In recent years, studies have focused on the combined ecological risks posed by microplastics and other organic pollutants. Although both microplastics and progestin residues are frequently detected in the aquatic environments, their ecological implications remain unknown. Adult zebrafish were exposed to polystyrene microplastics (PS, 200 nm, 200 μg/L), norethindrone (NET, 69.6 ng/L), and their mixture (200 μg/L PS + 63.1 ng/L NET) for 30 days. The results demonstrated that exposure to PS and NET resulted in gill damage. Notably, the PS and PS+NET exhibited a significant decrease in glutathione (GSH) and oxidized glutathione (GSSG) content, as well as reduced antioxidase activity in the gills. The oxidative stress in PS+NET primarily originated from PS. The PS, NET, or their mixture resulted in a decrease in testosterone (T) and estradiol (E2) levels in female. Furthermore, compared to NET, the PS+NET showed a significant reduction in E2 levels, thereby augmenting the inhibitory effect on reproductive ability mediated by NET. However, males showed an increase in 11-ketodihydrotestosterone (11-KT) content, accompanied by a significant decrease in spermatogonia (Sg) and increase in spermatocytes (Sc). Consequently, it can be inferred that PS enhances the androgenic effect of NET. In female fish brain, NET alone resulted in transcriptional down-regulation of partial hormone receptors; however, co-administration of PS effectively mitigated the interference effects. Furthermore, transcriptional downregulation of 17-alpha-hydroxylase (cyp17), hydroxysteroid 3-beta dehydrogenase (hsd3b), estrogen receptor 1 (esr1), and estrogen receptor 2a (esr2b) genes in the ovary was found to be associated with the androgenic activity induced by NET. Moreover, in comparison to PS or NET alone, PS+NET resulted in a notable decrease in Cetobacterium abundance and an increase in Aeromonas population, suggesting that the co-exposure of PS+NET may exacerbate intestinal burden. The findings highlight the importance of studying the combined toxicity of PS and NET.

Fluorene-9-bisphenol (BHPF) is an emerging contaminant. Presently, there is no report on its interaction with G protein-coupled estrogen receptor 1 (GPER). By using an integrated toxicity research scenario that combined theoretical study with experimental methods, BHPF was found to inhibit the GPER-mediated effect via direct receptor binding. Molecular dynamics simulations found that Trp2726.48 and Glu2756.51 be the key amino acids of BHPF binding with GPER. Moreover, the calculation indicated that BHPF was a suspected GPER inhibitor, which neither can activate GPER nor is able to form water channels of GPER. The role of two residues was successfully verified by following gene knockout and site-directed mutagenesis assays. Further in vitro assays showed that BHPF could attenuate the increase in intracellular concentration of free Ca2+ induced by G1-activated GPER. Besides, BHPF showed an enhanced cytotoxicity compared with G15, indicating that BHPF might be a more potent GPER inhibitor than G15. In addition, a statistically significant effect on the mRNA level of GPER was observed for BHPF. In brief, the present study proposes that BHPF be a GPER inhibitor, and its GPER molecular recognition mechanism has been revealed, which is of great significance for the health risk and assessment of BHPF.

Parabens (PBs), a group of widely used synthetic preservatives with potential endocrine disrupting activity, have been detected with increasing frequency in organisms and environmental matrices. This study assessed the hormone interference effects of four typical PBs, namely methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), and butylparaben (BuP), in zebrafish and elucidated the probable underlying mechanisms. Transcriptomic and metabolomic analyses showed that the differentially expressed genes and metabolites were associated with the tyrosine metabolism, arachidonate metabolism, and glycerophospholipid metabolism, indicating they were essential precursors of steroid hormone biosynthesis and metabolism. Histopathological analysis revealed impaired gonad development in the zebrafish exposed to PBs, as evidenced by the significantly increased vitellogenin (VTG) and estradiol (E2) levels. Furthermore, molecular dynamics simulation suggested that the four PBs could preferentially activate the zebrafish estrogen receptor, zfERβ2, to regulate the downstream pathways. Disruption of the amino acid metabolism and lipid metabolism, and activation of zfERβ2 signaling pathway were found to be the key mechanisms for the endocrine disrupting effects of PBs. The hormone interference effects of PBs were apparently dependent on the shared oxybenzene on their structures, with the degree of interference determined largely by the length of their alkyl chains. These findings provide new insights into the endocrine disrupting effects of PBs and could help better assess their risk to human health.

