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Alkylphenols, such as nonylphenol and 4-tert-octylphenol (OP), are byproducts of the biodegradation of alkylphenol ethoxylates and present substantial ecological and health risks in aquatic environments and higher life forms. In this context, our study aimed to explore the effect of OP on reproductive endocrine function in both female and male zebrafish. Over a period of 21 days, the zebrafish were subjected to varying concentrations of OP (0, 0.02, 0.1, and 0.5 μg/L), based on the lowest effective concentration (EC10 = 0.48 μg/L) identified for zebrafish embryos. OP exposure led to a pronounced increase in hepatic vitellogenin (vtg) mRNA expression and 17β-estradiol biosynthesis in both sexes. Conversely, OP exhibits anti-androgenic properties, significantly diminishes gonadal androgen receptor (ar) mRNA expression, and reduces endogenous androgen (testosterone and 11-ketotestosterone) levels in male zebrafish. Notably, cortisol and thyroid hormone (TH) levels demonstrated concentration-dependent elevations in zebrafish, influencing the regulation of gonadal steroid hormones (GSHs). These findings suggest that prolonged OP exposure may result in sustained reproductive dysfunction in adult zebrafish, which is largely attributable to the intricate reciprocal relationship between hormone levels and the associated gene expression. Our comprehensive biological response analysis of adult zebrafish offers vital insights into the reproductive toxicological effects of OP, thereby enriching future ecological studies on aquatic systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Combined oral contraceptives, comprising of both an oestrogen and a progestin component, are released in aquatic environments and potentially pose a risk to aquatic wildlife by their capacity to disrupt physiological mechanisms. In this study, the endocrine disruptive potential of two mixtures, 17α-ethinylestradiol (EE2), a synthetic oestrogen, or estetrol (E4), a natural oestrogen, with the progestin drospirenone (DRSP) have been characterised in three generations of zebrafish, according to an adapted Medaka Extended One Generation Reproduction Test. Zebrafish (Danio rerio) were exposed to a range of concentrations of EE2/DRSP and E4/DRSP (∼1×, ∼3×, ∼10× and ∼30× predicted environmental concentration, PEC). Survival, growth, hatching success, fecundity, fertilisation success, vitellogenin (VTG), gonad histopathology, sex differentiation, and transcriptional analysis of genes related to gonadal sex steroid hormones synthesis were assessed. In the F0 generation, exposure to EE2/DRSP at ∼10 and ∼30× PEC decreased fecundity and increased male VTG concentrations. The highest concentration of EE2/DRSP also affected VTG concentrations in female zebrafish and the expression of genes implicated in steroid hormones synthesis. In the F1 generation, sex determination was impaired in fish exposed to EE2/DRSP at concentrations as low as ∼3× PEC. Decreased fecundity and fertility, and abnormal gonadal histopathology were also observed. No effects were observed in the F2 generation. In contrast, E4/DRSP induced only minor histopathological changes and an increase in the proportion of males, at the highest concentration tested (∼30× PEC) in the F1 generation and had no effect on hatching success of F2 generation. Overall, this study suggests that the combination E4/DRSP has a more favourable environmental profile than EE2/DRSP., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Céline Gérard and Céline Delierneux are employees of Mithra Pharmaceuticals., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Perchlorate, a widespread environmental contaminant originating from various industrial applications, agricultural practices, and natural sources, poses potential risks to ecosystems and human health. While previous studies have highlighted its influence on the thyroid endocrine system and its impact on gonadal maturation, reproduction, and sex hormone synthesis, the specific interplay between thyroid and steroid hormones, in this context, remains largely unexplored. Therefore, this study was undertaken to investigate the adverse effects and underlying mechanisms triggered by exposure to sodium perchlorate (SP) on reproductive endocrine activity in zebrafish. For 21 d, the fish were exposed to test SP concentrations (0, 3, 30, 300 mg/L), which were determined based on the exposure concentrations that induced various toxic effects in the fish, considering naturally occurring concentrations. Exposure to SP, except at 3 mg/L in males, significantly decreased the production of thyroid hormone (TH) in both female and male zebrafish. Moreover, gonadal steroid levels were markedly reduced in both sexes. The expression of hepatic vitellogenin (VTG) mRNA in female zebrafish was significantly decreased, whereas aromatase activity in male zebrafish was significantly elevated in the SP exposure groups. The reduced levels of THs and gonadal steroid hormones were strongly correlated. Abnormal responses to SP exposure led to reduced reproductive success in the 300 mg/L SP exposure group. These findings indicate that prolonged and continuous exposure to a specific concentration of SP may lead to long-term reproductive problems in zebrafish, primarily through hormonal imbalances and suppression of hepatic VTG mRNA expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The specific functions and essentiality of type II vitellogenin (Vtg2) in early zebrafish development were investigated in this study. A vtg2-mutant zebrafish line was produced and effects of genomic disturbance were observed in F2 females and F3 offspring. No change in vtg2 transcript has been detected, however, Vtg2 abundance in F2 female liver was 5×, and in 1 hpf F3 vtg2-mutant embryos was 3.8× less than Wt (p < 0.05). Fecundity was unaffected while fertilization rate was more than halved in F2 vtg2-mutant females (p < 0.05). Hatching rate was significantly higher in F3 vtg2-mutant embryos in comparison to Wt embryos. Survival rate declined drastically to 29% and 18% at 24 hpf and 20 dpf, respectively, in F3 vtg2-mutant embryos. The introduced mutation caused vitelline membrane deficiencies, significant mortalities at early embryonic stages, and morphological abnormalities in the surviving F3 vtg2-mutant larvae. Overrepresentation of histones, zona pellucida proteins, lectins, and protein degradation related proteins in F3 vtg2-mutant embryos provide evidence to impaired mechanisms involved in vitellin membrane formation. Overall findings imply a potential function of Vtg2 in acquisition of vitellin membrane integrity, among other reproductive functions, and therefore, its essentiality in early zebrafish embryo development., (© 2023. The Author(s).)

