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Predicting the metabolic activation mechanism and potential hazardous metabolites of environmental endocrine-disruptors is a challenging and significant task in risk assessment. Here the metabolic activation mechanism of benzophenone-3 catalyzed by P450 1A1 was investigated by using Molecular Dynamics, Quantum Mechanics/Molecular Mechanics and Density Functional Theory approaches. Two elementary reactions involved in the metabolic activation of BP-3 with P450 1A1: electrophilic addition and hydrogen abstraction reactions were both discussed. Further conversion reactions of epoxidation products, ketone products and the formaldehyde formation reaction were investigated in the non-enzymatic environment based on previous experimental reports. Binding affinities analysis of benzophenone-3 and its metabolites to sex hormone binding globulin indirectly demonstrates that they all exhibit endocrine-disrupting property. Toxic analysis shows that the eco-toxicity and bioaccumulation values of the benzophenone-3 metabolites are much lower than those of benzophenone-3. However, the metabolites are found to have skin-sensitization effects. The present study provides a deep insight into the biotransformation process of benzophenone-3 catalyzed by P450 1A1 and alerts us to pay attention to the adverse effects of benzophenone-3 and its metabolites in human livers., 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 Elsevier Ltd. All rights reserved.)

High-throughput in vitro assays combined with in vitro-in vivo extrapolation (IVIVE) leverage in vitro responses to predict the corresponding in vivo exposures and thresholds of concern. The integrated approach is also expected to offer the potential for efficient tools to provide estimates of chemical toxicity to various wildlife species instead of animal testing. However, developing fish physiologically based toxicokinetic (PBTK) models for IVIVE in ecological applications is challenging, especially for plausible estimation of an internal effective dose, such as fish equivalent concentration (FEC). Here, a fish PBTK model linked with the IVIVE approach was established, with parameter optimization of chemical unbound fraction, pH-dependent ionization and hepatic clearance, and integration of temperature effect and growth dilution. The fish PBTK-IVIVE approach provides not only a more precise estimation of tissue-specific concentrations but also a reasonable approximation of FEC targeting the estrogenic potency of endocrine-disrupting chemicals. Both predictions were compared with in vivo data and were accurate for most indissociable/dissociable chemicals. Furthermore, the model can help determine cross-species variability and sensitivity among the five fish species. Using the available IVIVE-derived FEC with target pathways is helpful to develop predicted no-effect concentration for chemicals with similar mode of action and support screening-level ecological risk assessment.

In vitro to in vivo (IVIVE) leverages in vitro high-throughput biological responses to predict the corresponding in vivo exposures and further estimate the human safe dose. However, for phenolic endocrine disrupting chemicals (EDCs) linked with complicated biological pathways and adverse outcomes (AO), such as bisphenol A (BPA) and 4-nonylphenol (4-NP), plausible estimation of human equivalent doses (HED) by IVIVE approaches considering various biological pathways and endpoints is still challenging. To explore the capabilities and limitations of IVIVE, this study conducted physiologically based toxicokinetic (PBTK)-IVIVE approaches to derive pathway-specific HEDs using BPA and 4-NP as examples. In vitro HEDs of BPA and 4-NP varied in different adverse outcomes, pathways, and testing endpoints and ranged from 0.0013 to 1.0986 mg/kg bw/day and 0.0551 to 1.7483 mg/kg bw/day, respectively. In vitro HEDs associated with reproductive AOs initiated by PPARα activation and ER agonism were the most sensitive. Model verification suggested the potential of using effective in vitro data to determine reasonable approximation of in vivo HEDs for the same AO (fold differences of most AOs ranged in 0.14-2.74 and better predictions for apical endpoints). Furthermore, system-specific parameters of cardiac output and its fraction, body weight, as well as chemical-specific parameters of partition coefficient and liver metabolic were most sensitive for the PBTK simulations. The results indicated that the application of fit for-purpose PBTK-IVIVE approach could provide credible pathway-specific HEDs and contribute to high throughput prioritization of chemicals in a more realistic scenario., 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 B.V. All rights reserved.)

Thiamethoxam has emerged as an environmental contaminant detected in aqueous environments, and its endocrine-disrupting effect at chronic exposure in teleosts remains unknown. In the present study, a docking experiment and an in vivo test were integrated to systematically explore the toxic mechanisms of thiamethoxam in fish. Histological analysis, plasma VTG and hormone level (E2, 11-KT, T3 and T4) determinations, and HPG and HPT gene expression quantification were performed after Chinese rare minnow (Gobiocypris rarus) was exposed to thiamethoxam (0, 0.5, 5, and 50 μg/L) for 90 days. According to the docking study, thiamethoxam had different interactions with ERα, AR and TRα via hydrogen bonding. A decrease in body length and plasma T4 was observed in both genders. The histological damage in liver and delayed gonadal development were observed in both genders at 50 μg/L thiamethoxam treatment. In males, the following HPG axis genes were upregulated: gnrh and cyp19b in the brain; vtg and cyp19a in the liver; and cyp17 and cyp19a in the gonad. In females, erɑ in the liver was significantly upregulated with 0.5 μg/L thiamethoxam treatment, and cyp17 in the gonad was upregulated with all treatment. The suppression of cyp19a, gnrh, cyp11a, and ttr was observed at the concentration of 5 μg/L in the female liver. Taken together, the endocrine system of Chinese rare minnow might be disrupted after chronic exposure to thiamethoxam., (Copyright © 2019. Published by Elsevier Inc.)

