Deciphering the mechanisms and interactions of the endocrine disruptor bisphenol A and its analogs with the androgen receptor.

Bisphenol A (BPA) and its various forms used as BPA alternatives in industries are recognized toxic compounds and antiandrogenic endocrine disruptors. These chemicals are widespread in the environment and frequently detected in biological samples. Concerns exist about their impact on hormones, disrupting natural biological processes in humans, together with their negative impacts on the environment and biotic life. This study aims to characterize the interaction between BPA analogs and the androgen receptor (AR) and the effect on the receptor's normal activity. To achieve this goal, molecular docking was conducted with BPA and its analogs and dihydrotestosterone (DHT) as a reference ligand. Four BPA analogs exhibited higher affinity (-10.2 to -8.7 kcal/mol) for AR compared to BPA (-8.6 kcal/mol), displaying distinct interaction patterns. Interestingly, DHT (-11.0 kcal/mol) shared a binding pattern with BPA. ADMET analysis of the top 10 compounds, followed by molecular dynamics simulations, revealed toxicity and dynamic behavior. Experimental studies demonstrated that only BPA disrupts DHT-induced AR dimerization, thereby affecting AR's function due to its binding nature. This similarity to DHT was observed during computational analysis. These findings emphasize the importance of targeted strategies to mitigate BPA toxicity, offering crucial insights for interventions in human health and environmental well-being.

Chlorothalonil as a potential endocrine disruptor in male zebrafish (Danio rerio): Impact on the hypothalamus-pituitary-gonad axis and sperm quality.

Chlorothalonil is a broad-spectrum organochlorine fungicide widely employed in agriculture to control fungal foliar diseases. This fungicide enters aquatic environments through the leaching process, leading to toxicity in non-target organisms. Organic contaminants can impact organism reproduction as they have the potential to interact with the neuroendocrine system. Although there are reports of toxic effects of chlorothalonil, information regarding its impact on reproduction is limited. The aim of the present study was to evaluate the influence of chlorothalonil on male reproductive physiology using the zebrafish (Danio rerio) as ecotoxicological model. Zebrafish were exposed for 7 days to two concentrations of chlorothalonil (0.1 and 10 µg/L) along with a control group (with DMSO - 0.001%). Gene expression of hypothalamus-pituitary-gonad axis components (gnrh2, gnrh3, lhr, fshr, star, hsd17b1, hsd17b3, and cyp19a1), as well as hepatic vitellogenin concentration were assessed. In sperm cells, reactive oxygen species (ROS) content, lipid peroxidation (LPO), mitochondrial functionality, and membrane integrity and fluidity were evaluated. Results indicate that exposure to the higher concentration of chlorothalonil led to a reduction in brain gnr2 expression. In gonads, mRNA levels of lhr, star, and hsd17b1 were decreased at both chlorothalonil concentrations tested. Similarly, hepatic vitellogenin concentration was reduced. Regarding sperm cells, a decreased ROS level was observed, without significant difference in LPO level. Additionally, a higher mitochondrial potential and lower membrane fluidity were observed in zebrafish exposed to chlorothalonil. These findings demonstrate that chlorothalonil acts as an endocrine disruptor, influencing reproductive control mechanisms, as evidenced by changes in expression of genes HPG axis, as well as hepatic vitellogenin concentration. Furthermore, our findings reveal that exposure to this contaminant may compromise the reproductive success of the species, as it affected sperm quality parameters.

Prediction of the Interactions of a Large Number of Per- and Poly-Fluoroalkyl Substances with Ten Nuclear Receptors.

Per- and poly-fluoroalkyl substances (PFASs) are persistent, toxic chemicals that pose significant hazards to human health and the environment. Screening large numbers of chemicals for their ability to act as endocrine disruptors by modulating the activity of nuclear receptors (NRs) is challenging because of the time and cost of in vitro and in vivo experiments. For this reason, we need computational approaches to screen these chemicals and quickly prioritize them for further testing. Here, we utilized molecular modeling and machine-learning predictions to identify potential interactions between 4545 PFASs with ten different NRs. The results show that some PFASs can bind strongly to several receptors. Further, PFASs that bind to different receptors can have very different structures spread throughout the chemical space. Biological validation of these in silico findings should be a high priority.