In recent years, the widespread use of the pesticide cyprodinil has attracted attention due to its harmful effects on aquatic organisms. The purpose of this study was to evaluate the adverse effects of long-term exposure to cyprodinil on the reproductive system of female zebrafish. After the embryos had been treated with 0.1, 1 and 10 μg/L cyprodinil for 180 days, we observed that female fish treated with 1 and 10 μg/L cyprodinil showed decreased sexual attractiveness, a decreased proportion of primordial follicles in the ovary, an increased proportion of mature follicles, and increased egg production. Moreover, exposed females that mated with normal males produced offspring with increased rates of mortality and deformity (the F1 generation). In addition, the levels of gonadotropin and testosterone (T) were increased in females after cyprodinil exposure, especially in the 10 μg/L treated group. After cyprodinil treatment, some key genes in the hypothalamic-pituitary-gonad axis underwent significant changes. For example, gene expression of brain gonadotropin-releasing hormone receptors (gnrhr1, gnrhr2 and gnrhr4) was significantly downregulated after cyprodinil treatment. The study found that expression of the aromatase (cytochrome P450 family 19 subfamily A polypeptide 1a, cyp19a1a) responsible for converting T into estradiol was significantly downregulated after cyprodinil treatment, consistent with elevated T levels in the ovaries and muscles. In summary, these data provide a more comprehensive understanding of the toxicity of cyprodinil and may inform evaluation of the ecotoxicity of cyprodinil to female reproduction at environmentally relevant concentrations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The bioconcentration factor (BCF) is a key parameter for bioavailability assessment of environmental pollutants in regulatory frameworks. The comparative toxicology and mechanism of action of congeners are also of concern. However, there are limitations to acquire them by conducting field and laboratory experiments while machinelearning is emerging as a promising predictive tool to fill the gap. In this study, the Direct Message Passing Neural Network (DMPNN) was applied to predict logBCFs of bisphenol A (BPA) and its four analogues (bisphenol AF (BPAF), bisphenol B (BPB), bisphenol F (BPF) and bisphenol S (BPS)). For the test set, the Pearson correlation coefficient (PCC) and mean square error (MSE) were 0.85 and 0.52 respectively, suggesting a good predictive performance. The predicted logBCFs values by the DMPNN ranging from 0.35 (BPS) to 2.14 (BPAF) coincided well with those by the classical EPI Suite (BCFBAF model). Besides, estrogen receptor α (ERα) bioactivity of these bisphenols was also predicted well by the DMPNN, with a probability of 97.0 % (BPB) to 99.7 % (BPAF), which was validated by the extent of vitellogenin (VTG) induction in male zebrafish as a biomarker except BPS. Thus, with little need for expert knowledge, DMPNN is confirmed to be a useful tool to accurately predict logBCF and screen for estrogenic activity from molecular structures. Moreover, a gender difference was noted in the changes of three endpoints (logBCF, ER binding affinity and VTG levels), the rank order of which was BPAF > BPB > BPA > BPF > BPS consistently, and abnormal amino acid metabolism is featured as an omics signature of abnormal hormone protein expression., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In aquatic milieus, tris (2-chloroethyl) phosphate (TCEP) was detected as an emerging environmental contaminant. In this study, in vivo experiment and in-silico docking was integrated systematically to explore the toxic mechanisms of TCEP using zebrafish (Danio rerio). Fish (mean weight of 0.24 ± 0.02 g) were exposed to 100 and 1500 μg L -1 concentrations of TCEP for 28 days under the static renewal method. During chronic exposure, plasma steroid hormones such as testosterone (T) and 17β estradiol (E2), plasma vitellogenin (Vtg) and antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and lipid peroxidation (LPO) in gonads were significantly (P < 0.05) altered in TCEP exposed group (1500 μg L -1 ) compared to the control group. However, the alterations of these parameters were not significant on the 14th day (except Vtg and GR in testis) in 100 μg L -1 of TCEP exposed groups. There were no significant differences (p > 0.05) in the growth parameters comparing TCEP exposed groups with the control group. The gonads of fish exposed to TCEP showed significant histopathological changes when compared to the control groups. A docking study observed that TCEP possessed binding affinity with the estrogen receptor (ERβ) and androgen receptor (AR). These data indicate that TCEP at tested concentrations adversely affects the aquatic organisms., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Emerging contaminants (EC) such as benzotriazole are being released into the environment in various ways, therefore it is necessary to understand how organisms are affected by EC. In this study, we exposed medaka (Oryzias latipes) and zebrafish (Danio rerio) during their embryonic period (1 day after hatching) to benzotriazole to investigate its effects on oxidative stress (ROS, GSH, GST, SOD, CAT and MDA) and changes in gene expression patterns. In both medaka and zebrafish, the influence of oxidative stress was confirmed through an increased MDA level and changes in the ROS and GSH levels. Antioxidant enzymes such as GST, CAT, and SOD were affected by benzotriazole; however, medaka and zebrafish showed different patterns in the effects by benzotriazole. Results of oxidative stress genes expression showed that medaka had either no influence or had a decrease in the gene expression profile, whereas zebrafish had a statistically significant increase in the expression of some genes. The cyp1a gene expression was increased in both species. However, vtg gene expression was increased only in zebrafish but decreased in medaka, indicating no estrogenic effects in medaka. Apoptosis genes showed changes in expression in both the species but was these changes were not dose-dependent. However, zebrafish caspase-9 gene expression was increased in all of the exposed groups, suggesting the effects on the intrinsic pathway associated with caspase-9. In conclusion, the results indicate that the toxic effects of benzotriazole differ at various levels in the two small fish medaka and zebrafish embryos., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A wide range of health effects in fish have been reported for exposure to wastewater treatment work (WwTW) effluents including feminized responses in males. Most of these exposure studies, however, have assessed acute health effects and chronic exposure effects are less well established. Using an Estrogen Responsive Element-Green Fluorescent Protein (ERE-GFP)-Casper transgenic zebrafish, we investigated chronic health effects and life stage sensitivities for exposure to an estrogenic WwTW effluent and the synthetic estrogen 17α-ethinylestradiol (EE2). Exposure to the WwTW effluent (at full strength;100%) and to 10 ng/L (nominal) EE2 delayed testis maturation in male fish but accelerated ovary development in females. Exposure to 50% and 100% effluent, and to 10 ng/L EE2, also resulted in skewed sex ratios in favor of females. Differing patterns of green fluorescent protein (GFP) expression, in terms of target tissues and developmental life stages occurred in the ERE-GFP- zebrafish chronically exposed to 100% effluent and reflected the estrogenic content of the effluent. gfp and vitellogenin ( vtg ) mRNA induction were positively correlated with measured levels of steroidal estrogens in the effluent throughout the study. Our findings illustrate the importance of a fish's developmental stage for estrogen exposure effects and demonstrate the utility of the ERE-GFP zebrafish for integrative health analysis for exposure to estrogenic chemical mixtures., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cooper, David, Lange and Tyler.)

The thymus is present in all gnathostome vertebrates and is an essential organ for the adaptive immune system via the generation of functional mature T-cells. Over the life span of mammals, the thymus undergoes morphological and functional alterations, including an age-related involution, which in humans starts in early life. Life history tradeoffs have been suggested as possible reasons for thymus involution. While in teleost fish, only a few studies have investigated alterations of thymus structure and function over different life stages, resulting in a fragmented database. Here, we investigated the thymus growth of zebrafish (Danio rerio) from early life, throughout puberty and reproductive stage, up to 1-year-old. We assessed thymus growth by histological and morphometric analyses and thymocyte numbers. Thymus function was assessed by measuring the transcripts of the thymocyte marker genes, ikaros, tcrα, and tcrδ. Additionally, we analyzed gonad maturity and tail homogenate vitellogenin concentrations to align thymus status with the status of the reproductive system. Our results showed that the zebrafish thymus, in contrast to the human thymus, grew strongly during early life and puberty but started to undergo involution when the fish reached the reproductive age. The involution was characterized by reduced thymus area and thymocyte number, altered histoarchitecture, and decreasing thymocyte marker gene transcript levels. Our findings suggest that age-related changes of the zebrafish thymus do exist and could be partly explained in terms of resource tradeoffs, but also in terms of the ontogenetically late development of a functional adaptive immune system in teleosts., (© 2020 Wiley Periodicals LLC.)

A study was conducted to understand the potential for ibuprofen to impact the hypothalamus-pituitary-gonadal endocrine axis resulting in disruption of fish reproduction. The Good Laboratory Practice study was conducted according to the Organisation for Economic Co-operation and Development 229 Protocol, Fish Short-Term Reproduction Assay, and extended an additional 4 d to evaluate hatching success in the F1 generation. Test organisms were exposed to nominal test concentrations of 0.5, 2.4, 11.5, 55.3, and 265.4 µg ibuprofen/L and a negative control (dilution water). To strengthen the statistical power of the study, twice the number of replicates were used in the negative control versus individual treatment levels. A 21-d pre-exposure to identify groups of actively spawning fish was immediately followed by a 36-d exposure. Results for apical endpoints of survival, growth, and reproduction (fecundity and fertility), as well as the biomarker vitellogenin in the F0 generation and time to hatch and hatching success in the F1 generation are presented. Based on mean measured exposure concentrations and effects on fecundity in the F0 generation and hatching success in the F1 generation, overall no-observed-effect concentration and lowest-observed-effect concentration for the present study were 55.2 and 265.9 µg ibuprofen/L, respectively. Results from the present study indicate a lack of endocrine-mediated reproductive effects in zebrafish at environmentally relevant concentrations of ibuprofen. Environ Toxicol Chem 2020;39:1534-1545. © 2020 SETAC., (© 2020 SETAC.)

Sexual differences have been observed in the onset and prognosis of human cardiovascular diseases, but the underlying mechanisms are not clear. Here, we found that zebrafish heart regeneration is faster in females, can be accelerated by estrogen and is suppressed by the estrogen-antagonist tamoxifen. Injuries to the zebrafish heart, but not other tissues, increased plasma estrogen levels and the expression of estrogen receptors, especially esr2a. The resulting endocrine disruption induces the expression of the female-specific protein vitellogenin in male zebrafish. Transcriptomic analyses suggested heart injuries triggered pronounced immune and inflammatory responses in females. These responses, previously shown to elicit heart regeneration, could be enhanced by estrogen treatment in males and reduced by tamoxifen in females. Furthermore, a prior exposure to estrogen preconditioned the zebrafish heart for an accelerated regeneration. Altogether, this study reveals that heart regeneration is modulated by an estrogen-inducible inflammatory response to cardiac injury. These findings elucidate a previously unknown layer of control in zebrafish heart regeneration and provide a new model system for the study of sexual differences in human cardiac repair.

Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, our objective was to use a newly developed transgenic zebrafish line expressing eGFP under the control of the cyp19a1a promoter in the OECD Fish Short Term Reproduction Assay (TG 229) to provide additional mechanistic information on tested substances. For this purpose, we exposed adult transgenic zebrafish to a reference substance of the TG 229, i.e. prochloraz (PCZ; 1.7, 17.2 and 172.6 μg/L). In addition to "classical" endpoints used in the TG 229 (reproductive outputs, vitellogenin), the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. Our data revealed that 172.6 μg/L PCZ significantly decreased the number of eggs laid per female per day and the concentrations of vitellogenin in females, reflecting the decreasing E2 synthesis due to the inhibition of the ovarian aromatase activities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 17.2 and 172.6 μg/L. A similar profile was observed for the endogenous cyp19a1a expression measured by qPCR thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. Overall, this study illustrates the feasibility of using the cyp19a1a-eGFP transgenic line for assessing the effect of PCZ in an OECD test guideline while providing complementary information on the time- and concentration-dependent effects of the compound, without disturbing reproduction of fish. The acquisition of this additional mechanistic information on a key target gene through in vivo fluorescence imaging of the ovaries was realized without increasing the number of individuals., Competing Interests: Declaration of Competing Interest The authors declare there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2020 Elsevier B.V. All rights reserved.)

