Altered non-reproductive behavior and feminization caused by developmental exposure to 17α-ethinylestradiol persist to adulthood in three-spined stickleback (Gasterosteus aculeatus).

The synthetic estrogen 17α-ethinylestradiol (EE2), ubiquitous in the aquatic environment and commonly detected in sewage effluents, interferes with the endocrine system in multiple ways. Exposure during sensitive windows of development causes persistent effects on fertility, reproductive and non-reproductive behavior in mammals and fish. In the present study, three-spined stickleback (Gasterosteus aculeatus) were exposed to nominal 0 and 20 ng/L EE2 from fertilization to 7 weeks post-hatch. After 8 months of remediation in clean water three non-reproductive behaviors, not previously analyzed in developmentally EE2-exposed progeny of wild-caught fish, were evaluated. Chemical analysis revealed that the nominal 0 and 20 ng/L exposure contained 5 and 30 ng/L EE2, respectively. Therefore, the use of control fish from previous experiments was necessary for comparisons. Fish exposed during development showed significant concentration-dependent reduction in anxiety-like behavior in the scototaxis (light/dark preference) test by means of shorter latency to first entrance to the white compartment, more visits in white, and longer total time in white compared to unexposed fish. In the novel tank test, developmental exposure significantly increased the number of transitions to the upper half of the aquaria. Exposure to EE2 during development did not alter shoal cohesion in the shoaling test compared with unexposed fish but fish exposed to 30 ng/L EE2 had significantly longer latency to leave the shoal and fewer transitions away from the shoal compared to fish exposed to 5 ng/L EE2. Skewed sex ratio with more females, sex reversal in genetic males as well as intersex in males was observed after exposure to 30, but not 5 ng/L EE2. In conclusion, EE2 exposure during development in three-spined stickleback resulted in persistent effects on anxiety-like behaviors. These long-term effects from developmental exposure are likely to be of higher relevance for natural populations than are short-term effects from adult exposure.

Combinatory effects of low concentrations of 17α-etinylestradiol and citalopram on non-reproductive behavior in adult zebrafish (Danio rerio).

Sewage effluents contain pharmaceuticals, personal care products and industrial chemicals, exposing aquatic organisms to complex mixtures. The consequences of exposure to combinations of different classes of drugs in fish are largely unknown. In this study, we exposed adult zebrafish (Danio rerio) males and females for two weeks to low, environmentally relevant concentrations of the endocrine disrupting chemical 17α-etinylestradiol (EE2) and the selective serotonin re-uptake inhibitor (SSRI) citalopram, alone and in combination, and analyzed behaviors of importance for population fitness, scototaxis (light/dark preference), the novel tank test and shoal cohesion. Control water contained 0.4ng/L EE2 and the measured exposure concentrations were 0.9ng/L EE2 (nominal 0.1) and 1ng/L EE2 (nominal 0.5). The measured concentrations of citalopram were 0.1 (nominal 0.1) and 0.4µg/L (nominal 0.5). Both EE2 exposures increased anxiety in males in the scototaxis test, with significantly longer latency periods before entering and fewer visits to the white zone of the tank. The combined exposures (0.9ng/L EE2+0.1µg/L citalopram and 1ng/L EE2+0.4µg/L citalopram) resulted in abolishment of effects of EE2, with shorter latency period and more transitions to white than for fish exposed to EE2 alone. In the novel tank test, the results surprisingly indicated lower anxiety after both EE2 and citalopram exposure. Significantly more transitions to the upper half of the tank observed in males exposed to 0.1µg/L citalopram alone compared to control males. Males exposed to EE2 (0.9ng/L) had shorter latency period to the upper half. Combination exposure resulted in a longer latency and fewer transitions to the upper half compared to both control, EE2- and citalopram-exposed males. Males exposed to the combination spent significantly less time in the upper half than males EE2 or citalopram-exposed males. Females exposed to 1ng/L EE2 had fewer transitions to the upper half than the control group and females exposed to 0.4µg/L citalopram. In the shoaling test, males exposed to 0.1µg/L citalopram+0.9ng/L EE2 showed more transitions away from peers than males exposed to 0.1µg/L citalopram alone. In conclusion, low concentrations of EE2, closely above the predicted no effect concentration (NOEC) of 0.1ng/L, created anxiety-like behavior in zebrafish males. Citalopram showed marginal effects at these low concentrations but in the combination exposure the behavioral effects of EE2 were abolished. This is an initial effort to understand the effects of cocktails of anthropogenic substances contaminating aquatic environments.