The widely used surfactant nonylphenol ethoxylate (NPEO) and its raw material 4-n-nonylphenol (4-n-NP), as well as its degradation products, are recognized as endocrine disrupting chemicals. The USA Environmental Protection Agency (EPA) released an assessment that looked for safe alternatives to NPEO. Vanillin ethoxylate (VAEO) is a novel substitute for NPEO and is quite similar to NPEO in structure; there is a risk that it has similar endocrine disrupting effects to NPEO. However, their effects on various nuclear hormone receptors have not been thoroughly examined. In this study, the effects of NPEO, VAEO, 4-n-NP and Vanillin on the estrogen receptor α (ERα), androgen receptor (AR), thyroid hormone receptor (TR), retinoic X receptor β (RXRβ) and estrogen-related receptor γ (ERRγ) were determined and compared using a battery of recombined yeast strains expressing β-galactosidase. The results showed that NPEO and 4-n-NP acted as significant antagonists of ER, AR, TR and ERRγ. In addition, 4-n-NP also had antagonistic activity toward RXRβ. Moreover, VAEO was shown to be a very weak antagonist of TR and ERRγ, and Vanillin had no interaction with any nuclear receptors. For the first time, it was found that NPEO had AR, TR and ERRγ antagonistic effects and that 4-n-NP was an antagonist of RXRβ. The in vitro data indicated that NPEO, 4-n-NP and VAEO have the potential to act as endocrine disruptors involving more than one nuclear hormone receptor, but VAEO has much lower endocrine disrupting potential than NPEO. Thus, it is critical to find safe substitutes for NPEO and a substitute of NPEO with structural analogues should be carried out with caution. Furthermore, to look for preferable alternatives for NPEO, more in vivo and in vitro studies of the alternatives concerning endocrine disruption are needed, especially in vitro studies need to involve various target points, not only focus on their effects on ER but also take other nuclear hormone receptor pathways into consideration., (Copyright © 2019. Published by Elsevier Inc.)

In the present study, Chinese rare minnows (Gobiocypris rarus) were exposed to 1, 10, and 100 μg/L of carbamazepine (CBZ) under flow-through conditions for 28 d. A hepatic-specific custom microarray identified 111 and 71 differentially expressed genes in the livers of females and males, respectively, exposed to 100 μg/L of CBZ (ratio ≥ 2, p ≤ 0.05). The levels of five differentially expressed genes associated with the hypothalamic-pituitary-gonadal (HPG) axis were quantified by qPCR, and the results indicated the feasibility of screening endocrine-disrupting chemicals using a custom microarray. The mRNA levels of genes related to the HPG axis differed significantly in different organs of Chinese rare minnows (p < 0.05). Significant differences were observed in the 11-ketotestosterone and plasma vitellogenin levels in all treatments and in the 17β-estradiol (E2) levels in the 100 μg/L CBZ treatment. In contrast, the gonadosomatic index was significantly higher in females and slightly higher in males without significant differences. A pathological analysis determined that 10 and 100 μg/L of CBZ could lead to ova-testis in males and significantly promoted ovum maturation in females. Therefore, our results demonstrate that environmentally relevant concentrations of CBZ have homologous estrogenic activity and induce reproductive toxicity in Chinese rare minnows.

There is increasing evidence of activities of chlorinated by-products of bisphenol A (BPA) on retinoic acid system. Their agonistic and antagonistic activities to human retinoid X receptor (RXR) were assessed by a two-hybrid yeast assay. Aqueous solutions of 1mg/L BPA were chlorinated by sodium hypochlorite (NaClO). It showed that chlorination of BPA increased RXRβ antagonistic activity, while no agonistic activity was detected, showing chlorine might act as a toxic potentiator rather than a toxic deactivator in RXRβ disrupting effects. BPA and its byproducts including 2,2',6,6'-tetrachlorobisphenol A (TCBPA) and 2,4,6-trichlorophenol (TCP) were quantitatively determined by gas chromatography/mass spectrometry (GC/MS). BPA rapidly degraded. With the increasing of ICC and reaction time, concentration of formed TCBPA increased initially then decreased, while concentration of formed TCP increased stably. Using the toxic equivalent (TEQ) approach, the main contributors should be mono-, di- and tri- chlorobisphenol A at initial chlorine concentration (ICC) of 1mg/L. At ICC of 2 and 5mg/L, the main contributors were TCBPA and TCP, being 57.7%-70.7% and 45.3%-59.4%. Molecular docking showed BPA chlorination by-products might have the same mode of action with BPA, forming hydrogen bond and pi-pi interaction with their OH group or hydrophobic ring., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The adverse outcome pathway (AOP) framework could be helpful for chemical risk assessment and mechanistic research. The aim of the present study was to unravel the mechanism of chlordecone-induced endocrine disruption by illustrating the main molecular initiating event (MIE)/perturbations responsible for the observed effects. In silico simulations were performed to predict the MIE(s), and the results pointed to agonistic interaction with estrogen receptors (ERα, ERβ), androgen receptor (AR), cytochrome P450 (CYP19A) by chlordecone. In vivo endocrine disruptions were evaluated in rare minnow (Gobiocypris rarus) exposed to 0.01, 0.1, 1 and 10 μg L(-1) chlordecone from 2 h post-fertilization until sexually mature. In the females, increases of vitellogenin (vtg) mRNA levels in liver and gonad, plasma estradiol (E2), testosterone (T) and E2/T, and renalsomatic index confirmed the role of agonism of ER and CYP19A as MIEs, but the decreased gonadosomatic index, degenerated ovaries as well as the feed-forward response pointed to other potential but important MIEs and corresponding AOPs. In the males, increased E2/T ratio, increased testis vtg mRNA levels and occurrence of intersex confirmed the roles of agonism of ERα and CYP19A as main MIEs in chlordecone-induced endocrine disruptions. Our results also fetches out the limit of AOPs in predicting the adverse outcomes and explaining the mechanism of chemicals at present, thus reflected a critical need for expanding AOPs and AOP network before using it in chemical risk assessment., (Copyright © 2016. Published by Elsevier Ltd.)