2-bromo-4, 6-dinitroaniline (BDNA) is a mutagenic aromatic amine involved in the production and degradation of Disperse blue 79, one of the most extensively used brominated azo dyes. In our previous study, a multigenerational exposure of BDNA (0.5, 5, 50 and 500 μg/L) to zebrafish from F 0 adult to F 2 larvae including a recovery group in F 2 larvae was conducted. The effects on apical points observed in individuals and the long-term effects predicted on population were all related to reproduction. In this study, we performed molecular analysis to elucidate the underlying mechanisms of the reproductive toxicity of BDNA. In F 1 generation, measurement of vitellogenin and transcription levels of genes associated with hypothalamus-pituitary-gland (HPG) axis, estrogen receptor (ER) and androgen receptor (AR) were conducted. There was a decrease in VTG level in the blood of F 1 female fish and transcription of genes related to ER was more affected than that of genes related to AR. These results were consistent with adverse effects that sexual differentiation was biased towards males and fecundity was impaired in a concentration-dependent manner in adults of F 1 generation after 150 days exposure. In F 2 generation, global gene transcriptions of F 2 larvae were investigated. It was uncovered that processes related to apoptosis, development and DNA damage were strongly affected. Alterations to these biological pathways accounted for the irreversible parental influence on a significant decrease in hatchability and increase in abnormality of F 2 larvae. All evidence suggested that the multigenerational exposure of BDNA posed lasting effects transmitted from parents to offspring that persisted after exposure ceased., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Harmful cyanobacteria and their production of microcystins (MCs) exert significant toxicity on reproduction of fish, especially the process of oogenesis. Our previous studies demonstrated that MCs have negative impacts on the quantity and quality of mature oocytes in female zebrafish. However, the underlying mechanisms of MCs disrupting oocyte maturation (OM) have been rarely reported. In the present study, in vitro oocytes (immature) were separated from zebrafish and treated with 1, 10, 100 μg/L MC-LR. The serine/threonine protein phosphatase 2A (PP2A) activity was downregulated significantly in oocytes exposed to 10 and 100 μg/L MC-LR for both 2 and 4 h. The phosphorylation levels of mitogen-activated protein kinase (MAPK) were detected without noticeable change in all oocytes treated with MC-LR for 2 h, whereas the activated levels of MAPK subtypes (ERK, p38 and JNK) increased remarkably in the 100 μg/L MC-LR treatment of 4 h. In the oocytes exposed to 100 μg/L MC-LR for 4 h, germinal vesicle breakdown (GVBD) rates changed abnormally and maturation-promoting factor (MPF) activity increased significantly, in accordance with the upregulation of Cyclin B protein levels. Moreover, the MAPK inhibitors (10 μM) were applied to explore the role of MAPK subtypes during MC-LR influencing OM and results showed that ERK inhibitor U0126 and p38 inhibitor SB203580 mitigated the effects of 100 μg/L MC-LR-induced MAPK hyper-phosphorylation and elevated GVBD in the oocytes. In conclusion, the present study indicates that microcystins disrupt the meiotic maturation by the pathway of MC-PP2A-MAPK-OM due to the phosphorylation disorder in oocytes., (Copyright © 2019. Published by Elsevier B.V.)

Oviparous vertebrates produce multiple forms of vitellogenin (Vtg), the major source of yolk nutrients, but little is known about their individual contributions to reproduction and development. This study utilized clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing to assess essentiality and functionality of zebrafish (Danio rerio) type-I and type-III Vtgs. A multiple CRISPR approach was employed to knockout (KO) all genes encoding type-I vtgs (vtg1, 4, 5, 6, and 7) simultaneously (vtg1-KO), and the type-III vtg (vtg3) individually (vtg3-KO). Results of polymerase chain reaction (PCR) genotyping and sequencing, quantitative PCR, liquid chromatography-tandem mass spectrometry, and Western blot analysis showed that only vtg6 and vtg7 escaped Cas9 editing. In fish whose remaining type-I vtgs were incapacitated (vtg1-KO), and in vtg3-KO fish, significant increases in Vtg7 transcript and protein levels occurred in liver and eggs, revealing a heretofore-unknown mechanism of genetic compensation regulating Vtg homeostasis. Egg numbers per spawn were elevated more than 2-fold in vtg1-KO females, and egg fertility was approximately halved in vtg3-KO females. Substantial mortality was evident in vtg3-KO eggs/embryos after only 8 hr of incubation and in vtg1-KO embryos after 5 days. Hatching rate and timing were markedly impaired in embryos from vtg mutant mothers and pericardial and yolk sac/abdominal edema and spinal lordosis were evident in the larvae, with feeding and motor activities also being absent in vtg1-KO larvae. By late larval stages, vtg mutations were either completely lethal (vtg1-KO) or nearly so (vtg3-KO). These novel findings offer the first experimental evidence that different types of vertebrate Vtg are essential and have disparate requisite functions at different times during both reproduction and development., (© 2019 Wiley Periodicals, Inc.)

The purpose of this study was to investigate the bioaccumulation and biochemical responses exposed to one of the main organic ultraviolet (UV) pollutants in the environment, ethylhexyl methoxy cinnamate (EHMC), and its main transformation product, either alone or in combination in zebrafish (Danio rerio). Four-month-old zebrafish were exposed to EHMC (34.4, 344 nmol/L) solution for 14 days, the species and contents of EHMC transformation products in zebrafish were determined and 3,5-dichloro-2-hydroxyacetophenone (3,5DCl 2 HAcP) was the one with the highest concentration in transformation products. Then, zebrafish were exposed to EHMC, 3,5DCl 2 HAcP alone and mixed solution for 21 days. At 7, 14 and 21 d, the related indexes of antioxidant defense system were determined. Results showed that both EHMC and 3,5DCl 2 HAcP can lead to the increase of malondialdehyde (MDA) and glutathione (GSH) contents, superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) activities in visceral mass compared with the corresponding control group, thus produced oxidative stress effect in organism and 3,5DCl 2 HAcP even showed stronger oxidative stress than EHMC. The effects of the two lower concentration co-exposure groups were similar and more significant to that of single exposure groups, while excessive oxidative stress occurred at the highest co-exposure group indicated by the decrease of GSH content, SOD, CAT, GR activities and the continued increase of MDA content. At 21 d, estradiol (E 2 ), vitellogenin (Vtg) and testosterone (T) contents, estrogen receptor (Esr), progesterone receptor (Pgr), androgen receptor (Ar), Vtg1, P450 aromatase (Cyp19a1) and 17β-hydroxysteroid dehydrogenase (Hsd17b3) expression were all significantly increased when exposed to 3,5DCl 2 HAcP alone, showing complex estrogen and androgen effects. When exposed to EHMC alone, E 2 and Vtg contents, Esr, Pgr, Vtg1, Cyp19a1 and Hsd17b1 gene expression levels decreased significantly, and T content and Ar and Hsd17b3 expression increased significantly, indicated that EHMC can produce anti-estrogen and androgen effect. Last, the decrease of estrogen effect and increase of androgen effect in co-exposure group suggested that 3,5DCl 2 HAcP might weaken the estrogen effect and promote the androgen effect of EHMC., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 (CV N (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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this study, the vitellogenin (Vtg) modulation by genistein and polychlorinated biphenyl-126 (PCB-126) exposure in zebrafishes has been investigated. Both PCB-126 and genistein have been identified as aquatic pollutants and can further increase estrogenicity of waterways. Vtg is egg yolk precursor protein release by the hepatocytes during vitellogenesis. This process occurs normally in the hepatocytes in response to the activation with the estrogens such as 17-β-estradiol. Our immunohistochemical findings showed a Vtg expression that increases at 12 h and at 72 h in the liver of treated fishes with both PCB-126 and genistein, individually and in combination. Furthermore, for the first time, also hepatic stellate cells (HSC) in the liver parenchyma were strongly positive for vitellogenin.

