. The present work highlights the importance of using a combination of several biomarkers to study the effects of estrogenic compounds, especially when trying to link these results to potential population-level effects.

Low concentrations, potential ecological consequences: synthetic estrogens alter life-history and demographic structures of aquatic invertebrates.

Contraceptive drugs are nowadays found in aquatic environments around the globe. Particularly, 17α-ethinylestradiol (EE2) may act even at low concentrations, such as those recorded in natural ecosystems. We evaluated the physiological effects of EE2 on cyclopoids and calanoids, common copepods in both marine and freshwater communities. We used three EE2 concentrations and assessed its impact on activity of different physiological endpoints: Acetylcholinesterase (neurotransmission), Glutathione S-transferase (detoxifying system), and Caspase-3 (apoptosis). While EE2 exerts, distinctive effect on detoxifying and apoptotic systems, no effect on AChE was observed at environmental doses. Our results show that EE2 exposure affects differently copepod physiology endpoints, altering moulting process, adult recruitment in calanoids and calanoid to cyclopoid ratio. The ecological consequences of this underlying physiological process may affect since life history to population and community structures, and this represent a new aspects of this xenobiotic in natural systems.

Microscopic comparative study of the exposure effects of testosterone cypionate and ethinylestradiol during prenatal life on the prostatic tissue of adult gerbils.

There is an increasing variety of endocrine disrupting chemicals (EDCs) either with (anti)estrogenic or (anti)androgenic potential widely present in the environment. These xenosteroids may mimic endogenous steroid hormones disrupting the homeostasis of physiological pathways and leading to several disturbances, especially in tissues highly dependent on steroid hormones such as the prostate. Taking this into account, this comparative study aimed to verify the potential of ethinylestradiol (EE) and testosterone acting as ECDs on the prostate of both male and female adult gerbils exposed to these agents during the embryonic phase. Consequently, pregnant gerbils were treated either with 10 µg/kg/day of EE or with a single dose of 1 mg of testosterone cypionate. The pups that were born 6-8 days after testosterone exposure and the pups that were born after 3 days of EE exposure were allowed to grow but were sacrificed within 4 months. Serological, morphological, stereological, and immunohistochemical analyses were used. Overall, the results showed that both sexes exposed to testosterone and EE during gestation had a prostatic gland with an increased stromal and epithelial and a reduced luminal compartment. Moreover, we observed that glands affected with prostatic intraepithelial neoplasia showed intense stromal reshuffling. In conclusion, although these alterations were observed in both sexes, more relevant to this study was the differential responsiveness of males and females exposed to these different drugs. Whereas the EE affected males more, the testosterone was more harmful to the females.

Exposure to ethinylestradiol during prenatal development and postnatal supplementation with testosterone causes morphophysiological alterations in the prostate of male and female adult gerbils.

Steroids perform significant functions in prostatic development and growth, so that interferences of this equilibrium may predispose the gland to the development of diseases during the life. Embryonic and neonatal exposure to xenoestrogens, many of them with endocrine-disrupting potential, has been related to the induction of disturbances in reproductive system organs. Thus, this study aimed to analyse morphological and immunocytochemical aspects of prostate in both male and female adult gerbils either exposed to ethinylestradiol during the prenatal phase (pregnant females received 10 µg/kg, by gavage) (EE group) or exposed to testosterone (1 mg/kg) during the postnatal period (EE/T group). Serological analysis revealed a rise in estradiol levels in adult males and females of the EE group. A higher incidence of prostatic intraepithelial neoplasia (PIN) was observed in the male and female prostate of the treated groups, besides an increase in collagen and reticular fibres. Immunocytochemistry showed an increase in prostatic epithelial cells immunoreactive to AR and a presence of a smooth muscle layer, evidenced by α actin, in injured regions this way absent in prostatic epithelial buds. These pieces of evidence suggest that the alterations verified in the prostate in adulthood of both sexes may be due to the high oestrogen levels. Either males or females of the EE/T group showed normalized estradiol levels, although prostatic lesions could be observed. While the prostatic gland of male gerbils was more affected than the female prostate, this study showed that the exposure to EE during this critical period of development disrupts the prostate of both sexes in terms of prostatic lesions.