2,4-Dichloro-6-nitrophenol (DCNP) is an environmental transformation product of 2,4-dichlorophenol that has been identified as widespread in effluent wastewater, but little is known about its toxicity because this compound is not regulated. Therefore, to investigate the endocrine disruption potency of DCNP in Chinese rare minnows (Gobiocypris rarus), adult and juvenile fish were exposed to various concentrations of DCNP (2, 20, and 200 μg/L) for 28 d. After 28 d exposure, the plasma vitellogenin (VTG) levels were reduced in females while increased in males and juvenile fish considerably, as compared with the control. These results suggested that DCNP affects the HPG-axis in a sex-dependent way. Testosterone (T) levels in the plasma were significantly lower in adult and juvenile fish and were accompanied by an increased estradiol (E2)/T ratio. Histopathological observation revealed hypertrophy of the hepatocytes and nuclear pyknosis in the liver, the inhibition of spermatogenesis in the testes, and the degeneration of oocytes in the ovaries after DCNP exposure. The expression pattern of selected genes indicated that the nuclear receptor, steroidogenesis and gonadotropin regulation pathways were perturbed after DCNP exposure. Above all, our results demonstrated that DCNP clearly had anti-androgenic activity in both adult and juvenile fish and can therefore be considered as an endocrine-disrupting chemical., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Estrogen receptor (ER) antagonistic chemicals in aquatic environments are believed to influence the binding of both endogenous and exogenous estrogens to ERs in aquatic organisms. Although the combined effects of estrogenic compounds have attracted much scientific concern, little work has been done on the influence of such antiestrogens on the biological effects of estrogens. This study focused on how the presence of different amounts of antagonists affects the results of ER agonist activity tests. To achieve this, three questions were stated and answered in sequence. A two-hybrid recombinant yeast assay mediated by ER was adopted, providing a single mode of action and single target of action for this study. Mixtures created by an ER agonist and three antagonists following the fixed-ratio principle were assessed. The concentration of 17β-estradiol causing maximum induction was set as the fixed dose of estrogen in the antagonist activity test (question 1). When the two classes of chemicals coexisted, antiestrogens, which as a whole behaved according to the concentration addition model (question 2), decreased the response of estrogen and compressed the concentration-response curves along the y-axis in the agonist activity test (question 3). This may cause the estradiol equivalent to be underestimated and potentially mask the action of estrogenic effects in toxicity evaluation of environmental samples., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Benzotriazole (BT), an anticorrosive agent, is widely used in industrial applications and household dishwashing agents. Despite its reported toxicity to aquatic organisms, little is known about its endocrine disrupting effects. In this study, adult Chinese rare minnows (Gobiocypris rarus) were exposed to 0.05, 0.5, and 5 mg L(-1) BT for 28 d. The pathological damage in liver was associated with hypertrophy of the hepatocytes, nuclei pyknosis and vacuolization at 5 mg L(-1) groups. Additionally, the degeneration of the ovary and the stimulation of spermatogenesis were observed at 5 mg L(-1) groups. The plasma 17β-estradiol level was significantly increased in the males but decreased in the females at 5 mg L(-1) (p<0.05). In the brain, the up-regulation of CYP19B, GnRHs, and LHβ mRNA was detected across all doses (p<0.05). In the gonad, the transcriptional levels of StAR, CYP11A, 3βHSD, CYP17, 17βHSD, and CYP19A were generally decreased in the males at 5 mg L(-1) (p<0.05), whereas these genes, except for 3βHSD, were significantly increased in females at all concentrations (p<0.05). Moreover, the expression level of VTG in the livers from all exposure groups was significantly increased compared with controls (p<0.05). Taken together, our results indicate that BT could adversely affect the rare minnows in a sex-dependent manner., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Assessing protein responses to endocrine disrupting chemicals is critical for understanding the mechanisms of chemical action and for the assessment of hazards. In this study, the response of the liver proteome of male rare minnows (Gobiocypris rarus) treated with 17β-estradiol (E2) and females treated with 17α-methyltestosterone (MT) were analyzed. A total of 23 and 24 proteins were identified with differential expression in response to E2 and MT, respectively. Pyruvate carboxylase (PC) was the only common differentially expressed protein in both males and females after E2- and MT-treatments. The mRNA as well as the protein levels of PC were significantly down-regulated compared with that of the controls (p < 0.05). Our results suggest that endocrine disruptors interfere with genes and proteins of the TCA cycle and PC may be a sensitive biomarker of exposure to exogenous steroid chemicals in the liver of fish., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Detection of estrogenic disrupting compounds (EDCs) in drinking waters around China has led to rising concerns about health risks associated with these compounds. There is, however, a paucity of studies on the occurrence and identification of the main compounds responsible for this pollution in the source waters. To fill this void, we screened estrogenic activities of 23 source water samples from six main river systems in China, using a recombinant two-hybrid yeast assay. All sample extracts induced significant estrogenic activity, with E2 equivalents (EEQ) of raw water ranging from 0.08 to 2.40 ng/L. Additionally, 16 samples were selected for chemical analysis by gas chromatography-mass spectrometry. The EDCs of most concern, including estrone (E1), 17beta-estradiol (E2), 17alpha-ethinylestradiol (EE2), estriol (E3), diethylstilbestrol (DES), estradiol valerate (EV), 4-t-octylphenol (4-t-OP), 4-nonylphenols (4-NP) and bisphenol A (BPA), were determined at concentrations of up to 2.98, 1.07, 2.67, 4.37, 2.52, 1.96, 89.52, 280.19 and 710.65 ng/L, respectively. Causality analysis, involving comparison of EEQ values from yeast assay and chemical analysis identified E2, EE2 and 4-NP as the main responsible compounds, accounting for the whole estrogenic activities (39.74% to 96.68%). The proposed approach using both chemical analysis and yeast assay could be used for the identification and evaluation of EDCs in source waters of China.