Transgenerational effects of 17α-ethinyl estradiol on anxiety behavior in the guppy, Poecilia reticulata.

Environmental contaminants can cause alterations that can be transgenerationally transmitted to subsequent generations. Estrogens are among those contaminants shown to induce heritable changes that persist over generations in mammals. Results in other vertebrates are few. We have analyzed the effects on anxiety of 17α-ethinyl estradiol (EE2) in the F1 and F2 generations in guppies, Poecilia reticulata, obtained from F0 fish maternally exposed to 0 or 20ng/L EE2 until birth. F0 males and females were bred with fish of the same treatment but different families producing F1 offspring. Behavior in the novel tank test at 6months revealed that males with EE2-exposed parents had significantly longer latency to the upper half of the tank than control males, while no EE2 effects were observed in females. Also in F2, obtained from F1 as above, males in the EE2 group had longer latency time compared to control males, with no differences due to EE2-exposure of F0 observed in females. In the scototaxis (light/dark preference) test, latency to first transition to black compartment and total transitions to black were significantly altered in females due to EE2 exposure of F0 while the total time in black was higher in males with EE2-exposed F0 compared with controls. The increased anxiety in the F2 generation demonstrates a transgenerational anxiety phenotype and shows that non-reproductive behavior can be transgenerationally modified by estrogens in fish.

Developmental exposure of zebrafish (Danio rerio) to 17α-ethinylestradiol affects non-reproductive behavior and fertility as adults, and increases anxiety in unexposed progeny.

Exposure to estrogenic endocrine disruptors (EDCs) during development affects fertility, reproductive and non-reproductive behavior in mammals and fish. These effects can also be transferred to coming generations. In fish, the effects of developmental EDC exposure on non-reproductive behavior are less well studied. Here, we analyze the effects of 17α-ethinylestradiol (EE2) on anxiety, shoaling behavior and fertility in zebrafish after developmental treatment and remediation in clean water until adulthood. Zebrafish embryos were exposed from day 1 to day 80 post fertilization to actual concentrations of 1.2 and 1.6ng/L EE2. After remediation for 82days non-reproductive behavior and fertilization success were analyzed in both sexes. Males and females from the 1.2ng/L group, as well as control males and females, were bred, and behavior of the untreated F1 offspring was tested as adults. Developmental treatment with 1.2 and 1.6ng/L EE2 significantly increased anxiety in the novel tank test and increased shoaling intensity in both sexes. Fertilization success was significantly reduced by EE2 in both sexes when mated with untreated fish of opposite sex. Progeny of fish treated with 1.2ng/L EE2 showed increased anxiety in the novel tank test and increased light avoidance in the scototaxis test compared to control offspring. In conclusion, developmental exposure of zebrafish to low doses of EE2 resulted in persistent changes in behavior and fertility. The behavior of unexposed progeny was affected by their parents' exposure, which might suggest transgenerational effects.

Short-term treatment of adult male zebrafish (Danio Rerio) with 17α-ethinyl estradiol affects the transcription of genes involved in development and male sex differentiation.