Bioaccumulation and thyroid endcrione disruption of 2-ethylhexyl diphenyl phosphate at environmental concentration in zebrafish larvae.

2-ethylhexyl diphenyl phosphate (EHDPP) strongly binds to transthyretin (TTR) and affects the expression of genes involved in the thyroid hormone (TH) pathway in vitro. However, it is still unknown whether EHDPP induces endocrine disruption of THs in vivo. In this study, zebrafish (Danio rerio) embryos (< 2 h post-fertilization (hpf)) were exposed to environmentally relevant concentrations of EHDPP (0, 0.1, 1, 10, and 100 µg·L-1) for 120 h. EHDPP was detected in 120 hpf larvae at concentrations of 0.06, 0.15, 3.71, and 59.77 µg·g-1 dry weight in the 0.1, 1, 10, and 100 µg·L-1 exposure groups, respectively. Zebrafish development and growth were inhibited by EHDPP, as indicated by the increased malformation rate, decreased survival rate, and shortened body length. Exposure to lower concentrations of EHDPP (0.1 and 1 µg·L-1) significantly decreased the whole-body thyroxine (T4) and triiodothyronine (T3) levels and altered the expressions of genes and proteins involved in the hypothalamic-pituitary-thyroid axis. Downregulation of genes related to TH synthesis (nis and tg) and TH metabolism (dio1 and dio2) may be partially responsible for the decreased T4 and T3 levels, respectively. EHDPP exposure also significantly increased the transcription of genes involved in thyroid development (nkx2.1 and pax8), which may stimulate the growth of thyroid primordium to compensate for hypothyroidism. Moreover, EHDPP exposure significantly decreased the gene and protein expression of the transport protein transthyretin (TTR) in a concentration-dependent manner, suggesting a significant inhibitory effect of EHDPP on TTR. Molecular docking results showed that EHDPP and T4 partly share the same mode of action of binding to the TTR protein, which might result in decreased T4 transport due to the binding of EHDPP to the TTR protein. Taken together, our findings indicate that EHDPP can cause TH disruption in zebrafish and help elucidate the mechanisms underlying EHDPP toxicity.

Environmental bisphenol A disrupts methylation of steroidogenic genes in the ovary of Paradise threadfin Polynemus paradiseus via abnormal DNA methylation: Implications for human exposure and health risk assessment.

Bisphenol A, endocrine-disrupting chemicals (EDCs) impacting disease development via epigenetic modifications, is crucial in transcriptional regulation. However, ecotoxicology's limited exploration of epigenetics prompted our study's objective: examining the extended exposure of riverine Bisphenol A (BPA), a potent EDC, on DNA methylation during female paradise threadfin (Polynemus paradiseus) reproductive maturation. Assessing BPA contamination in riverine water, we collected fish samples from two locations with distinct contamination levels. In the highly contaminated region (Hc), we observed elevated DNA methylation in aromatase (7.5-fold), 20ß-HSD (3-fold), and FSHR (2-fold) genes. Hormone receptor investigation highlighted an escalating connection between transcriptional hyper-methylation and contamination levels. Additionally, our study revealed a positive correlation between oocyte growth and global DNA methylation, suggesting BPA's potential to modify DNA methylation in female paradise threadfins. This effect likely occurs through changes in hormone receptor expression, persisting throughout oocyte maturation. Notably, our research, the first of its kind in estuarine areas, confirmed BPA contamination in paradise threadfins, raising concerns about potential health risks for humans.

Systematic transcriptome-wide meta-analysis across endocrine disrupting chemicals reveals shared and unique liver pathways, gene networks, and disease associations.

