Antiestrogenic property of 9,9-bis[4-(2-hydroxyethoxy)phenyl]fluorene (BPEF) and its effects on female development in CD-1 mice.

Identifying chemicals with endocrine disrupting properties linked to disease outcomes is a key concern, as stated in the WHO-UNEP 2012 report on endocrine-disrupting chemicals. The chemical 9,9-bis[4-(2-hydroxyethoxy)phenyl]fluorene (BPEF) is widely and increasingly applied in synthesizing fluorene-based cardo polymers with superior optical, thermal and mechanical properties for various uses. However, little toxicological information is available regarding its safety. Here, we studied the endocrine disrupting property of BPEF by multiple toxicological tools and investigated its effects on female development in adolescent mice. Using the yeast two-hybrid bioassay, BPEF showed strong antiestrogenicity which was similar to that of tamoxifen, an effective antiestrogenic drug. In adolescent CD-1 mice, BPEF significantly decreased the uterine weight at relatively low doses and induced marked endometrial atrophy. Immunohistochemical staining and transcriptome analyses of the mice uteri revealed that BPEF could repressed the expressions of estrogen-responsive genes. Molecular simulation indicated that BPEF could be docked into the antagonist pocket of human estrogen receptor α, and the formation of hydrogen bonds and hydrophobic interactions between BPEF and the active site of receptor maintained their strong binding. All of the data demonstrated that BPEF possessed strong antiestrogenic property and might disrupt female development, suggesting it should be avoided in making products that might directly expose to people, particularly immature women.

Intestinal epithelial deletion of the glucocorticoid receptor NR3C1 alters expression of inflammatory mediators and barrier function.

Glucocorticoids (GCs) are important hormones involved in the regulation of multiple physiologic functions. GCs are also widely used in anti-inflammatory/immunosuppressant drugs. GCs are synthesized by the adrenal cortex as part of the hypothalamus-pituitary-adrenal axis and also by intestinal epithelial cells, among other peripheral sites. GCs are one of the main therapy choices for the exacerbations of inflammatory bowel disease, but they are not useful to prolong remission, and development of tolerance with secondary treatment failure is frequent. Thus, GC actions at the intestinal epithelial level are of great importance, both physiologically and pharmacologically. We generated a tamoxifen-inducible nuclear receptor subfamily 3 group C member 1 (NR3C1)ΔIEC mouse model to study the effects of GCs on epithelial cells in vivo. Nr3c1 deletion in epithelial cells of the small intestine and colon was associated with limited colonic inflammation at 1 wk postdeletion, involving augmented epithelial proliferation and mucus production, plus local and systemic immune/inflammatory changes. This phenotype regressed substantially, but not completely, after 2 wk. The mechanism may involve augmented inflammatory signaling by epithelial cells or defective barrier function. We conclude that the epithelial GC receptor plays a significant role in colonic homeostasis in basal conditions, but its deficiency can be compensated in the short term. Future studies are required to assess the impact of Nr3c1 deletion in other conditions such as experimental colitis.-Aranda, C. J., Arredondo-Amador, M., Ocón, B., Lavín, J. L., Aransay, A. M., Martínez-Augustin, O., Sánchez de Medina, F. Intestinal epithelial deletion of the glucocorticoid receptor NR3C1 alters expression of inflammatory mediators and barrier function.

Tamoxifen-induced, intestinal-specific deletion of Slc5a6 in adult mice leads to spontaneous inflammation: involvement of NF-κB, NLRP3, and gut microbiota.

The sodium-dependent multivitamin transporter (SMVT; SLC5A6) is involved in intestinal absorption of vitamin B7 (biotin). We have previously shown that mice with an embryonic intestinal-specific SMVT knockout (KO) develop biotin deficiency and severe spontaneous intestinal inflammation in addition to growth retardation, developmental delays, and death within the first 6-7 wk of life. The profound morbidity and mortality associated with the SMVT-KO has limited our ability to further characterize the intestinal inflammation and other sequelae of this deletion in adult mice with a mature gut microbiota. To overcome this limitation, we generated an intestine-specific, tamoxifen-inducible, conditional SMVT-KO (SMVT-icKO). Our results showed that adult SMVT-icKO mice have reduced body weight, biotin deficiency, shorter colonic length, and bloody diarrhea compared with age- and sex-matched control littermates. All SMVT-icKO mice also developed spontaneous intestinal inflammation associated with induction of calprotectin (S100a8/S100a9), proinflammatory cytokines (IL-1ß, TNF-α, IFN-γ, and IL-6), and an increase in intestinal permeability. Additionally, the intestines of SMVT-icKO showed activation of the NF-κB pathway and the nucleotide-binding domain and leucine-rich repeat pyrin 3 domain (NLRP3) inflammasome. Notably, administration of broad-spectrum antibiotics reduced lethality and led to normalization of intestinal inflammation, proinflammatory cytokines, altered mucosal integrity, and reduced expression of the NLRP3 inflammasome. Overall, these findings support our conclusion that the biotin transport pathway plays an important role in the maintenance of intestinal homeostasis, and that NF-κB and the NLRP3 inflammasome, as well as gut microbiota, drive the development of intestinal inflammation when SMVT is absent.NEW & NOTEWORTHY This study demonstrates that deletion of the intestinal biotin uptake system in adult mice leads to the development of spontaneous gut inflammation and that luminal microbiota plays a role in its development.