The synthetic estrogen 17α-ethinyl estradiol (EE2) disturbs reproduction and causes gonadal malformation in fish. Effects on the transcription of genes involved in gonad development and function that could serve as sensitive biomarkers of reproductive effects in the field is, however, not well known. We have studied mRNA expression in testes and liver of adult zebrafish (Danio rerio) males treated with 0, 5 or 25 ng/L EE2for 14 days. qPCR analysis showed that the mRNA expression of four genes linked to zebrafish male sex determination and differentiation, Anti-Mullerian Hormone, Double sex and mab-related protein, Sry-related HMG box-9a and Nuclear receptor subfamily 5 group number 1b were significantly decreased by 25 ng/L, but not 5 ng/L EE2 compared with the levels in untreated fish. The decreased transcription was correlated with a previously shown spawning failure in these males (Reyhanian et al., 2011. Aquat Toxicol 105, 41-48), suggesting that decreased mRNA expression of genes regulating male sexual function could be involved in the functional sterility. The mRNA level of Cytochrome P-45019a, involved in female reproductive development, was unaffected by hormone treatment. The transcription of the female-specific Vitellogenin was significantly induced in testes. While testicular Androgen Receptor and the Estrogen Receptor-alpha mRNA levels were unchanged, Estrogen receptor-beta was significantly decreased by 25 ng/L EE2. Hepatic Estrogen Receptor-alpha mRNA was significantly increased by both exposure concentrations, while Estrogen Receptor-beta transcription was unaltered. The decreased transcription of male-predominant genes supports a demasculinization of testes by EE2 and might reflect reproductive disturbances in the environment.

Reprint of "Effects of the SSRI citalopram on behaviours connected to stress and reproduction in Endler guppy, Poecilia wingei".

Psychoactive drugs, such as selective serotonin reuptake inhibitors (SSRI) have been identified in high levels in effluents from Swedish sewage treatment plants (STP) at concentrations high enough to give pharmacological effects in fish. In humans SSRIs are used in the treatment of depression and they have anxiolytic effects. In the present study we exposed Endler guppy (Poecilia wingei) of both sexes to citalopram that showed the highest concentrations of SSRIs in STP effluents and studied reproductive and non-reproductive behaviour. Male courting behaviours were not affected compared to control fish after 14-28 days exposure to 1 µgL(-1). In two experiments exposing both sexes to 0.2, 2.3 or 15 µgL(-1) for 21 days, fish exposed to the two highest doses showed anxiolytic effects when placed in a novel environment (novel tank diving test, NT). Males were only affected by exposure to 15 µgL(-1). They had significantly longer latency to explore the upper half of the aquarium, more visits and longer time spent in the upper half, and showed less bottom freezing behaviour, all markers of anxiolytic behaviour. In females exposure to 2.3 or 15 µgL(-1) significantly increased freezing behaviour, while no effects on other behaviour variables were observed. No effects on shoaling behaviour could be discerned. These results show that citalopram have anxiolytic effects on guppy fish and thus affect ecologically relevant behaviours of importance to survival of fish.

Effects of the SSRI citalopram on behaviours connected to stress and reproduction in Endler guppy, Poecilia wingei.

Psychoactive drugs, such as selective serotonin reuptake inhibitors (SSRI) have been identified in high levels in effluents from Swedish sewage treatment plants (STP) at concentrations high enough to give pharmacological effects in fish. In humans SSRIs are used in the treatment of depression and they have anxiolytic effects. In the present study we exposed Endler guppy (Poecilia wingei) of both sexes to citalopram that showed the highest concentrations of SSRIs in STP effluents and studied reproductive and non-reproductive behaviour. Male courting behaviours were not affected compared to control fish after 14-28 days exposure to 1 µg L(-1). In two experiments exposing both sexes to 0.2, 2.3 or 15 µg L(-1) for 21 days, fish exposed to the two highest doses showed anxiolytic effects when placed in a novel environment (novel tank diving test, NT). Males were only affected by exposure to 15 µg L(-1). They had significantly longer latency to explore the upper half of the aquarium, more visits and longer time spent in the upper half, and showed less bottom freezing behaviour, all markers of anxiolytic behaviour. In females exposure to 2.3 or 15 µg L(-1) significantly increased freezing behaviour, while no effects on other behaviour variables were observed. No effects on shoaling behaviour could be discerned. These results show that citalopram have anxiolytic effects on guppy fish and thus affect ecologically relevant behaviours of importance to survival of fish.

Brain circuit imprints of developmental 17α-Ethinylestradiol exposure in guppies (Poecilia reticulata): persistent effects on anxiety but not on reproductive behaviour.