Cardiometabolic disorders (CMD) are a growing public health problem across the world. Among the known cardiometabolic risk factors are compounds that induce endocrine and metabolic dysfunctions, such as endocrine disrupting chemicals (EDCs). To date, how EDCs influence molecular programs and cardiometabolic risks has yet to be fully elucidated, especially considering the complexity contributed by species-, chemical-, and dose-specific effects. Moreover, different experimental and analytical methodologies employed by different studies pose challenges when comparing findings across studies. To explore the molecular mechanisms of EDCs in a systematic manner, we established a data-driven computational approach to meta-analyze 30 human, mouse, and rat liver transcriptomic datasets for 4 EDCs, namely bisphenol A (BPA), bis(2-ethylhexyl) phthalate (DEHP), tributyltin (TBT), and perfluorooctanoic acid (PFOA). Our computational pipeline uniformly re-analyzed pre-processed quality-controlled microarray data and raw RNAseq data, derived differentially expressed genes (DEGs) and biological pathways, modeled gene regulatory networks and regulators, and determined CMD associations based on gene overlap analysis. Our approach revealed that DEHP and PFOA shared stable transcriptomic signatures that are enriched for genes associated with CMDs, suggesting similar mechanisms of action such as perturbations of peroxisome proliferator-activated receptor gamma (PPARγ) signaling and liver gene network regulators VNN1 and ACOT2. In contrast, TBT exhibited highly divergent gene signatures, pathways, network regulators, and disease associations from the other EDCs. In addition, we found that the rat, mouse, and human BPA studies showed highly variable transcriptomic patterns, providing molecular support for the variability in BPA responses. Our work offers insights into the commonality and differences in the molecular mechanisms of various EDCs and establishes a streamlined data-driven workflow to compare molecular mechanisms of environmental substances to elucidate the underlying connections between chemical exposure and disease risks.

In vivo and in silico toxicity assessment of four common liquid crystal monomers to Daphnia magna: Novel endocrine disrupting chemicals in crustaceans?

Liquid crystal monomers (LCMs) are widely used in liquid crystal displays (LCDs) and are proposed to be a new generation of environmentally persistent, bioaccumulative and toxic (PBT) substances that are increasingly detected in rivers and seas. However, there is a lack of in vivo data that characterize adverse responses and toxic mechanisms of LCMs on aquatic organisms. The aim of this study was to comprehensively investigate the effect of four typical LCMs on the lethality, growth, molting, and reproductive capacity of Daphnia magna (D. magna), a highly studied aquatic species in environmental toxicology. Whole body and enzymatic biomarkers (i.e., body length, chitobiase, acetylcholinesterase, antioxidant defense) were measured to assess the toxicity of LCMs. The 48 h mortality rate and observations of disrupted thorax development and inhibition of ecdysis indicate that D. magna are sensitive to LCMs exposure. Oxidative stress, impaired neurotransmission, and disruptions in molting were observed in short-term biomarker tests using LCMs. A 21 day exposure of D. magna to LCMs resulted in reduced growth, reproduction, and population intrinsic growth rate. In addition, chitobiase and 20-hydroxyecdysone, enzymes important for the molting process, were altered at 7, 14 and 21 d. This is hypothesized to be related to endocrine imbalance resulting from LCM exposure. Based on molecular docking simulations, there is evidence that LCMs bind directly to ecdysteroid receptors; this may explain the observed endocrine disrupting effects of LCMs. These data support the hypothesis that LCMs are endocrine disrupting chemicals in aquatic species, impacting the process of molting. This may subsequently lead to lower reproduction and unbalanced population dynamics.

Pharmaceuticals and endocrine disrupting compounds modulate adverse effects of climate change on resource quality in freshwater food webs.