2,4-Dichlorophenol (2,4-DCP) is known as an important chemical intermediate and an environmental endocrine disruptor. There is no paper dealing with the predicted no-effect concentration (PNEC) of 2,4-DCP, mainly due to shortage of chronic and site-specific toxicity data. In the present study, toxicity data was obtained from the tests using six Chinese native aquatic species. The HC(5) (hazardous concentration for 5% of species) was derived based on the constructed species sensitivity distribution (SSD), which was compared with that derived from literature toxicity data of non-native species. For invertebrates, the survival no-observed effect concentrations (NOECs) were 0.05 and 1.00 mg L(-1) for Macrobrachium superbum and Corbicula fluminea, respectively. NOECs based on fishes' growth were 0.10, 0.20 and 0.40 mg L(-1) for Mylopharyngodon piceus, Plagiognathops microlepis and Erythroculter ilishaeformis, respectively. For aquatic plant Soirodela polyrhiza, NOEC based on concentration of chlorophyll was 1.00 mg L(-1). A final PNEC calculated using the SSD approach with a 50% certainty based on different taxa ranged between 0.008 and 0.045 mg L(-1). There is no significant difference between HC(5) derived from native and that from non-native taxa., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: To measure the endocrine disrupting chemicals (EDCs) in wastewater and evaluate the EDCs removal efficiencies in the municipal wastewater treatment plants (WWTP)., Methods: A battery of in vitro recombinant yeast bioassays incorporated with exogenous metabolic activation system (rat liver preparation, S9 mix) was conducted to assess the estrogen receptor (ER), androgen receptor (AR), progesterone receptor (PR), and thyroid receptor (TR) ant/agonistic activities of effluents collected from Datansha WWTP., Results: The indirect estrogenic, anti-androgenic, anti-progesteronic, and anti-thyroidic activities were observed in the influent. The removal efficiencies of EDCs were above 74%, suggesting that the present wastewater treatment processes were good enough to remove most of these indirect endocrine disrupting chemicals., Conclusion: The incorporation of exogenous metabolic capacity into the test system was valid for the study of indirect effects on ER, AR, PR, and TR., (Copyright © 2011 The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier B.V. All rights reserved.)

It is generally known that there are many endocrine disrupting compounds (EDCs) in the effluents from wastewater treatment plants (WWTPs). Most research has focused on the occurrence of estrogenic or androgenic activities, while ignoring that there are environmental chemicals disrupting thyroid system, which is essential for growth and development in both humans and animals. In the present work, a two-hybrid yeast assay was conducted to evaluate the removal efficiencies of agonistic or antagonistic thyroid receptor (TR) mediated effects in different treatment processes of three WWTPs located in Beijing. We found no TR agonistic, but TR antagonistic activities in all processes from the WWTPs. The TR antagonistic activities in organic extracts of water samples were then calibrated regarding to a known TR-inhibitor, amiodarone hydrochloride (AH). The observed concentration of TR disrupting substances ranged from 2.35 x 10(-8) to 6.19 x 10(-7) mol/L AH in Gaobeidian WWTP, 3.76 x 10(-8) to 8.75 x 10(-8) mol/L AH in Lugouqiao WWTP, and 4.80 x 10(-9) to 2.55 x 10(-8) mol/L AH in Beixiaohe WWTP. Of the three WWTPs, the removal rates were 92.7%, 42.2%, and 23.1% respectively. Industrial sewage may contain more TR disrupting substances compared with domestic sewage. The recipient waters were found to contain considerable concentrations of TR disrupting substances that may cause adverse effects on the exposed organisms.