Ras activation in retinal progenitor cells induces tumor formation in the eye.

The RAS gene family members, H-RAS, K-RAS, and N-RAS, are frequently mutated in human cancer. A subset of retinal tumors displays K-RAS mutations; however, the specific role of RAS activation on retinal tumor formation is unclear. To examine the role of RAS in retinal development, we overexpressed the mutant H-RAS gene (G12V) in retinal progenitor cells (RPCs), a multipotent progenitor cell population that gives rise to all six neuron types in the retina and to the Muller glia. The Msi1CreER mouse strain was used to induce mosaic activation of Ras (RasV12) in the RPCs of the postnatal retina. RAS-activated RPCs translocated to the basal part of the retina, differentiated into cells with glial characteristics, and underwent apoptosis. We next induced RAS activation in a large population of RPCs in the embryonic retina using the Pax6Cre mouse strain. In contrast to the phenotype observed in Msi1CreER;RasV12 mice, Ras-activated cells retained their apical attachment. Basal translocation was partially suppressed in the retina of Pax6Cre;RasV12 mice, indicating that basal translocation of Ras-activated cells was not cell autonomous. Notably, RAS-activated retinal cells were highly proliferative and promoted the formation of eye tumors in Pax6Cre;RasV12 mice. Together, our data indicate that the tumorigenicity of RAS activation in RPCs is context dependent, with tumor formation occurring when RAS activity is present in a large cluster of embryonic RPCs.

Assessment of endocrine-disrupting activities of alternative chemicals for bis(2-ethylhexyl)phthalate.

Plastic products are closely intertwined with modern life. Some plasticizers used in making plastics, such as phthalates, are reported to be endocrine-disrupting chemicals. Plasticizers can be released into the environment, and health risks related to plasticizer exposure have been reported. In addition, due to plastic waste that flows into the ocean, microplastics have been found in marine products, including non-biological seawater products such as sea salt. Plastics can affect the body via a variety of pathways, and therefore safer alternative chemicals are needed. Three chemicals were evaluated: acetyl tributyl citrate (ATBC), triethyl 2-acetylcitrate (ATEC), and trihexyl O-acetylacitrate (ATHC), replacing bis(2-ethylhexyl)phthalate (DEHP), a typical plasticizer. The endocrine-disrupting activities of each chemical, including estrogenic or anti-estrogenic activity (test guideline (TG) No. 455), androgenic or anti-androgenic activity (TG No. 458), steroidogenesis (TG No. 456), and estrogenic properties via a short-term screening test using the uterotrophic assay (TG No. 440), were assessed in accordance with the Organisation for Economic Co-operation and Development guidelines for chemical testing. Our results showed that DEHP, ATBC, ATEC, ATHC possess no estrogenic activity, whereas DEHP, ATBC and ATHC demonstrate anti-estrogenic activity and ATBC anti-androgenic activity. DEHP and ATHC exhibited a disruption in steroidogenesis activities. Additional tests are necessary, but our results suggest that ATEC is a good candidate plasticizer providing a suitable alternative to DEHP.

Naturally occurring mixtures of Alternaria toxins: anti-estrogenic and genotoxic effects in vitro.