Freshwater biodiversity, ecosystem functions and services are changing at an unprecedented rate due to the impacts of vast number of stressors overlapping in time and space. Our study aimed at characterizing individual and combined impacts of pollution with pharmaceuticals (PhACs) and endocrine disrupting compounds (EDCs) and increased water temperature (as a proxy for climate change) on primary producers and first level consumers in freshwaters. We conducted a microcosm experiment with a simplified freshwater food web containing moss (Bryophyta) and shredding caddisfly larvae of Micropterna nycterobia (Trichoptera). The experiment was conducted with four treatments; control (C), increased water temperature + 4 °C (T2), emerging contaminants' mix (EC = 15 PhACs & 5 EDCs), and multiple stressor treatment (MS = EC + T2). Moss exhibited an overall mild response to selected stressors and their combination. Higher water temperature negatively affected development of M. nycterobia through causing earlier emergence of adults and changes in their lipidome profiles. Pollution with PhACs and EDCs had higher impact on metabolism of all life stages of M. nycterobia than warming. Multiple stressor effect was recorded in M. nycterobia adults in metabolic response, lipidome profiles and as a decrease in total lipid content. Sex specific response to stressor effects was observed in adults, with impacts on metabolome generally more pronounced in females, and on lipidome in males. Thus, our study highlights the variability of both single and multiple stressor impacts on different traits, different life stages and sexes of a single insect species. Furthermore, our research suggests that the combined impacts of warming, linked to climate change, and contamination with PhACs and EDCs could have adverse consequences on the population dynamics of aquatic insects. Additionally, these findings point to a potential decrease in the quality of resources available for both aquatic and potentially terrestrial food webs.

Assessment of endocrine disruptor impacts on lipid metabolism in a fatty acid-supplemented HepaRG human hepatic cell line.

The incidence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing worldwide. This disease encompasses several stages, from steatosis to steatohepatitis and, eventually, to fibrosis and cirrhosis. Exposure to environmental contaminants is one of the risk factors and an increasing amount of evidence points to a role for endocrine disrupting compounds (EDCs). This study assesses the impact of selected EDCs on the formation of lipid droplets, the marker for steatosis in a hepatic model. The mechanisms underlying this effect are then explored. Ten compounds were selected according to their obesogenic properties: bisphenol A, F and S, butyl-paraben, cadmium chloride, p,p'-DDE, DBP, DEHP, PFOA and PFOS. Using a 2D or 3D model, HepaRG cells were exposed to the compounds with or without fatty acid supplementation. Then, the formation of lipid droplets was quantified by an automated fluorescence-based method. The expression of genes and proteins involved in lipid metabolism and the impact on cellular respiration was analyzed. The formation of lipid droplets, which is revealed or enhanced by oleic acid supplementation, was most effectively induced by p,p'-DDE and DEHP. Experiments employing either 2D or 3D culture conditions gave similar results. Both compounds induced the expression of PLIN2. p,p'-DDE also appears to act by decreasing in fatty acid oxidation. Some EDCs were able to induce the formation of lipid droplets, in HepaRG cells, an effect which was increased after supplementation of the cells with oleic acid. A full understanding of the mechanisms of these effects will require further investigation. The novel automated detection method described here may also be useful in the future as a regulatory test for EDC risk assessment.

Assessment of developmental toxicity and the potential mode of action underlying single and binary exposure to estrogenic endocrine disrupting chemicals in zebrafish (Danio rerio).