A battery of in vitro recombinant yeast bioassays was conducted to assess the estrogen receptor, androgen receptor, progesterone receptor and thyroid receptor ant/agonistic activities of effluents collected from Datansha wastewater treatment plant (WWTP), furthermore to evaluate the removal efficiencies of endocrine disrupting chemicals in the WWTP. The results showed that estrogenic, anti-androgenic, anti-progesteronic and anti-thyroidic activities were observed in influent. The removal efficiencies of these compounds were more than 80%, which suggested that the present wastewater treatment processes were good enough to remove most of all kinds of endocrine disruption chemicals.

Background: Hydroxylated polybrominated diphenyl ethers (HO-PBDEs) may disrupt thyroid hormone status because of their structural similarity to thyroid hormone. However, the molecular mechanisms of interactions with thyroid hormone receptors (TRs) are not fully understood., Objectives: We investigated the interactions between HO-PBDEs and TRbeta to identify critical structural features and physicochemical properties of HO-PBDEs related to their hormone activity, and to develop quantitative structure-activity relationship (QSAR) models for the thyroid hormone activity of HO-PBDEs., Methods: We used the recombinant two-hybrid yeast assay to determine the hormone activities to TRbeta and molecular docking to model the ligand-receptor interaction in the binding site. Based on the mechanism of action, molecular structural descriptors were computed, selected, and employed to characterize the interactions, and finally a QSAR model was constructed. The applicability domain (AD) of the model was assessed by Williams plot., Results: The 18 HO-PBDEs tested exhibited significantly higher thyroid hormone activities than did PBDEs (p < 0.05). Hydrogen bonding was the characteristic interaction between HO-PBDE molecules and TRbeta, and aromaticity had a negative effect on the thyroid hormone activity of HO-PBDEs. The developed QSAR model had good robustness, predictive ability, and mechanism interpretability., Conclusions: Hydrogen bonding and electrostatic interactions between HO-PBDEs and TRbeta are important factors governing thyroid hormone activities.

Some phenols have been suspected to modulate the endocrine systems of wildlife and humans, but less is known about their interactions with different types of nuclear receptors. In this study, the ability of 2-tert-butylphenol, 2-isopropylphenol, 4-tert-octylphenol (4-t-OP), 2,4-dichlorophenol (2,4-DCP), 3,4-dichlorophenol (3,4-DCP), pentachlorophenol (PCP), bisphenols A (BPA), tetrabromobisphenol A (TBBPA), tetrachlorobisphenol A (TCBPA) and 4-phenylphenol to activate estrogen receptor (ER), androgen receptor (AR), progesterone receptor (PR) and estrogen-related receptor (ERR) were determined using a set of recombined yeast strains. It was found that 4-t-OP, 3,4-DCP, PCP, BPA, TBBPA, TCBPA and 4-phenylphenol were ERalpha agonists, while 4-t-OP, PCP and 4-phenylphenol showed ERalpha antagonistic activities. 2-tert-Butylphenol, 4-t-OP, 2-isopropylphenol, 2,4-DCP, 3,4-DCP, BPA, TCBPA and 4-phenylphenol were antagonists for AR, whereas none of the compounds studied were found to be an AR agonist. TCBPA, TBBPA and PCP were PR antagonists, and 2-tert-butylphenol, 3,4-DCP, 4-t-OP, 4-phenylphenol and 2-isopropylphenol were weak inhibitors on expression under control of the PR. None of the phenols were PR agonists. 2-tert-Butylphenol, 4-t-OP and PCP were ERRgamma inverse agonists, while 2,4-DCP, 3,4-DCP, PCP, BPA, TBBPA and TCBPA exhibited the ability to reverse the ERR inhibition induced by 4-hydroxytamoxifen. Based on the functional agonistic or antagonistic receptor-mediated effects, we further discussed the possible action mechanisms of these phenols as endocrine disrupting chemicals.

It is supposed that the estrogenic effects of estrogenic chemicals will be balanced by the presence of other endocrine-disrupting chemicals (EDCs) with different properties. However, little convincing research into this issue has been carried out to date. In the present study, the combined effects of EDCs with estrogenic and antiestrogenic activities were assessed using a 21-day reproduction assay with the Japanese medaka (Oryzias latipes) as an in vivo model. Natural estrogen (17beta-estradiol, E2) and the pharmaceutical substances letrozole and tamoxifen, which are both model antiestrogens, were tested as reference compounds individually and in combination. Exposure to E2 (200 ng/L) alone resulted in significant biological changes in paired fish, including impaired reproductive capacity and plasma vitellogenin induction. Upon coexposure with tamoxifen or letrozole (10, 50 and 250 microg/L), the effects of estrogen on some biomarkers (e.g., plasma vitellogenin concentration) could be neutralized to some extent, but the impairments in reproductive performance were hardly ameliorated or even became more severe. Since reproductive performance is a more holistic parameter with population-level relevance, the possibility that the effects of estrogenic chemicals can be cancelled out by the presence of antiestrogens in aquatic organisms should be questioned. Our observations also illustrate the importance of considering multiple endpoints at different biological levels of organisms for accurate and adequate assessment of the integrated effects of EDCs, especially in environmentally relevant exposure situations.