Estrogens and androgens are typical steroid hormones and often occur together in contaminated aquatic environments, but their mixed effects in aquatic organisms have been less well reported. In this study, the endocrine disrupting effects of binary mixtures of 17β-estradiol (E2) and testosterone (T) in western mosquitofish (Gambusia affinis) were assessed by analyzing the sex ratio, secondary sex characteristics, gonadal histology, and transcriptional expression of target genes related to the hypothalamic-pituitary-gonadal (HPG) axis in G. affinis (from embryos) continuously exposed to E2 (50 ng/L), T (T1: 50 ng/L; T2: 200 ng/L), and mixtures of both (E2 + T1: 50 + 50 ng/L; E2 + T2: 50 + 200 ng/L) for 119 d. The results showed that exposure to E2 + T1 and E2 + T2 reduced the length ratio of ray 4/6 ratio in male G. affinis, suggesting feminized phenomenon in male G. affinis. Furthermore, 16.7-38.5 % of female G. affinis showed masculinized anal fins and hemal spines when exposed to T alone and in combination with E2. Importantly, the transcriptional levels of certain target genes related to the HPG axis were significantly altered in G. affinis following exposure to E2 and T alone and in combinations. Moreover, exposure to E2 and T in combinations can lead to combined effects (such as synergistic and antagonistic effects) on the transcriptional levels of some genes. These results collectively suggest that exposure to environmentally relevant concentrations of E2 and T alone and in mixtures can impact the endocrine system of G. affinis, and may pose potential risks in aquatic systems.

Over the past decades, the synthetic glucocorticoids (GCs) have been widely used in clinical practice and animal husbandry. Given the health hazard of these toxic residues in food, it is necessary to explore the detailed interaction mechanisms of typical GCs and their main target glucocorticoid receptor (GR). Hence, this work compared the GR binding and agonist activities of typical GCs. Fluorescence polarization assay showed that these GCs were potent ligands of GR. Their GR binding affinities were in the order of methylprednisolone>betamethasone≈prednisolone>dexamethasone, with IC50 values of 1.67, 2.94, 2.95, and 5.58 nM. Additionally, the limits of detection of dexamethasone, betamethasone, prednisolone, and methylprednisolone were 0.32, 0.14, 0.19, and 0.09 μg/kg in fluorescence polarization assay. Reporter gene assay showed that these GCs induced GR transactivation in a dose-dependent manner, confirming their GR agonist activities. Among which, dexamethasone at the concentration of 100 nM produced a maximal induction of more than 11-fold over the blank control. Molecular docking and molecular dynamics simulations suggested that hydrogen-bonding and hydrophobic interactions played an important role in stabilizing the GC-GR-LBD complexes. In summary, this work might help to understand the GR-mediated endocrine disrupting effects of typical GCs.

In context of testing, screening and monitoring of endocrine-disrupting (ED) type of environmental pollutants, tunicates could possibly represent a particularly interesting group of bioindicator organisms. These primitive chordates are already important model organisms within developmental and genomics research due to their central position in evolution and close relationship to vertebrates. The solitary ascidians, such as the genus Ciona spp. (vase tunicates), could possibly be extra feasible as ED bioindicators. They have a free-swimming, tadpole-like larval stage that develops extremely quickly (<20 h under favorable conditions), has a short life cycle (typically 2-3 months), are relatively easy to maintain in laboratory culture, have fully sequenced genomes, and transgenic embryos with 3D course data of the embryo ontogeny are available. In this article, we discuss possible roles of Ciona spp. (and other solitary ascidians) as ecotoxicological bioindicator organisms in general but perhaps especially for effect studies of contaminants with presumed endocrine disrupting modes of action.

Studies on cow\'s milk have mainly focused on analyzing specific chemical groups and natural components. Therefore, in this study, we evaluated if effect-based in vitro methods could be used as a screening tool to monitor chemical hazards in milk. In total, 32 milk samples were collected from a Swedish dairy company throughout one year. These samples included conventional and organic semi-skimmed as well as raw milk. The milk samples were tested in five in vitro methods covering eight endpoints. These endpoints included cytotoxicity, endocrine disruption (estrogen/androgen induction/inhibition), aryl hydrocarbon receptor activity, oxidative stress and DNA damage. Estrogen and androgen receptor inhibition, in addition to aryl hydrocarbon receptor activity, were the most responsive endpoints, where 10 to 13 out of the 32 milk samples were bioactive. Organic and conventional milk showed no major differences. Overall, no or only low activities were observed in milk samples in the remaining in vitro assays, which is a promising result with regard to applying effect-based methods as a screening tool. Concerning the most responsive assays, more research is needed to understand the normal background variations before they can be used as a screening tool for chemical hazards in milk.