The current study investigated the effect of single and binary exposure to distinct xenoestrogens, including diethylstilbestrol (DES) and zearalenone (ZEN), on zebrafish embryos subjected to continuous exposure for 4 days starting from 4 h post fertilization. Noteworthy impact on cumulative mortality, hatchability, spinal and tail curvature, pericardial edema, and reduction in blood circulation were observed in DES-treated embryos, with lower incidence and intensity shown for ZEN at the same nominal concentration (3 µM). An interactive effect was seen for the combined exposure to DES and ZEN, in which deformities and circulatory failure mediated by DES were mitigated by co-treatment with low concentrations of ZEN. Similarly, ZEN-induced spinal and tail curvature, pericardial edema, and blood flow reduction declined dramatically following DES co-exposure at low concentrations. A significant counteracting effect has been observed against DES- and ZEN-induced developmental anomalies following co-treatment with an estrogen receptor (ER) antagonist, fulvestrant (FUL). The assessment of the aromatase gene (CYP19A1b) showed that DES strongly upregulated mRNA expression of CYP19A1b with a lower EC50 (1.1 × 10-3 nM) than a natural estrogen, 17ß-estradiol (2.5 nM). Similarly, ZEN induced CYP19A1b mRNA expression with an EC50 of 57 nM. Exposure to 10 or 20 µM FUL inhibited the expression of CYP19A1b induced by a single treatment of DES or ZEN. Overall, the competitive action against ER could be the main mechanism underlying the developmental toxicity induced by DES and ZEN.

Ovarian follicular fluid levels of phthalates and benzophenones in relation to fertility outcomes.

BACKGROUND: Many endocrine disrupting chemicals (EDCs), for instance phthalates and benzophenones, are associated with adverse fertility outcomes and semen quality parameters. OBJECTIVE: To evaluate if concentrations of selected phthalate metabolites and benzophenones measured in follicular fluid are associated with fertility outcomes (i.e., reproductive hormones, antral follicle count, detected heartbeat at gestational week 7, and live birth) and, in a supplementary study, if measured concentrations of chemicals in follicular fluid can exert biological effects on human spermatozoa. METHODS: Overall, 111 couples from a fertility clinic in Denmark contributed with 155 follicular fluid samples. Concentrations of 43 metabolites from 19 phthalates and phthalate substitutes and six benzophenones were measured in follicular fluid using liquid chromatography-tandem mass spectrometry. Multiple linear and logistic regression with an applied generalized estimating equation model allowing more than one measurement per woman assessed the association between follicular EDC levels and fertility outcomes. The assessment of biological effects of individual and mixtures of EDCs on human spermatozoa was conducted through a human sperm cell based Ca2+-fluorimetric assay. RESULTS: Benzophenone-3 (BP-3) and seven metabolites of five phthalates were detectable in follicular fluid. Women with metabolites of dibutyl phthalate isomers in the highest tertiles had lower antral follicle count (MiBP: ß = -5.35 [95 % CI: -9.06; -2.00], MnBP: ß = -5.25 [95 % CI: -9.00; -2.00]) and lower odds for detecting a heartbeat at gestational week 7 (MiBP: OR = 0.35 [95 % CI: 0.14; 0.91], MnBP: OR = 0.39 [95 % CI: 0.13; 1.15]). Mixtures of the measured concentrations of BP-3 and the seven phthalate metabolites induced a small significant increase in the intracellular calcium ion concentration in human spermatozoa from healthy donors (n = 3). DISCUSSION: Phthalate metabolites and BP-3 were detectable in follicular fluid and high concentrations of some phthalate metabolites were linked with lower chance of successful fertility treatment outcomes. Chemical mixture concentrations in follicular fluid induced a calcium response in human spermatozoa highlighting possible biological effects at physiologically relevant concentrations.

Ethylene and ROS mediate root growth inhibition induced by the endocrine disruptor bisphenol A (BPA).

Bisphenol A (BPA) functions as a detrimental substance that disrupts the endocrine system in animals while also impeding the growth and development of plants. In our previous study, we demonstrated that BPA hinders the growth of roots in Arabidopsis by diminishing cell division and elongation, which is ascribed to the increased accumulation and redistribution of auxin. Here, we examined the mediation of ROS and ethylene in BPA-induced auxin accumulation and root growth inhibition. BPA enhanced ROS levels, and ROS increased auxin contents but reduced cell division activity and the expression of EXPA8 involved in root elongation. ROS scavenger treatment reversed BPA-triggered root growth retardation, auxin accumulation, and cell division inhibition. In addition, BPA induced ethylene, and ethylene synthesis inhibitor treatment reversed BPA-triggered root growth retardation and auxin accumulation. Taken together, ROS and ethylene are involved in BPA-inhibited cell elongation and cell division by mediating auxin accumulation and redistribution.