Environmental estrogens are a serious concern worldwide due to their ubiquity and adverse ecotoxicological and health effects. Chemical structure of these substances is highly diverse, therefore estrogenicity cannot be predicted on the basis of molecular structure. Furthermore, estimation of estrogenicity of environmental samples based on chemical analytics of suspects is difficult given the complex interaction of chemicals and the impact on estrogenicity. The full estrogenic impact of an environmental sample can thus only be revealed by a series of sensitive in vitro and in vivo ecotoxicological tests. Herein we describe a vitellogenin reporter transgenic zebrafish line (Tg(vtg1:mCherry)) that enables the detection of estrogenicity in the environmentally relevant, low concentration ranges in embryonic tests that are in accordance with 3Rs and relevant animal welfare regulations. The transgene construct used for the development of Tg(vtg1:mCherry) carried a long (3.4 kbp) natural vitellogenin-1 promoter sequence with a high number of ERE sites. A test protocol was developed based on our finding that the endogenous vitellogenin and the reporter show similar spatial expression pattern and both endogenous and vitellogenin reporter is only produced in the left hepatic lobe of 5 dpf zebrafish embryos. Seven generations of Tg(vtg1:mCherry) have been established, and the estrogen responsiveness was tested with different estrogenic substances and wastewater samples. Embryos were exposed from 3 to 5 days post fertilization (dpf). Fluorescence in embryos could be detected upon treatment with 17-ß-estradiol from a concentration of 100 ng/L, 17-α-ethynilestradiol from 1 ng/L, zearalenone from 100 ng/L and bisphenol-A from 1 mg/L. In the adult stage transgene activity appeared to be more sensitive to estrogen treatment, with detectable transgene activity from 5 ng/L 17-ß-estradiol concentration. The transgenic line Tg(vtg1:mCherry) was also suitable for the direct measurement of estrogenicity in wastewater samples without sample extraction. The detection of estrogenic activity using the reporter line was confirmed by the bioluminescent yeast estrogen screen., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Polychlorinated biphenyls (PCBs) are a class of industrial chemicals that cause endocrine changes, since they are able to bind to estrogen receptors and interfere with estrogen-regulated processes, such as fish vitellogenesis. Therefore, the present work aimed to assess potential endocrine effects of PCB-77 exposure during zebrafish (Danio rerio) gonadal differentiation. To achieve that, zebrafish juveniles were exposed to increasing concentrations of PCB-77 for 14 days during a critical window of gonad differentiation (30-44 days post-fertilization). Vitellogenin (Vtg) levels and several endpoints such as survival, growth, gonadosomatic index (GSI) and hepatosomatic index were recorded at the end of exposure and/or after 3 months in clean medium. The results obtained showed a reduction of Vtg-like proteins in juveniles, just after exposure to PCB-77 accompanied, after 3 months, by a decrease in gonadal Vtg levels and GSI of females. These results suggest that exposure to PCB-77 during the critical window of gonadal differentiation decreased vitellogenesis in juvenile zebrafish which lasted until adulthood affecting the normal development of female gonad, which might have further implications in reproduction success.

Ethylhexyl methoxy cinnamate (EHMC) is one of the major organic ultraviolet (UV) filter pollutants in the environment. The purpose of this study was to investigate the parental transfer of EHMC and induced biochemical responses in zebrafish (Danio rerio). Zebrafish embryos were exposed to EHMC solution (1, 10, and 100 μg/L) for 4 months until sexual maturation. Then male and female parents were paired to lay eggs. F1 generations were divided into 2 categories: with and without continued EHMC exposure. EHMC was detected in both F0 parents and F1 eggs, indicating that EHMC can accumulate in zebrafish and transfer to offspring through reproduction. The hatching rate decreased and malformation rate increased significantly among parents and progeny embryos in the high concentration exposure group. For 40 dpf (days post-fertilisation) F0 generations, estradiol hormone and vitellogenin (Vtg) contents, the expression levels of Vtg1, P450 aromatase (Cyp19a and Cyp19b), 17β-hydroxysteroid dehydrogenase (Hsd17b1, Hsd17b3), estrogen receptor-alpha and progesterone receptor in all concentration groups decreased significantly, while androgen receptor increased significantly in 10 and 100 μg/L exposure groups compared with the corresponding control group, showing anti-estrogen and androgen effects. For 120 dpf F0 generations, acetylcholinesterase activity was significantly decreased and glutathione and malondialdehyde levels, superoxide dismutase, catalase and glutathione reductase activities were significantly increased in all treatment groups compared with the corresponding control group. In addition, F1 offspring with or without continued exposure to EHMC suffered similar or stronger oxidative stress compared with their parents. DNA breakage and apoptosis also occurred in 120 dpf parental liver cells in all treatment groups as a result of oxidative damage. Results suggested that EHMC have transfer effects between parents and offspring, which may cause negative effects on growth and development of zebrafish and induce biochemical responses in both parents and offspring., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Scrutiny of the zebrafish (Danio rerio) genomic database confirmed eight functional vitellogenin (vtg) genes, each with one or two transcript variants, and the encoded Vtg polypeptides were structurally and functionally characterized in detail by in silico and experimental analyses. There were five type I (vtgs1, 4, 5, 6, and 7), two type II (vtg2 and vtg8), and one type III (vtg3) vtg gene(s) encoding three major types of Vtg protein based on subdomain structure (Vtg-I, Vtg-II, and Vtg-III, respectively). Among various tissues of mature zebrafish, transcripts of the eight vtg genes were detected by RNA-Seq only in liver and intestine, with liver being the main site of vtg expression. All vtg transcripts except vtg8 were also detected in mature female liver by RT-qPCR. The relative abundances of Vtg proteins and their variants were quantified by LC-MS/MS in the liver of mature females and in eggs. The Vtgs were generally several fold more abundant in eggs, but profiles of abundance of the 19 different forms of Vtg evaluated were otherwise similar in liver and eggs, suggesting that yolk protein composition is determined largely by hepatic Vtg synthesis and secretion. Based on transcript and protein levels, Vtg-I is, by far, the dominant type of Vtg in zebrafish, followed by Vtg-II and then Vtg-III. When relative abundances of the different forms of Vtg were evaluated by LC-MS/MS in egg batches of good versus poor quality, no differences in the proportional abundance of individual forms of Vtg, or of different Vtg types, attributable to egg quality were observed.