Phthalate esters (PAEs) are widely exposed in the environment as plasticizers in plastics, and they have been found to cause significant environmental and health hazards, especially in terms of endocrine disruption in humans. In order to investigate the processes underlying the endocrine disruption effects of PAEs, three machine learning techniques were used in this study to build an adverse outcome pathway (AOP) for those effects on people. According to the results of the three machine learning techniques, the random forest and XGBoost models performed well in terms of prediction. Subsequently, sensitivity analysis was conducted to identify the initial events, key events, and key features influencing the endocrine disruption effects of PAEs on humans. Key features, such as Mol.Wt, Q+, QH+, ELUMO, minHCsats, MEDC-33, and EG, were found to be closely related to the molecular structure. Therefore, a 3D-QSAR model for PAEs was constructed, and, based on the three-dimensional potential energy surface information, it was discovered that the hydrophobic, steric, and electrostatic fields of PAEs significantly influence their endocrine disruption effects on humans. Lastly, an analysis of the contributions of amino acid residues and binding energy (BE) was performed, identifying and confirming that hydrogen bonding, hydrophobic interactions, and van der Waals forces are important factors affecting the AOP of PAEs\' molecular endocrine disruption effects. This study defined and constructed a comprehensive AOP for the endocrine disruption effects of PAEs on humans and developed a method based on theoretical simulation to characterize the AOP, providing theoretical guidance for studying the mechanisms of toxicity caused by other pollutants.

Alkylphenols (APs) represent one of the highest exposure levels among endocrine disrupting chemicals (EDCs) in the South China Sea (SCS) due to their extensive use as plastic additives. The concerns about EDCs, including APs, have been reiterated since the surge in plastic waste from the COVID-19 response, but far less is known about the response of AP loadings in the SCS to emerging public policies and activities, including the COVID-19 pandemic. Here, we used cetaceans as bioindicators for monitoring two major APs, 4-nonylphenol (4-NP) and 4-tert-octylphenol (4-t-OP), in nine stranded cetacean species (n = 110) in the SCS between 2004 and 2021. Prior to the COVID-19, APs loads showed decreasing temporal trends for finless porpoises and humpback dolphins, most likely due to China\'s restrictions on AP use or a shift in dominant prey species. Unexpectedly, AP loads continued to decline after the COVID-19 outbreak, probably due to a temporal-lag response of marine AP fluxes to the pandemic. The health risk assessments based on hormone biomarkers and toxicity thresholds suggest the potential adverse effects of APs on cetaceans, while recent declines in APs, though limited, may mitigate the detrimental impacts.

As a promiscuous xenobiotic receptor, pregnane X receptor (PXR) has been confirmed to participate in numerous physiological process. In addition to the conventional estrogen/androgen receptor, PXR also serves as an alternative target for environmental chemical contaminants. In this work, the PXR-mediated endocrine disrupting effects of typical food contaminants were explored. Firstly, the time-resolved fluorescence resonance energy transfer assays confirmed the PXR binding affinities of 2,2\',4,4\',5,5\'-hexachlorobiphenyl, bis(2-ethylhexyl) phthalate, dibutyl phthalate, chlorpyrifos, bisphenol A, and zearalenone, with IC50 values ranging from 1.88 to 4284.00 nM. Then their PXR agonist activities were assessed by PXR-mediated CYP3A4 reporter gene assays. Subsequently, the regulation of gene expressions of PXR and its targets CYP3A4, UGT1A1, and MDR1 by these compounds was further investigated. Intriguingly, all the tested compounds interfered with these gene expressions, confirming their endocrine disrupting effects via PXR-mediated signaling. The compound-PXR-LBD binding interactions were explored by molecular docking and molecular dynamics simulations to unravel the structural basis of their PXR binding capacities. The weak intermolecular interactions are key players in stabilizing these compound-PXR-LBD complexes. During the simulation process, 2,2\',4,4\',5,5\'-hexachlorobiphenyl remained stable while the other 5 compounds underwent relatively severe disturbances. In conclusion, these food contaminants might exhibit endocrine disrupting effects via PXR.