Bisphenol A exposure decreases sperm production and male fertility through inhibition PCBP2 expression.

Growing evidence suggests that the exposure of bisphenol A (BPA), an endocrine disruptor that commonly present in the environment, can impair reproduction. However, conflicting results have been reported, and the underlying mechanism has not been fully understood. In this study, 3-week-old male mice were oral exposed to 50 mg/kg/d BPA or equivalent corn oil for 28 days. Their testis and epididymis were then collected for morphology examination by HE stains. The number of sperm was counted, and the morphology was analyzed by PNA (peptide nucleic acid) and pap staining. Fertilization capacity and successful rate were analyzed after mating with wide-type females. Spermatid DNA damage and apoptosis were evaluated by DFI, γH2AX stain, and TUNEL assay. RNA sequencing analysis was conducted to identify differentially expressed genes in testicular tissue of mice exposed to BPA. RNA interference was used to verify the regulatory mechanism of BPA exposure on gene expression in GC-2 cells. Our data showed that the total number of sperm was decreased and the morphology was impaired in BPA-exposed mice. In addition, the serum testosterone level and fertilization efficiency were also reduced. Mechanism studies showed that BPA could suppress the expression of PCBP2, a key regulatory gene in spermatid development, by activating the EZH2/H3K27me3. In conclusion, we found that BPA exposure can impair spermatid development via affecting key gene expression that is at least partially due to epigenetic modification.

The endocrine disruptor DE-79 alters oxytocinergic transmission and sexual behavior expression in male rats.

Polybrominated diphenyl ethers (PBDEs), used as flame retardants are persistent organic pollutants exerting important health effects. PBDEs with >5 bromide substitutions were considered less harmful and therefore extensively used commercially. DE-79 was a widely used PBDE mixture of hexa-, hepta-, octa- and nona-brominated compounds that increases vasopressin (AVP) production. AVP and oxytocin (OT) are both produced in neurons of the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei projecting to the neurohypophysis and to brain regions involved in copulatory behavior. OT plays an important role in male copulation. Since DE-79 alters AVP expression in the SON and PVN, it might also modify OT content and alter male sexual behavior. We analyzed if repeated DE-79 exposure of adult male rats affected OT content and OT receptor (OTR) density in the SON, PVN, medial preoptic area (mPOA), ventral tegmental area, nucleus accumbens, and amygdala, and if male copulatory behavior was affected. We show that DE-79 exposure produces a generalized decrease in brain OT immunoreactivity, increases OTR density in all brain regions analyzed but the mPOA, and reduces the ejaculatory threshold after a first ejaculation. The documented ejaculation-induced OT release might participate in this last effect. Thus, one-week DE-79 exposure alters the OT-OTR system and modifies male rat sexual performance. Based on the literature it could be speculated that these effects are related to the putative endocrine disrupting actions of DE-79, ultimately altering brain OT levels and OTR expression that might affect copulation and other important OT-mediated brain functions.

A battery of in silico models application for pesticides exerting reproductive health effects: Assessment of performance and prioritization of mechanistic studies.

Given the high attention to endocrine disrupting chemicals (EDC), there is an urgent need for the development of rapid and reliable approaches for the screening of large numbers of chemicals with respect to their endocrine disruption potential. This study aimed at the assessment of the correlation between the predicted results of a battery of in silico tools and the reported observed adverse effects from in vivo reproductive toxicity studies. We used VirtualToxLab (VTL) software and the EndocrineDisruptome (ED) online tool to evaluate the binding affinities to nuclear receptors of 17 pesticides, 7 of which were classified as reprotoxic substances under Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures (CLP). Then, we aligned the results of the in silico modelling with data from ToxCast assays and in vivo reproductive toxicity studies. We combined results from different in silico tools in two different ways to improve the characteristics of their predictive performance. Reproductive toxicity can be caused by various mechanisms; however, in this study, we demonstrated that the use of a battery of in silico tools for assessing the binding to nuclear receptors can be useful for identifying hazardous compounds and for prioritizing further studies.