This study was conducted to investigate the effects of embryonic short-term exposure to benzo(a)pyrene (BaP), a model polycyclic aromatic hydrocarbon, on ovarian development and reproductive capability in adult female zebrafish. In 1-year-old fish after embryonic exposure to BaP for 96 h, the gonadosomatic indices and the percentage of mature oocytes were significantly decreased in the 0.5, 5 and 50 nmol/L treatments. The spawned egg number, the fertilization rate and the hatching success were significantly reduced, while the malformation rate of the F1 unexposed larvae were increased. The mRNA levels of follicle-stimulating hormone, luteinizing hormone, ovarian cytochrome P450 aromatase cyp19a1a and cyp19b, estrogen receptor esr1 and esr2, and hepatic vitellogenin vtg1 and vtg2 genes, were down-regulated in adult female zebrafish that were exposed to BaP during embryonic stage. Both 17β-estradiol and testosterone levels were reduced in the ovary of adult females. The methylation levels of the gonadotropin releasing hormone (GnRH) gene gnrh3 were significantly increased in the adult zebrafish brain, and those of the GnRH receptor gene gnrhr3 were elevated both in the larvae exposed to BaP and in the adult brain, which might cause the down-regulation of the mRNA levels of gnrh3 and gnrhr3. This epigenetic change caused by embryonic exposure to BaP might be a reason for physiological changes along the brain-pituitary-gonad axis. These results suggest that short-term exposure in early life should be included and evaluated in any risk assessment of pollutant exposure to the reproductive health of fish., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Pharmaceutical residues and other micro-contaminants may enter aquatic environments through effluent from sewage treatment plants (STPs) and could cause adverse effects in wild fish. One strategy to alleviate this situation is to improve wastewater treatment by ozonation. To test the effectiveness of full-scale wastewater effluent ozonation at a Swedish municipal STP, the added removal efficiency was measured for 105 pharmaceuticals. In addition, gene expression, reproductive and behavioral endpoints were analyzed in zebrafish (Danio rerio) exposed on-site over 21 days to ozonated or non-ozonated effluents as well as to tap water. Ozone treatment (7 g O 3 /m 3 ) removed pharmaceuticals by an average efficiency of 77% in addition to the conventional treatment, leaving 11 screened pharmaceuticals above detection limits. Differences in biological responses of the exposure treatments were recorded in gene expression, reproduction and behavior. Hepatic vitellogenin gene expression was higher in male zebrafish exposed to the ozonated effluent compared to the non-ozonated effluent and tap water treatments. The reproductive success was higher in fish exposed to ozonated effluent compared to non-ozonated effluent and to tap water. The behavioral measurements showed that fish exposed to the ozonated STP effluent were less active in swimming the first minute after placed in a novel vessel. Ozonation is a capable method for removing pharmaceuticals in effluents. However, its implementation should be thoroughly evaluated for any potential biological impact. Future research is needed for uncovering the factors which produced the in vivo responses in fish., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Tributyltin (TBT), an organotin acting as aromatase (Cyp19a1) inhibitor, has been found to disrupt gametogenesis and reproductive behaviors in several fish species. However, few studies addressing the mechanisms underlying the impaired gametogenesis and reproduction have been reported. In this study, female adults of zebrafish (Danio rerio) were continuously exposed to two nominal concentrations of TBT (100 and 500 ng/L, actual concentrations: 90.8 ± 1.3 ng/L and 470.3 ± 2.7 ng/L, respectively) for 28 days. After exposures, TBT decreased the total egg number, reduced the hatchability and elevated the mortality of the larvae. Decreased gonadosomatic index (GSI) and altered percentages of follicles in different developmental stages (increased early-stage follicles and reduced mid/late-stage follicles) were also observed in the ovary of TBT-treated fish. TBT also lowered the plasma level of 17β-estradiol and suppressed the expressions of cyp19a1a in the ovary. In treated fish, up-regulated expressions of aldhla2, sycp3 and dmc1 were present in the ovary, indicating an enhanced level of meiosis. The mRNA level of vtg1 was dramatically suppressed in the liver of TBT-treated fish, suggesting an insufficient synthesis of Vtg protein, consistent with the decreased percentage of mid/late-stage follicles in the ovaries. Moreover, TBT significantly suppressed the reproductive behaviors of the female fish (duration of both sexes simultaneously in spawning area, the frequency of meeting and the visit in spawning area) and down-regulated the mRNA levels of genes involved in the regulation of reproductive behaviors (cyp19a1b, gnrh-3 and kiss 2) in the brain. In addition, TBT significantly suppressed the expressions of serotonin-related genes, such as tph2 (encoding serotonin synthase), pet1 (marker of serotonin neuron) and kiss 1 (the modulator of serotonin synthesis), suggesting that TBT might disrupt the non-reproductive behaviors of zebrafish. The present study demonstrated that TBT may impair the reproductive success of zebrafish females probably through disrupting oogenesis, disturbing reproductive behaviors and altering serotonin synthesis. The present study greatly extends our understanding on the reproductive toxicity of TBT on fish., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Microcystis aeruginosa, a primary species in cyanobacterial blooms, is ubiquitously distributed in water. Microcystins (MCs) purified from M. aeruginosa can exert reproductive toxicity in fish. However, the effects of M. aeruginosa at environmentally relevant levels on the reproductive and endocrine systems of zebrafish are still unknown. The present study investigated the reproductive and endocrine-disrupting toxicity of M. aeruginosa on female zebrafish (Danio rerio) by short-term exposure (96 h). After exposure, marked histological lesions in the liver or gonads, such as nuclear pyknosis and deformation, were observed, and the fertilization rate and hatchability of eggs spawned from treated females were both significantly lower than they were in females in the control group, suggesting the possibility of transgenerational effects of M. aeruginosa exposure. Moreover, M. aeruginosa exposure decreased the concentration of 17β-estradiol (E2) and testosterone (T) in female zebrafish. Interestingly, the vtg1 transcriptional level significantly decreased in the liver, whereas plasma vitellogenin (VTG) protein levels increased. The present findings indicate that M. aeruginosa could modulate endocrine function by disrupting transcription of hypothalamic-pituitary-gonadal-liver (HPGL) axis-related genes, and impair the reproductive capacity of female zebrafish, suggesting that M. aeruginosa causes potential adverse effects on fish reproduction in Microcystis bloom-contaminated aquatic environments., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Given the importance of bisphenol A (BPA) as a xenoestrogen and its potential effects on human and animal health, we evaluated BPA exposure's short-term effects on follicular development, yolk protein vitellogenin (VTG) production and aromatase expression in female zebrafish. Histological modifications were observed along with increased presence of atretic follicles. Whole-body VTG concentration increased with the dose of BPA exposure. In contrast, expression of Cyp19a mRNA in the ovaries of BPA-exposed fish exhibited an apparent non-monotonic response curve, marked by downregulation at 1 μg/L BPA, upregulation at 10 μg/L BPA, and a return to downregulation at 100 μg/L BPA and higher doses. Ovaries only exhibited significant increases in follicular atresia and VTG concentration after exposure to 100 μg/L BPA and higher doses. Ovarian histopathology, aromatase Cyp19a transcript levels and whole-body VTG protein abundance may be good biomarkers for early detection of environmental BPA exposure., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

By measuring the vitellogenin 1 (vtg1) expression through quantitative PCR and in situ hybridization, we used the zebrafish embryo as an in vivo model to access the estrogenic or anti-estrogenic effects of several endocrine disrupting chemicals (EDCs), such as natural estrogen 17β-estradiol (E2), estriol (E3), synthetic hormones including diethylstilbestrol (DES), 4-octyl phenol (OP), bisphenol A (BPA), tamoxifen (TMX) and 3-(2,3-dibromopropyl) isocyanurate (TBC). According to our data, the estrogenic effect of the tested chemicals was ranked as: DES>E 2 >E 3 >OP>BPA, which is consistent with various in vivo and in vitro models. Therefore, the measurement of vtg1 gene expression in zebrafish embryos would be a valuable method for screening EDCs including both environmental estrogens and anti-estrogens., (Copyright © 2017. Published by Elsevier Inc.)

The binding affinity of bisphenol A (BPA) to estrogen receptors (ERs) is much lower than that of 17β-estradiol (E 2 ), and whether there are other molecular mechanisms responsible for the estrogenic action of BPA in vivo currently remains unknown. The objective of this study was to explore the potential association between the estrogenic effect induced by bisphenol A in vivo and changes of endogenous E 2 and gene specific DNA methylation levels. After a waterborne exposure of male zebrafish to 500, 1000, or 1500μg/L of BPA for 21d, vitellogenin (VTG) concentration in whole body homogenate, plasma E 2 and testosterone levels, hepatic ERs mRNA expressions, gonadal cyp19a1a and cyp17a1 mRNA expressions, and methylation levels of hepatic esr1 and gonadal cyp19a1a's promoters were determined. Our results indicated that for the 500 and 1500μg/L treatment groups, VTG might be induced mainly by the elevated E 2 levels; increases of E 2 levels could be partly explained by the up-regulated expression of gonadal aromatase, mRNA levels of which were found to be negatively related to the methylation levels of both its promoter and one CpG site. In addition, upon BPA exposure, hepatic esr1 mRNA levels were also negatively related to the methylation levels of both its promoter and one CpG site. These observations provide evidence for the non-ERs mediated mechanisms underlying the estrogenic action of BPA on male zebrafish., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Bisphenol A (BPA) is a widely used chemical that has been extensively studied as an endocrine-disrupting chemical (EDC). Other bisphenols sharing close structural features with BPA, are increasingly being used as alternatives, increasing the need to assess associated hazards to the endocrine system. In the present study, the estrogenic activity of BPA, bisphenol S (BPS) and bisphenol F (BPF) was assessed by using a combination of zebrafish-specific mechanism-based in vitro and in vivo assays. The three bisphenols were found to efficiently transactivate all zebrafish estrogen receptor (zfER) subtypes in zebrafish hepatic reporter cell lines (ZELH-zfERs). BPA was selective for zfERα while BPS and BPF were slightly more potent on zfERβ subtypes. We further documented the estrogenic effect in vivo by quantifying the expression of brain aromatase using a transgenic cyp19a1b-GFP zebrafish embryo assay. All three bisphenols induced GFP in a concentration-dependent manner. BPS only partially induced brain aromatase at the highest tested concentrations (>30µM) while BPA and BPF strongly induced GFP, in an ER-dependent manner, at 1-10µM. Furthermore, we show that BPF strongly induced vitellogenin synthesis in adult male zebrafish. Overall, this study demonstrates the estrogenic activity of BPA, BPF and BPS in different cell- and tissue-contexts and at different stages of development. Differences between in vitro and in vivo responses are discussed in light of selective ER activation and the fate of the compounds in the models. This study confirms the relevance of combining cellular and whole-organism bioassays in a unique model species for the hazard assessment of candidate EDCs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Both diethylstilbestrol (DES, an environmental estrogen) and flutamide (FLU, an anti-androgen) are found to impair spermatogenesis by disrupting hypothalamic-pituitary-gonadal (HPG) axis and altering androgen levels through different mechanisms/modes of action in fish with poorly understood underlying mechanisms. Furthermore, it is not known whether and how a combined exposure of DES and FLU has a stronger effect than the compounds alone. In this study, male zebrafish adults were exposed to DES, FLU and their combination (DES+FLU) for 30days, and their effects on histological structure and sperm count in testis, androgen level in plasma, as well as the mRNA levels of genes involved in HPG axis, meiotic regulation and apoptosis were analyzed. After exposure, DES and FLU disrupted spermatogenesis in zebrafish, and their combination resulted in even more severe impairment, indicating the inhibitory roles of these chemicals on spermatogenesis and their additive effects on zebrafish. The different regulation of vtg1 expression in the liver in response to DES and FLU further confirmed the different modes of action of these drugs. Gene expression and plasma steroid level analyses demonstrated the suppressed mRNA levels of the key genes (such as gnrh3, fshβ and lhβ in brain and dmrt1, sf1, cyp17a1 and cyp11b2 in testis) in HPG axis and decreased 11-ketotestosterone (11-KT) levels in plasma. The declined level of 11-KT was thus supposed to be closely related to the down-regulation of cyp26a1 (encoding the catabolic enzyme of retinoic acid) and suppression of genes involved in meiotic regulation (nanos1, dmc1 and sycp3). In fish exposed to DES and DES+FLU, enhanced apoptosis (elevated bax/bcl-2 expression ratio) was also observed. The suppression of meiotic regulation in response to all the exposures and enhanced apoptosis in response to DES were thus supposed to result in the spermatogenic impairment in zebrafish. The present study greatly extends our understanding on the mechanisms underlying of reproductive toxicity of environment estrogens and anti-androgens in fish., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Nucleotide excision repair (NER) removes helix-distorting DNA lesions such as UV-induced pyrimidine dimers and cisplatin-induced strand crosslinking. Our earlier studies have identified low-molecular-weight proteins homologous to the 150-kDa vitellogenin 1 (Vg1) as UV-damaged DNA-binding factors expressed in developing zebrafish (Danio rerio). This present study explored if Vg1-like proteins also participated in NER in zebrafish. Immunoblot analysis of affinity-captured 12 h post-fertilization (hpf) zebrafish extract proteins showed a transient binding of a 30-kDa Vg1-like polypeptide to UV-damaged DNA. A transcription-based in vitro repair assay revealed a significant up-regulation of UVC or cisplatin-suppressed transcriptional activity of a marker cDNA driven by a SP6 RNA polymerase-regulated promotor after incubating the damaged plasmid with the extracts of 12 hpf embryos or 96 hpf larvae. The up-regulation of UV or cisplatin-suppressed transcription was abolished in the presence of a monoclonal anti-zebrafish Vg1 antibody. The differential sensitivity of UV-induced repair in 12 and 96 hpf zebrafish extracts to exogenous ATP suggested a development-dependent expression of Vg1-like NER factors. A T 4 endonuclease V digestion assay showed no inhibition of the anti-Vg1 antibody on the excision of UV-induced cyclobutane pyrimidine dimers. Our results identified the participation of Vg1-like factors in NER in developing zebrafish, and these factors may function at post-incison steps of NER.