The occurrence of estrogens in the aquatic environment attracts increasing attention because of their strong endocrine disrupting potency. In the present work, concentrations of six estrogens including diethylstilbestrol (DES), estrone (E1), beta-estradiol (E2), estriol (E3), 17alpha-ethynylestradiol (EE2) and beta-estradiol 17-valerate (EV) in surface water and sediment sampled from three rivers in Tianjin area, northern China, were determined by gas chromatography-mass spectrometry (GC-MS). The concentrations of all six estrogens (Sigma6ES) ranged from 0.64 to 174 ng L(-1) in waters and from 0.98 to 51.6 ng g(-1) dry weight (dw) in sediments, and varied for each river. Among these estrogens, E1 was the most abundant and could be detected in all samples. DES and EV could be detected either in river water or in sediment, but in the concentration below 10 ng L(-1) and 10 ng g(-1) for water and sediment, respectively. The relationships between concentration of estrogens and organic carbon content in sediments and the relationships between sediment-water partition coefficient (logK(oc)) and octanol-water partition coefficient (logK(ow)) were examined. The results showed that the contents of the Sigma6ES correlated significantly with the contents of organic carbon (OC%). It indicated that sediments with high organic carbon were more likely to retain estrogens than those with lower organic carbon levels in the natural aquatic environment. Furthermore, the linear correlations between logK(ow) and logK(oc) were obtained for each river, which indicated that sediment-water partitioning of estrogens in three rivers could be predicted by their hydrophobic properties.

Some organochlorine pesticides (OCPs) are suspected of modulating the endocrine systems of humans. Aspects of neuro-endocrine system modulation include interactions such as agonism or antagonism of estrogen receptor (ER) binding. However, less is known about their interactions with other nuclear receptors (NRs). The objectives of this study were to determine and compare the ability of p,p'-dichlorodiphenylethane (p,p'-DDE), p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT), hexachlorobenzene (HCB) and r-hexachlorocyclohexane (r-HCH) to interact with ERalpha, androgen receptor (AR), progesterone receptor (PR) and estrogen-related receptor (ERRgamma) using a set of recombined yeast strains expressing beta-galactosidase, under control of ERalpha, AR, PR or ERRgamma. The results showed that p,p'-DDE was an ERalpha agonist, AR and PR antagonist (PR>AR), while p,p'-DDT was an ERalpha agonist and AR antagonist. HCB and r-HCH were antagonists for AR and ERRgamma, while r-HCH was a PR antagonist and a weak antagonist of ERRgamma, and was able to reverse the ERRgamma inhibition induced by 4-hydroxytamoxifen. All the results suggested that, for the tested OCPs, their ability to act as endocrine disruptors involves more than one mechanism, their (anti-)agonistic effects on different receptors should not be overlooked, and the potential for additive or synergistic effects must be taken into consideration in the risk assessment process.

The aim of this study is to investigate the chemical retinoic acid (RA) disruption at the level of retinoid X receptor (RXR) functioning. This assay makes use of recombined human RXR gene and reporter gene yeast, which specifically expresses beta-galactosidase when incubated with exogenous 9-cis retinoic acid (9-cis RA). Agonistic and antagonistic actions of chemicals including a series of phenols, phthalates, organochlorine pesticides (OCPs) were tested in the absence and presence of 5 x 10(-6)mol/L 9-cis RA, at which maximal beta-galactosidase activity could be induced. The results obtained reveal that some chemicals, e.g., 2-t-butylphenol, 2-isopropylphenol, 2,4-dichlorophenol (2,4-DCP), 3,4-dichlorophenol (3,4-DCP), 4-tert-octylphenol (4-t-OP) and hexachlorobenzene (HCB), are RXR agonists. Especially, bisphenol A (BPA) showed high induction activity to RXR when tested with metabolization. The 20% relative inhibitory concentration (RIC20) values of r-hexachlorocyclohexane (HCH), p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT) and 2,4-DCP with metabolization were lower than 1 x 10(-6)mol/L. These results suggest that BPA, HCH, p,p'-DDT and 2,4-DCP are chemicals that pose a threat to hRXR functioning. Altogether the results of the present study show that the newly developed, yeast two-hybrid assay can be used as a valuable tool for identification and quantification of compounds active in disturbing retinoid homeostasis at the level of RXR.