The effects of endocrine disruptors may vary with the timing of exposure. The physiological implications of adult exposure are present during and shortly after exposure while embryonic exposure can imprint changes manifested in adulthood. In this study, guppy (Poecilia reticulata) embryos were exposed to 2 and 20 ng/L of 17α-ethinylestradiol during development via the mother and reared in clean water from gestation until 6 months of age. As adults, fish exposed to 20 ng/L during development showed significantly altered behaviour in the Novel Tank test, where anxiety is determined as the tendency to remain at the bottom upon introduction into an unfamiliar tank. 17α-ethinylestradiol treatment increased the latency time before swimming to the upper half of the tank and decreased the number of transitions to the upper half. In control females the basal stress behaviour responses were significantly higher than in males, as indicated by longer latency period and fewer and shorter visits to the upper half, supporting the importance of gonadal hormones for the behaviour. The anxiety increased, however, with treatment in both sexes, suggesting that the observed response is not entirely due to feminisation of the males. Shoaling behaviour, analysed as tendency to leave a shoal of littermates, was neither sex-differentiated nor changed by treatment. Also male reproductive behaviour, brain aromatase activity and testes histology, previously shown to respond to oestrogen exposure in adult guppy, were unaffected by the developmental treatment. This suggests that the stress system in the guppy is very sensitive to 17α-ethinylestradiol, which possibly causes an early organisational imprint on the brain circuit that regulates stress reactions.

Long-term 17alpha-ethinyl estradiol treatment decreases cyclin E and cdk2 expression, reduces cdk2 kinase activity and inhibits S phase entry in regenerating rat liver.

BACKGROUND/AIMS: The synthetic estrogen 17alpha-ethinyl estradiol (EE), a potent tumor promoter in rat liver, stimulates growth during short-term treatment but inhibits hepatocyte proliferation upon prolonged treatment. To identify the molecular targets of the mitoinhibitory effect of EE, the expression of proteins regulating G(1)- and S-progression were analyzed during the first cell cycle in EE-treated female Wistar rats. METHODS: Long-term (60 days) EE treatment. Immunohistochemical staining for proliferation cell nuclear antigen (PCNA) to detect cells in S phase and quantification of mitosis. Western blot to monitor protein expression. Cdk2 kinase assay to examine histone H1 phosphorylation. RESULTS: EE reduced the number of cells in S phase and mitosis by about 70%. Cyclin D1 and D3 were unaffected, while cdk4 was moderately decreased. Cyclin E and cdk2 were markedly decreased with concomitant marked reduction of cdk2 kinase activity. EE also decreased cyclin A and increased G1 levels of p53 and p21. CONCLUSIONS: EE causes a cell cycle block before S-phase. The reduction of the cdk2 kinase activity, essential for G1/S-transition, might be involved in the cell cycle block. Also, EE treatment results in p53 activation and upregulation of the cdk inhibitor p21 that might contribute to the G1 arrest.

Mitoinhibitory effects of the tumor promoter 2-acetylaminofluorene in rat liver: loss of E2F-1 and E2F-3 expression and cdk 2 kinase activity in late G1.

BACKGROUND/AIMS: Examine the mitoinhibitory effect of the liver tumor promoter 2-acetylaminofluorene (2-AAF) in vivo, with focus on the proteins regulating G1- and S progression. METHODS: cdk 2 kinase assay to examine histone H1 phosphorylation. cdk 4 kinase assay to examine whether active cdk 4/cyclin D complexes, capable of phosphorylating Rb, are formed. Western blot to monitor protein expression. RESULTS: cdk 4 kinase-mediated Rb phosphorylation was increased by AAF treatment. Nuclear expression of the transcription factors E2F-1 and E2F-3 was downregulated, while E2F-4 was decreased. 2-AAF treatment also markedly reduced cdk 2 kinase activity/histone H1 phosphorylation during G1/S-transition. Western blot showed loss of nuclear as well as cytoplasmic cyclin A and cyclin B protein after 2-AAF treatment, while the Rb protein level was markedly increased during G1. The cell cycle dependent elevation of nuclear p107 protein, seen in control livers, was not observed in AAF-treated animals. CONCLUSIONS: Effects of 2-AAF; Very low cdk 2 kinase activity that could possibly block G1/S-transition; increased pRb level together with diminished levels of transcription factors E2F-1 and -3, that could be responsible for reducing the expression of E2F target genes such as cyclin A and E2F-1.