In the past few decades, extensive application of azoxystrobin has led to great concern regarding its adverse effects on aquatic organisms. The objective of the present study was to evaluate the reproductive toxicity of azoxystrobin to zebrafish. After adult zebrafish of both sexes were exposed to 2, 20 and 200 μg/L azoxystrobin for 21 days, egg production, the fertilization rate, the gonadosomatic index (GSI) and hepatosomatic index (HSI), 17β-estradiol (E2), testosterone (T) and vitellogenin (Vtg) concentrations, and histological alterations in the gonads and livers were measured. Meanwhile, expression alterations of genes encoding gonadotropins and gonadotropin receptors (fshb, lhb, fshr and lhr), steroid hormone receptors (era, er2b and ar), steroidogenic enzymes (cyp11a, cyp11b, cyp17, cyp19a, cyp19b, hsd3b and hsd17b) in the hypothalamic-pituitary-gonad (HPG) axis and vitellogenin (vtg1 and vtg2) in the livers were also investigated. The results showed that reduced egg production and fertilization rates were observed at 200 μg/L azoxystrobin. In female zebrafish, reduced E2 and Vtg concentrations, decreased GSI, increased T concentrations, and histological alterations in the ovaries and livers were observed at 200 μg/L azoxystrobin, along with significant down-regulation of lhb, cyp19b, lhr, cyp19a, vtg1 and vtg2, and up-regulation of cyp17, hsd3b and hsd17b. In male zebrafish, increased E2 and Vtg concentrations, reduced T concentration and GSI, and histological alterations in the testes and livers were observed after exposure to 20 and 200 μg/L azoxystrobin, along with significant up-regulations of cyp19b, cyp11a, cyp17, cyp19a, hsd3b and hsd17b, vtg1 and vtg2. Moreover, cyp11a, hsd3b, cyp19a, vtg1 and vtg2 in male zebrafish were significantly up-regulated after treatment with 2 μg/L azoxystrobin. The results of the present study indicate that azoxystrobin led to reproductive toxicity in zebrafish and male zebrafish were more sensitive to azoxystrobin than female zebrafish., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Gender is one of the factors influencing the intestinal microbial composition in mammals, but whether fish also have gender-specific intestinal microbial patterns remains unknown. In this decade, endocrine disrupting chemicals in surface and ground water of many areas and increasing observation of freshwater male fish displaying female sexual characteristics have been reported. Here we identified the difference in intestinal microbiota between male and female zebrafish, and revealed the influence of endocrine disrupting chemicals on zebrafish intestinal microbiota by using high-throughput sequencing. The results indicated that Fusobacteria, Bacteroidetes and Proteobacteria were dominant in the gut of zebrafish and there were no obvious gender-specific intestinal microbial patterns. Two endocrine disrupting chemicals, Estradiol (E2) and Bisphenol A (BPA), were selected to treat male zebrafish for 5 weeks. E2 and BPA increased vitellogenin expression in the liver of male zebrafish and altered the intestinal microbial composition with the abundance of the phylum CKC4 increased significantly. Our results suggested that because of the developmental character and living environment, gender did not influence the assembly of intestinal microbiota in zebrafish as it does in mammals, but exposure extra to endocrine disrupting chemicals disturbed the intestinal microbial composition, which may be related to changes in host physiological metabolism., Competing Interests: The authors have declared that no competing interests exist.

In this study, zebrafish (Danio rerio) were exposed to a UV-filter-octocrylene (OCT) with elevated concentrations for 28 d. The total body accumulation of OCT in zebrafish was found to reach 2321.01 ("L" level), 31,234.80 ("M" level), and 70,593.38 ng g(-1) ("H" level) when the average OCT exposure concentration was controlled at 28.61, 505.62, and 1248.70 μg L(-1), respectively. Gross and histological observations as well as RT-qPCR analysis were conducted to determine the effects of OCT accumulation on zebrafish. After exposure, the gonad-somatic index and percentage of vitellogenic oocytes were found to increase significantly in the ovaries of female zebrafish at the H accumulation level. Significant up-regulation of esr1 and cyp19b were observed in the gonads, as well as vtg1 in the livers for both female and male zebrafish. At M and H accumulation levels, apparent down-regulation of ar was observed in the ovaries and testis of the female and male zebrafish, respectively. Although the extent of the effects on zebrafish differed at different accumulation levels, the induction of vtg1 and histological changes in the ovaries are indications of estrogenic activity and the inhibition of esr1 and ar showed antiestrogenic and antiandrogenic activity, respectively. Thus, as OCT could easily accumulate in aquatic life such as zebrafish, one of its most of concern hazards would be the disturbance of the histological development and its multiple hormonal activities., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The present study aimed to determine the suitable development period for zebrafish to evaluate estrogenic activities accurately. An enzyme-linked immunosorbent assay was developed and used to detect the vitellogenin (Vtg)-derived yolk proteins and newly produced Vtg, and 9 d to 56 d posthatching was determined as the Vtg-blank period. Juveniles in this period were found to have lower baseline Vtg levels than adult males and were considered an alternative test organism for detecting environmental estrogens. Environ Toxicol Chem 2016;35:1783-1787. © 2015 SETAC., (© 2015 SETAC.)