Over the last few years, increasing evidence has become available that some chemicals may have thyroid hormone- disrupting potencies. The effects exerted via thyroid hormone receptors (TR) have not been studied thoroughly. The present study investigates chemical thyroid hormone disruption at the level of TR functioning. To this end the (ant)agonistic action of a series of xenobiotics was tested in the newly developed yeast two-hybrid assay. This assay makes use of recombined TRbeta gene and reporter gene yeast, which specifically expresses beta-galactosidase when incubated with exogenous 3,3',5-triiodo-l-thyronine (T(3)). Agonistic and antagonistic actions were studied in the absence and presence of 5 x 10(-6) mol/L T(3), which induced maximal beta-galactosidase activity. The compounds tested included the specific TR-antagonist amiodarone, as well as a series of phenols, phthalate, organochlorine pesticides (OCPs), polyhalogenated aromatic hydrocarbons (PHAHs) with structural similarity to T(3), and 3,3',5,5'-tetraiodo-l-thyronine (T(4)). The results obtained reveal that only 2-t-butylphenol and 2-isopropylphenol are specific agonists. Interestingly, some compounds showed potentiated antagonistic effects when tested in combination with T(3), which suggests that TR-mediated transcription may be disrupted in vivo. The 20% relative inhibitory concentration (RIC20) values of phenols, ester and OCPs were more than 10(-7) mol/L, but the values for PHAHs with metabolization were lower than 5 x 10(-7) g/L. These results suggest that PHAHs pose a serious threat to the human thyroid system. Altogether the results of the present study show that the newly developed, yeast two-hybrid assay can be used as a valuable tool for identification and quantification of compounds active in disturbing thyroid hormone homeostasis at the level of TR. The results are further evidence of thyroid-related effects of environmentally relevant PHAHs.

Letrozole is a synthetic aromatase inhibitor and interferes in the committed step in the synthesis of endogenous estrogens from androgens. To evaluate potential effects on the early life stages of Japanese medaka, larvae and fertilized eggs were exposed to letrozole for 96 h and 14 days, respectively. No larvae died and no adverse effects were found on embryonic development at concentrations up to 3125 microg/L. Reproductive effects were assessed by exposing adults to 1, 5, 25, 125 and 625 microg/L letrozole for 21 days. A dose-dependent decrease in fecundity (>25 microg/L) and fertility (>5 microg/L) accompanied by histological changes suggested the inhibition of oocyte growth and possibly maturation. At 625 microg/L, the fish ceased spawning during the last week of exposure. Letrozole (>5 microg/L) reduced plasma vitellogenin levels in females in a dose-dependent manner. Transgenerational effects were evaluated by removing freshly-laid F1 eggs from letrozole-contaminated water and raising them to 15 days post-hatching in control water. Hatchability and time to hatching were detrimentally affected (>5 microg/L), but no morphological deformities were observed. Furthermore, a dose-dependent increase in the proportion of genotypic F1 males was found (>5 microg/L).

Pentachlorophenol (PCP) is widely used to control termites and protect wood from fungal-rot and wood-boring insects, and is often detected in the aquatic environment. Few studies have evaluated PCP as an environmental endocrine disruptor. In the present work, Japanese medaka (Oryzias latipes) was exposed to PCP for 28 days (F0 generation) with subsequent measurements of vitellogenin (VTG), hepatic 7-ethoxyresorufin-O-deethylase (EROD), and reproductive endpoints. Plasma VTG significantly increased in male fish treated with PCP concentrations lower than 200 microg/l and decreased in male and female animals exposed to 200 microg/l. Hepatic EROD from female fish increased when PCP exposure concentrations exceeded 20 microg/l, but decreased in the 200 microg/l PCP treatment group. Fecundity and mean fertility of female medaka decreased significantly in the second and third week following exposure concentrations greater than 100 microg/l, and testis-ova of male medaka was observed at PCP concentrations greater than 50 microg/l. Histological lesions of liver and kidney occurred when exposure concentrations exceeded 50 microg/l. In F1 generations, the hatching rates and time to hatch of offspring were significantly affected in fish exposed to 200 microg/l. These results indicated that PCP exposure caused responses consistent with estrogen and aryl hydrocarbon receptor activation as well as reproductive impairment at environmentally relevant concentrations.

ABSTRACT Dicamba is a benzoic acid herbicide that has been detected in surface and ground water. The herbicide has been shown to have cytogeneic and DNA damaging effects and to cause organ damage in mammals; however, little is known about the endocrine disrupting effects of dicamba in fish. In this study, histological changes, plasma vitellogenin (VTG) and sex hormone levels, and mRNA expression of sex steroid hormone-related genes were determined in adult rare minnow exposed to environmentally relevant concentrations of dicamba (0, 0.05, 0.5, 5, and 50 μg/L) for 40 days. The results showed inhibition of spermatogenesis in male testes and ovarian degeneration in females. Plasma 17β-estradiol (E2) levels were significantly increased in both genders, and plasma VTG levels were significantly increased in males ( p < 0.05). These results indicate that sex hormone homeostasis and normal reproduction of fish could be affected by dicamba. Moreover, the mRNA levels of vtg were significantly upregulated in the livers and gonads of both male and female rare minnows ( p < 0.05). The downregulation of cytochrome P450c19a ( cyp19a) and steroidogenic acute regulatory protein ( star) mRNA levels, and the upregulation of cytochrome P450c17 ( cyp17) mRNA levels were observed in the livers and ovaries ( p < 0.05). The results of the mRNA analysis suggest that changes in steroid hormone-related gene expression could serve as a regulatory mechanism to maintain sex hormone homeostasis. Overall, dicamba exposure could result in histological lesions, plasma VTG increases, changes in sex hormone levels, and alterations of hormone-related gene expression. Therefore, dicamba should be considered to be a potential endocrine disruptor. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 693-703, 2015. [ABSTRACT FROM AUTHOR]