Are changes in vitellogenin concentrations in fish reliable indicators of chemical-induced endocrine activity?

Vitellogenin (VTG), a biomarker for endocrine activity, is a mechanistic component of the regulatory assessment of potential endocrine-disrupting properties of chemicals. This review of VTG data is based on changes reported for 106 substances in standard fish species. High intra-study and inter-laboratory variability in VTG concentrations was confirmed, as well as discrepancies in interpretation of results based on large differences between fish in the dilution water versus solvent control, or due to the presence of outlier measurements. VTG responses in fish were ranked against predictions for estrogen receptor agonist activity and aromatase inhibition from bioactivity model output and ToxCast in vitro assay results, respectively. These endocrine mechanisms explained most of the VTG responses in the absence of systemic toxicity, the magnitude of the VTG response being proportional to the in vitro potency. Interpretation of the VTG data was sometimes confounded by an alternative endocrine mechanism of action. There was evidence for both false positive and negative responses for VTG synthesis, but overall, it was rare for substances without endocrine activity in vitro to cause a concentration-dependent VTG response in fish in the absence of systemic toxicity. To increase confidence in the VTG results, we recommend improvements in the VTG measurement methodologies and greater transparency in reporting of VTG data (including quality control criteria for assay performance). This review supports the application of New Approach Methodologies (NAMs) by demonstrating that endocrine activity in vitro from mammalian cell lines is predictive for in vivo VTG response in fish, suggesting that in vitro mechanistic data could be used more broadly in decision-making to help reduce animal testing.

Chronic exposure to cortisone induces thyroid endocrine disruption and retinal dysfunction in adult female zebrafish (Danio rerio).

Cortisone has a large content in rivers because of its wide range of medical applications and elimination by organisms that naturally secrete it. As a steroid hormone, cortisone is recognized as a novel endocrine disruptor. Although ecotoxicological effects of the reproductive endocrine system have mainly been reported recently, thyroid endocrine in fish remains relatively less understood. Here, adult female zebrafish were exposed to cortisone at 0.0 (control), 3.2, 38.7, and 326.9 ng/L for 60 days. Evidence in this study came from fish behavior, hormone levels, gene expression, histological and morphological examinations. The results showed that THs (thyroid hormone) level disruption and pathohistological changes occurred in the thyroid gland, which may account for the gene expression changes in the hypothalamus-pituitary-thyroid gland axis. Specifically, more conversion of T4 (thyroxine) to T3 (triiodothyronine) led to an increased TSH (thyroid stimulating hormone) level in plasma. Severe thyroid tissue damage mainly occurred in the zebrafish exposed to 326.9 ng/L of cortisone. Meanwhile, consistent with the THs trend, the fish locomotion activity displayed more anxiety and excitement, the partial blockage of GABA (γ - aminobutyric acid) synthetic pathway genes might be the explanation of the underlying mechanism. Cortisone affected the gene expressions in the visual cycle and the circadian rhythm network also suggested interactions between thyroid endocrine disruption, retinal dysfunction, and abnormal behaviors of zebrafish. In summary, these findings suggest chronic exposure to cortisone induced various adverse effects in adult female zebrafish, which may help us better understand the risk of cortisone to fish in the wild.

Genetic and Epigenetic Evidence for Nonestrogenic Disruption of Otolith Development by Bisphenol A in Zebrafish.