Vitellogenin (Vtg) in zebrafish (Danio rerio) is a core biomarker for screening environmental estrogens in test guidelines of the Organization for Economic Cooperation and Development. To accurately quantify zebrafish Vtg, lipovitellin (Lv), the main Vtg-derived yolk protein, was used as the antigen to establish a sandwich enzyme-linked immunosorbent assay (ELISA). The purified Lv was a phospholipoglycoprotein with apparent molecular weight of ~445kDa, and separated into three polypeptides corresponding to ~117, ~102, and ~23.8kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunological analysis confirmed the specificity of the anti-Lv antibody for Vtg and the immunological similarity between Vtg and Lv. Using the purified Lv and anti-Lv antibody, a sandwich ELISA with a detection limit of 4.3ng/mL and a detection range from 7.8 to 250ng/mL was developed. The intra- and inter-assay coefficients of variation were both below 10%. Moreover, the Lv standard curve was nearly identical to the Vtg standard curve, and paralleled serial whole-body homogenate dilutions of male zebrafish exposed to 17β-estradiol, demonstrating that the Lv-based ELISA could be used for quantification of zebrafish Vtg. Zebrafish Lv showed high stability during purification process, heat treatment, -80°C storage, and repeated freeze/thaw cycles. Additionally, the standard curve of Lv stored at -80°C for 3months exhibited higher robustness than that of Vtg stored under the same conditions. Finally, the usefulness of the ELISA for detecting estrogenic activity was verified by quantifying Vtg inductions in zebrafish exposed to monocrotophos., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Tamoxifen is a widely used anticancer drug with both an estrogen agonist and antagonist effect. This study focused on its endocrine disrupting effect, and overall environmental significance. Zebrafish embryos were exposed to different concentrations (0.5, 5, 50 and 500 µg l(-1) ) of tamoxifen for 96 h. The results showed a complex effect of tamoxifen on zebrafish embryo development. For the 500 µg l(-1) exposure group, the heart rate was decreased by 20% and mild defects in caudal fin and skin were observed. Expressions of a series of genes related to endocrine and morphological changes were subsequently tested through quantitative real-time polymerase chain reaction. Bisphenol A as a known estrogen was also tested as an endocrine-related comparison. Among the expression of endocrine-related genes, esr1, ar, cyp19a1b, hsd3b1 and ugt1a1 were all increased by tamoxifen exposure, similar to bisphenol A. The cyp19a1b is a key gene that controls estrogen synthesis. Exposure to 0.5, 5, 50 and 500 µg l(-1) of tamoxifen caused upregulation of cyp19a1b expression to 152%, 568%, 953% and 2024% compared to controls, higher than the effects from the same concentrations of bisphenol A treatment, yet vtg1 was suppressed by 24% from exposure to 500 µg l(-1) tamoxifen. The expression of metabolic-related genes such as cyp1a, cyp1c2, cyp3a65, gpx1a, gstp1, gsr and genes related to observed morphological changes such as krt17 were also found to be upregulated by high concentrations of tamoxifen. These findings indicated the potential environmental effect of tamoxifen on teleost early development. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Perfluoroalkyl acids (PFAAs) are a group of anthropogenic compounds that have been widely used in consumer products for over 50 years. One of the most dominant PFAAs is perfluorononanoate (PFNA), a compound detected ubiquitously in aquatic ecosystems. While PFNA is suspected of being an endocrine disruptor, the mechanisms behind PFNA-induced reproductive disorders are poorly understood. The aim of this study was to investigate the reproduction-related effects and possible mechanisms of PFNA on adult zebrafish (Danio rerio) following 180 days of exposure at different concentrations (0.01, 0.1, 1mg/L). PFNA concentration in the gonads of zebrafish was tested by HPLC-MS/MS after chronic exposure to study possible inconsistent accumulation between the genders. The results showed that the accumulation of PFNA in the male gonads was almost one-fold higher than that in the female gonads, indicating a possible higher PFAA gonad burden for male zebrafish. Significant reductions in the male gonadosomatic index (GSI) and female egg production were observed. In addition, the decreased 72h hatching rate displayed an evident dosage effect, indicating that maternal exposure to PFNA might impair offspring developmental success. To investigate how PFNA exposure affects the hypothalamic-pituitary-gonadal-liver axis (HPGL axis), the transcriptional levels of genes were measured by real-time PCR. The disrupted expression of genes, such as ERα, ERβ, FSHR, LHR, StAR, and 17βHSD, indicated the possible interference of PFNA on the HPGL axis function and sex hormone synthesis. Furthermore, testosterone (T) and estradiol (E2) levels in serum and VTG content in the liver were detected to clarify the influences of PFNA on sex hormone levels. Except for the increase in serum estrogen levels, as an estrogen analogue, PFNA also induced the synthesis of biomarker protein vitellogenin (VTG) in the adult male liver. The results of this study indicate that chronic exposure to PFNA can lead to dysfunction in the HPGL axis and sex hormone synthesis and cause adverse effects on fish reproduction., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Bisphenol AF (BPAF) is widely used in food-contact products, electronic devices, and as a cross-linking reagent in fluoroelastomers. There are growing concerns about its toxicity and endocrine-disrupting effects based on its structural similarity with bisphenol A (BPA). The endocrine-disrupting effects of BPAF were studied by exposing 2-month-old zebrafish to 0, 0.05, 0.25, or 1 mg/L BPAF for 28 days and evaluating the effect on growth, histopathology, hormone levels, enzyme activity, and gene expression. The overall fitness was not significantly affected. There were no apparent alterations in the gills and intestine tissues of both sexes after BPAF exposure. However, exposure to 1 mg/L BPAF caused damage to the liver in the male fish, characterized by hepatocellular swelling and vacuolation. There was no obvious effect in the liver of female fish, suggesting that the hepatic toxicity of BPAF is gender dependent. Gonadal examination indicated that exposure to 1 mg/L BPAF caused induction of acellular areas in the testis and retardation of oocyte development in the ovary. BPAF exposure increased free triiodothyronine levels of females in a dose-dependent manner. In males, the testosterone levels decreased in a concentration-dependent manner. In contrast, estradiol levels increased in a concentration-dependent manner and were significantly higher in males exposed to 1 mg/L BPAF compared with the controls. In females, 0.05 and 0.25 mg/L BPAF caused an increase in testosterone levels. Furthermore, the estradiol levels increased in females exposed to 0.05 and 1 mg/L. We observed an upregulation of hepatic vitellogenin in both sexes and significantly higher levels in males exposed to 1 mg/L BPAF and females exposed to 0.25 mg/L BPAF, suggesting that BPAF has an estrogenic activity. Our results indicate that BPAF is an endocrine-disrupting chemical that exerts reproductive toxicity and estrogenic effects on zebrafish., (© 2014 Wiley Periodicals, Inc.)

Microcystin-LR (MC-LR) has been found to cause reproductive and developmental impairments as well as to disrupt sex hormone homeostasis of fish during acute and sub-chronic toxic experiments. However, fish in natural environments are continuously exposed to MC-LR throughout their entire life cycle as opposed to short-term exposure. Here, we tested the hypothesis that the mechanism by which MC-LR harms female fish reproduction and development within natural water bodies is through interference of the reproductive endocrine system. In the present study, zebrafish hatchlings (5 d post-fertilization) were exposed to 0, 0.3, 3 and 30 μg/L MC-LR for 90 d until reaching sexual maturity. Female zebrafish were selected, and the changes in growth and developmental indicators, ovarian ultrastructure as well as the levels of gonadal steroid hormones and vitellogenin (VTG) were examined along with the transcription of related genes in the hypothalamic-pituitary-gonadal-liver axis (HPGL-axis). The results showed for the first time, a life cycle exposure to MC-LR caused growth inhibition, decreased ovary weight and ovarian ultra-pathological lesions. Decreased ovarian testosterone levels indicated that MC-LR disrupted sex steroid hormone balance. Significantly up-regulated transcription of brain FSHβ and LHβ along with ovarian ERα, FSHR and LHR suggested positive feedback regulation in the HPGL-axis was induced as a compensatory mechanism for MC-LR damage. It was also noted that ovarian VTG content and hepatic ERα and VTG1 expression were all down-regulated, which might be responsible for reduced vitellus storage noted in our histological observations. Our findings indicate that a life cycle exposure to MC-LR impairs the development and reproduction of female zebrafish by disrupting the transcription of related HPGL-axis genes, suggesting that MC-LR has potential adverse effects on fish reproduction and thus population dynamics in MCs-contaminated aquatic environment., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Semicarbazide (SMC), a member of the hydrazine family, has various toxic effects and has been detected in organisms, aquatic environments, and food. SMC exposure inhibited the transcription of hepatic vitellogenin and estrogen receptors in female zebrafish (Danio rerio), suggesting that it had anti-estrogenic properties. In order to elucidate the mechanisms underlying these, we exposed female zebrafish to SMC and used enzyme-linked immunosorbent assays to examine plasma 17β-estradiol (E2) and testosterone (T) levels. Gonad histology was analyzed and the mRNA expression of genes involved in the reproductive axis, the gamma-aminobutyric acid (GABA) shunt, and leptin was quantified by real-time PCR. Zebrafish were exposed to 1, 10, 100, or 1000μg/L SMC in a semi-static system for 96hours or 28 days. Plasma E2 levels were significantly decreased and ovarian maturation was inhibited by SMC, suggesting that its anti-estrogenic effect was exerted by reducing endogenous E2 levels. This was likely due to the SMC-mediated inhibition of cytochrome P450 (CYP) 19A mRNA levels, because this enzyme catalyzes the conversion of T to E2 in the gonads. In addition, down-regulation of the mRNA expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, steroidogenic acute regulatory protein, CYP17, and 17beta-hydroxysteroid dehydrogenase was observed; this was predicted to reduce T concentrations and further contribute to the reduced E2 levels. SMC-induced changes in the expression of these steroidogenic genes correlated with decreased transcription of gonadotropic hormones (follicle-stimulating hormone and luteinizing hormone) and significantly elevated leptin expression. Furthermore, SMC also altered expression of the key enzyme in gamma-aminobutyric acid (GABA) synthesis, GABA receptors, and salmon gonadotropin-releasing hormone, thus affecting gonadotropin expression. Overall, SMC acted at multiple sites related to reproduction to reduce plasma E2 levels, consequently exerting an anti-estrogenic effect in female zebrafish. These effects were observed at environmentally relevant concentrations and highlight the importance of controlling SMC contamination., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The chemical ultraviolet (UV) filter benzophenone-3 (BP-3) is suspected to be an endocrine disruptor based on results from in vitro and in vivo testing. However, studies including endpoints of endocrine adversity are lacking. The present study investigated the potential endocrine-disrupting effects of BP-3 in zebrafish (Danio rerio) in the Fish Sexual Development Test (Organisation for Economic Co-operation and Development TG 234) and a 12-d adult male zebrafish study. In TG 234, exposure from 0 d to 60 d posthatch caused a monotone dose-dependent skewing of the phenotypic sex ratio toward fewer males and more female zebrafish (no observed effect concentration [NOEC]: 191 μg/L, lowest observed effect concentration [LOEC]: 388 μg/L). Besides, gonad maturation was affected in both female fish (NOEC 191 μg/L, LOEC 388 μg/L) and male fish (NOEC 388 μg/L, LOEC 470 μg/L). Exposure to BP-3 did not affect the vitellogenin concentration in TG 234. After 12 d exposure of adult male zebrafish, a slight yet significant increase in the vitellogenin concentration was observed at 268 μg/L but not at 63 μg/L and 437 μg/L BP-3. Skewing of the sex ratio is a marker of an endocrine-mediated mechanism as well as a marker of adversity, and therefore the conclusion of the present study is that BP-3 is an endocrine-disrupting chemical in accordance with the World Health Organization's definition., (© 2015 SETAC.)