Polychlorinated naphthalenes (PCNs) are dioxin-like environmental contaminants. There is growing concern over the endocrine-disrupting effects of PCNs, but very few studies have investigated the effect of PCNs on the thyroid system. This study used a yeast two-hybrid assay, which included the recombinant human thyroid receptor(TR)-β and reporter genes, to characterize the TRβ-disrupting effects of five individual PCN congeners, five PCN Halowax mixtures, and naphthalene. Their agonist and antagonist effects were studied in the absence and presence of 5x10-7 mol/L 3,3',5-triiodo-L-thyronine, which induced submaximal β-galactosidase activity. Naphthalene, 1,2,3,4,5,6,7,8-octachloronaphthalene and all of the Halowax mixtures (Halowax 1000, 1001, 1013, 1014 and 1099) showed no agonist or antagonist activity on TRβ at the concentrations tested (up to 10-2 g/L). The lighter PCN congeners, namely 1-chloronaphthalene, 2-chloronaphthalene, 1,4-dichloronaphthalene and 1,2,3,4-tetrachloronaphthalene showed no agonist activity but showed significant antagonist activity on TRβ. The 20% relative inhibitory concentrations of these PCNs were less than 9.13 x 10-3 g/L. Thus, bioaccumulation of these lighter PCN congeners may disrupt the thyroid hormone system and inhibit TR-mediated cellular responses. Studies in the future should investigate the possible associations between the presence PCNs and adverse health outcomes. [ABSTRACT FROM AUTHOR]

As the typical aryl-organophosphate flame retardants (OPFRs), triphenyl phosphate (TPhP) and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) were reported to be estrogen disruptors. However, estrogen receptor α (ERα) binding experiments could not explain their biological effects. In this study, their action on ERα, G protein-coupled estrogen receptor (GPER) and the synthesis of 17β-estradiol (E2) were investigated using in vitro assays and molecular docking. The results showed that TPhP acted as an ERα agonist and recruited steroid receptor co-activator 1 (SRC1) and 3 (SRC3), which was found for the first time. Unlike TPhP, TDCIPP acted as an ERα antagonist. However, both TPhP and TDCIPP activated the estrogen pathway by GPER in SKBR3 cells which were lack of ERα. Although molecular docking results revealed that both TPhP and TDCIPP could dock into ERα and GPER, their substituent groups and combination mode might affect the receptor activation. In addition, by using estrogen biosynthesis assay in H295R cells, both of TPhP and TDCIPP were found to promote E2 synthesis and E2/T ratio involving their different alteration on levels of progesterone, testosterone and estrone, and expression of various key genes. Our data proposed estrogen-disrupting mechanism frameworks of TPhP and TDCIPP. Moreover, our results will contribute to future construction of adverse outcome pathway (AOP) framework of endocrine disruptors. • TPhP acted as an ERα agonist, while TDCIPP acted as an ERα antagonist. • First evidence on TPhP-ERα recruiting SRC1 and SRC3. • TPhP and TDCIPP activated the GPER-mediated cAMP pathway. • The substituent groups of TPhP and TDCIPP might affect the estrogen receptors activation. • TPhP and TDCIPP promoted E2 synthesis by different effects on various key enzymes. [ABSTRACT FROM AUTHOR]

4-n-nonylphenol (4-n-NP), a typical endocrine disrupting chemical, has been so far frequently detected in various environmental mediums and editable food. However, the specific metabolic pathways in human and potential adverse effects of metabolites have not been elucidated yet. Here, metabolic profiling of 4-n-NP in human liver microsome (HLM) was comprehensively characterized by integrated approaches of testing and assessment. A total of 21 metabolites were identified using nontarget analysis with high-resolution mass spectrum, including three groups of unique phase I metabolites first determined in HLM. Seven various metabolic pathways of 4-n-NP were identified by both in silico and in vitro, and CYP1A2, 2C19, and 2D6 were the mainly participating enzymes. Two secondary metabolites with carbonyl groups on side chains (M4, M7) presented most abundant in HLM, which were also predicted to have high binding affinities towards HPG-axis-related receptors (AR, ER, and PR). ESRs (estrogen receptors) were shared core protein targets for all metabolites revealed by protein-protein interaction networks. Biological functions enrichment analysis indicated that 4-n-NP metabolites might primarily involve in ESR-mediated signaling, GPCR ligand binding, Class A/1 (Rhodopsin-like receptors) and metabolism-related pathways. These findings of 4-n-NP metabolites, pathways, and biological effects provide insightful information for its environmental exposure and risk assessment. [Display omitted] • Metabolic profiling of 4-n-NP was revealed by integrated approaches. • Seven metabolic pathways of 4-n-NP were identified by both in silico and in vitro. • Three groups of unique phase I metabolites were first identified in HLM. • ESRs were shared core protein targets of all 4-n-NP metabolites. • Secondary metabolites had stronger binding affinities to HPG-axis-related receptors. [ABSTRACT FROM AUTHOR]