Bisphenol A (BPA) is a well-known endocrine-disrupting chemical (EDC) that has estrogenic activities. In addition to disrupting reproductive development and function via estrogenic signaling pathways, BPA can also interfere with nonreproductive functions through nonestrogenic pathways; however, the mechanisms underlying such nonestrogenic activities are not well understood. In this study, we demonstrated that BPA could disrupt otolith formation during the early development of zebrafish with long-lasting ethological effects. Using multiple mutants of estrogen receptors, we provided strong genetic evidence that the BPA-induced otolith malformation was independent of estrogen signaling. Transcriptome analysis revealed that two genes related to otolith development, otopetrin 1 (otop1) and starmaker (stm), decreased their expression significantly after BPA exposure. Knockout of both otop1 and stm genes could phenocopy the BPA-induced otolith malformation, while microinjection of their mRNAs could rescue the BPA-induced abnormalities of otolith formation. Further experiments showed that BPA inhibited the expression of otop1 and stm by activating the MEK/ERK-EZH2-H3K27me3 signaling pathway. Taken together, our study provided comprehensive genetic and molecular evidence that BPA induced the otolith malformation through nonestrogenic pathway during zebrafish early development and its activities involved epigenetic control of key genes (e.g., otop1 and stm) participating in otolith formation.

Single and mixture toxicity evaluation of avobenzone and homosalate to male zebrafish and H295R cells.

Avobenzone and homosalate are widely used in sunscreens to provide ultraviolet (UV) protection, either as single compounds or in combination. Some UV filters exhibit estrogenic or anti-androgenic activities, however, studies regarding their interactions and toxicity in mixtures are limited. In this study, the effect of the toxicity of a binary mixture comprising avobenzone (0.72 µg L-1) and homosalate (1.02 and 103 µg L-1) on steroid hormone biosynthesis were investigated using male zebrafish and human adrenocortical carcinoma (H295R) cells. In fish exposed to homosalate, a significant decrease in the gonadosomatic index, testosterone level, and transcription of several genes (e.g, hsd3b2, cyp17a1, and hsd17b1) and a significant increase in the hepatosomatic index, liver steatosis, 17ß-estradiol level, and transcription of vtg gene were observed. These results suggest that estrogenic and anti-androgenic effects of homosalate were mediated by the steroidogenic pathway. The presence of 0.72 µg L-1 of avobenzone augmented the anti-androgenic responses in male fish. The testosterone level in the H295R cells were significantly decreased after they were exposed to homosalate alone or in combination with avobenzone, which is consistent with observations in male zebrafish. Further studies need to be conducted to understand the endocrine disrupting properties of long-term exposure to substances typically used in sunscreens.

Effects of perchlorate and exogenous T4 on growth, development and tail resorption of Rana chensinensis.

Endocrine disruptors have been demonstrated to exert adverse effects on growth and development of amphibians by disrupting hormone levels. Tail resorption, which is one of the most remarkable events during amphibian metamorphosis, is closely associated with thyroid hormones levels. However, limited research has been conducted on the effects of endocrine disruptors on tail resorption in amphibians. This study explored the effects of NaClO4 and T4 on the growth, development and tail resorption during the metamorphosis of Rana Chensinensis. The results demonstrated that exposure to NaClO4 led to an increase in body size and a delay in metamorphosis of R. Chensinensis tadpoles. Histological analysis revealed that both NaClO4 and exogenous T4 exposure resulted in thyroid gland injury, and NaClO4 treatment delayed the degradation of notochord and muscles, thereby delaying tail resorption. Moreover, transcriptome sequencing results showed that apoptosis-related genes (APAF1, BAX and CASP6) and cell component degradation-related genes (MMP9 and MMP13) were highly expressed in the T4 exposure group, and the expression of oxidative stress-related genes (SOD and CAT) was higher in the NaClO4 exposure group. Taken together, both NaClO4 and exogenous T4 affect tail resorption in R. Chensinensis, thereby affecting their adaptation to terrestrial life. The present study will not only provide a reference for future experimental research on the effects of other endocrine disruptors on the growth, development and tail resorption of amphibians but will also provide insights into environmental protection and ecological risk assessment.