The aim of the present study was to investigate the persistence of endocrine effects by prochloraz, a fungicide known to have multiple effects on the endocrine system of vertebrates. Since discontinuous exposure is particularly relevant in aquatic ecosystems, an exposure scenario with an exposure phase and a subsequent recovery period was chosen to assess the potential for reversibility of effects by prochloraz on the sexual development of zebrafish (Danio rerio). Zebrafish were exposed to different concentrations of prochloraz (10-300 μg/L) until 60 days post hatch (dph), which includes the period of sexual differentiation. For the subsequent 40 days, fish were either held in clean water for depuration or under further continuous exposure. Histological investigations of the gonads revealed persistent effects on sexual differentiation. The sex ratio was skewed towards males and significantly more intersex individuals were found after exposure to prochloraz at 60 dph. No intersex fish, but masculinized sex ratios were still present after the depuration period, documenting that prochloraz irreversibly affects the sexual development of zebrafish.

The diets of populations in industrialized nations have shifted to dramatically increased consumption of ω6 polyunsaturated fatty acids (PUFA), with a corresponding decrease in the consumption of ω3 PUFA. This dietary shift may be related to observed increases in obesity, chronic inflammation, and comorbidities in the human population. We examined the effects of ω3:ω6 fatty acid ratios in the context of constant total dietary lipid on the growth, total body fat, and responses of key inflammatory markers in adult zebrafish (Danio rerio). Zebrafish were fed diets in which the ω3:ω6 PUFA ratios were representative of those in a purported ancestral diet (1:2) and more contemporary Western diets (1:5 and 1:8). After 5 mo, weight gain (fat free mass) of zebrafish was highest for those that received the 1:8 ratio treatment, but total body fat was lowest at this ratio. Measured by quantitative real-time RT-PCR, mRNA levels from liver samples of 3 chronic inflammatory response genes (C-reactive protein, serum amyloid A, and vitellogenin) were lowest at the 1:8 ratio. These data provide evidence of the ability to alter zebrafish growth and body composition through the quality of dietary lipid and support the application of this model to investigations of human health and disease related to fat metabolism.

Bisphenol AF (BPAF) is one of the analogues of bisphenol A (BPA) and is widely used as a raw material in the plastics industry. The potential toxicity to fish from exposure to BPAF in the aquatic environment is largely unknown. In this study, zebrafish (Danio rerio) were exposed to BPAF at 5, 25 and 125 μg L(-1), from 4 hour-post-fertilization (hpf) to 120 day-post-fertilization (dpf), representing the period from embryo to adult. The levels of plasma hormones were measured and the expression of selected representative genes along the hypothalamus-pituitary-gonad (HPG) axis and liver were examined. The concentration of 17β-estradiol (E2) was significantly increased in male and female fish and a significant decrease of testosterone (T) was observed in male fish. The mRNA expression of genes along the HPG axis and in liver tissues in F0 generation fish demonstrated that the steroid hormonal balances of zebrafish were modulated through the alteration of steroidgenesis. The significant decrease of egg fertilization among offspring indicates the possibility of sperm deterioration of parent following exposure to BPAF. The higher occurrence of malformation and lower survival rate in the offspring from the exposure group suggested a possibility of maternal transfer of BPAF, which could be responsible for the increased prevalence of adverse health signs in the offspring. The hatching delay in 5 μg L(-1) BPAF indicated that parental exposure to environmentally relevant concentration of BPAF would result in delayed hatching of the offspring. A potential consequence of adverse effects in the offspring by BPAF deserves further investigation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The proteome profile of Danio rerio embryos grown in the medium containing doxorubicin, included in the phospholipid transport nanosystem (doxolip) has been investigated using combination of 1D-electrophoresis with subsequent MALDI-TOF-PMF mass spectrometry. Cultivation of growing of D. rerio embryos in the medium with doxolip caused a substantial increase in expression of the cytoskeletal proteins, a decrease in the number of nuclear proteins involved in DNA and RNA synthesis and disappearance of vitellogenin 2 in comparison with control (the cultivation medium containing the phospholipid transport nanosystem). Analysis of the proteomic profiles of doxolip-treated embryos suggests lower toxicity of doxorubicin incorporated in the phospholipid nanosystem.

Using 17α-ethinyl estradiol (EE2) and dibutyl phthalate (DBP) as a typical estrogen and phthalate ester, respectively, their combined in vivo effects on zebrafish (Danio rerio) were investigated from the juvenile state to the adult stage. The authors spiked EE2 (5 ng/L and 20 ng/L) and DBP (0.1 mg/L and 0.5 mg/L) either individually or in mixture. At 45 d postfertilization (dpf), the survival rate of zebrafish was comparable in all treatments. Dibutyl phthalate did not induce vitellogenin (VTG) synthesis, and no interaction was found between EE2 and DBP on VTG induction. At 90 dpf, both liver and gill were subject to more severe damage (lipid vacuoles of hepatocytes, amalgamation of gill lamellae, and clubbing at the tips of the secondary lamellae) when coexposed to these 2 chemicals, compared with single exposure. At 115 dpf, generally none of the binary mixture groups showed significantly different growth and sex ratios compared with the corresponding EE2 alone groups. In conclusion, no obvious interactions were detected between EE2 and DBP on the growth, VTG induction, or sex ratio of zebrafish, and they may act independently. However, the influence on morphology of gonad, liver, and gill induced by exposure to the mixture of EE2 and DBP was generally more potent than that by single exposure to EE2 or DBP, indicating the combined long-term harmful effects of EE2 and DBP on the development of zebrafish. Environ Toxicol Chem 2015;34:518-526. © 2014 SETAC., (© 2014 SETAC.)

The persistent organic pollutant p,p'-DDE, the major metabolite of the insecticide DDT, has displayed evidence of endocrine disruption through the inhibition of androgen binding to androgen receptors in different species. Although p,p'-DDE was continuously detected in wild fish with abnormal gonad development such as intersex, little is known about its mode of action during gonad development in fish. To elucidate the potential endocrine effects of this pollutant in zebrafish (Danio rerio), juveniles (30 days post hatch) were exposed to p,p'-DDE during the critical window of sexual differentiation. Fish were exposed to sublethal concentrations ranging from 0.01 to 20 µg l(-1) over 14 days and were maintained in control water for an additional 4 months. As core endpoints, the vitellogenin (vtg) concentration was measured at the end of exposure, and sex ratio and the gonadosomatic index were assessed 4 months after the end of exposure. An increase in vtg production in whole body homogenate was observed in fish exposed to 0.2 and 2.0 µg l(-1) p,p'-DDE. No significant differences were displayed in morphological parameters such as the gonadosomatic index of males and females or sex ratio. However, exposed females presented histopathological changes that include the reduction of the number of mature oocytes, which might impair their successful reproduction. These results demonstrate the ability of p,p'-DDE to cause endocrine disruption in zebrafish exposed during gonad differentiation of juvenile specimens. Furthermore, vtg induction by p,p'-DDE in juvenile zebrafish arises as a predictive marker for adverse effects of this DDT metabolite on the ovarian function of female zebrafish., (Copyright © 2014 John Wiley & Sons, Ltd.)