Alternaria molds can produce a variety of different mycotoxins, often resulting in food contamination with chemical mixtures, posing a challenge for risk assessment. Some of these metabolites possess estrogenic properties, an effect whose toxicological relevance is questioned in the light of the strong genotoxic and cytotoxic properties of co-occurring toxins. Thus, we tested a complex extract from A. alternata for estrogenic properties in Ishikawa cells. By assessing alkaline phosphatase activity, we did not observe estrogen receptor (ER) activation at non-cytotoxic concentrations (≤ 10 µg/ml). Furthermore, an extract stripped of highly genotoxic perylene quinones also did not mediate estrogenic effects, despite diminished genotoxic properties in the comet assay (≥ 10 µg/ml). Interestingly, both extracts impaired the estrogenicity of 17ß-estradiol (E2) at non-cytotoxic concentrations (5-10 µg/ml), indicating anti-estrogenic effects which could not be explained by the presence of known mycoestrogens. A mechanism for this unexpected result might be the activation of the aryl hydrocarbon receptor (AhR) by Alternaria metabolites, as indicated by the induction of CYP1A1 transcription. While a direct influence on the metabolism of E2 could not be confirmed by LC-MS/MS, literature describing a direct interplay of the AhR with estrogenic pathways points to a corresponding mode of action. Taken together, the present study indicates AhR-mediated anti-estrogenic effects as a novel mechanism of naturally co-occurring Alternaria toxin mixtures. Furthermore, our results confirm their genotoxic activity and raise questions about the contribution of still undiscovered metabolites to toxicological properties.

Is frying oil a dietary source of an endocrine disruptor? Anti-estrogenic effects of polar compounds from frying oil in rats.

The objective was to investigate endocrine-disrupting effects of polar compounds from oxidized frying oil. Estrogenicity of polar compounds was tested with a rat uterotrophic bioassay. Dietary oxidized frying oil (containing 51% polar compounds) or polar compounds isolated from it were incorporated into feed (in lieu of fresh soybean oil) and fed to ovariectomized rats, with or without treatment with exogenous ethynyl estradiol. Exogenous estrogen restored uterine weight, and caused histological abnormalities (stratified epithelia and conglomerate glands) as well as proliferation of uterine epithelial cells. However, tamoxifen or polar compounds reduced these effects. Furthermore, tamoxifen or polar compounds down-regulated uterine mRNA expression of estrogen receptor (ER)-target genes, implicating reduced ER activity in this hypo-uterotrophic effect. Inhibition of ER signaling and mitosis by polar compounds were attributed to reduced MAPK and AKT activation, as well as a reduced ligand binding domain-transactivity of ERα/ß. We concluded polar compounds from frying oil are potential endocrine-disrupting chemicals, with implications for food and environmental safety.

Cardiovascular Effects of PCB 126 (3,3',4,4',5-Pentachlorobiphenyl) in Zebrafish Embryos and Impact of Co-Exposure to Redox Modulating Chemicals.

The developing cardiovascular system of zebrafish is a sensitive target for many environmental pollutants, including dioxin-like compounds and pesticides. Some polychlorinated biphenyls (PCBs) can compromise the cardiovascular endothelial function by activating oxidative stress-sensitive signaling pathways. Therefore, we exposed zebrafish embryos to PCB126 or to several redox-modulating chemicals to study their ability to modulate the dysmorphogenesis produced by PCB126. PCB126 produced a concentration-dependent induction of pericardial edema and circulatory failure, and a concentration-dependent reduction of cardiac output and body length at 80 hours post fertilization (hpf). Among several modulators tested, the effects of PCB126 could be both positively and negatively modulated by different compounds; co-treatment with α-tocopherol (vitamin E liposoluble) prevented the adverse effects of PCB126 in pericardial edema, whereas co-treatment with sodium nitroprusside (a vasodilator compound) significantly worsened PCB126 effects. Gene expression analysis showed an up-regulation of cyp1a, hsp70, and gstp1, indicative of PCB126 interaction with the aryl hydrocarbon receptor (AhR), while the transcription of antioxidant genes (sod1, sod2; cat and gpx1a) was not affected. Further studies are necessary to understand the role of oxidative stress in the developmental toxicity of low concentrations of PCB126 (25 nM). Our results give insights into the use of zebrafish embryos for exploring mechanisms underlying the oxidative potential of environmental pollutants.

Anti-estrogenic activity of tris(2,3-dibromopropyl) isocyanurate through disruption of co-activator recruitment: experimental and computational studies.

As a potential endocrine disruptor, tris(2,3-dibromopropyl) isocyanurate (TBC) has previously been demonstrated to reduce expression of estrogen-dependent vitellogenin (vtg) mRNA in adult zebrafish. However, the underlying toxicity pathways and molecular mechanisms involved in TBC-induced endocrine disruption remain elusive. In the current study, E-Screen and MVLN assays were employed to explore the potential anti-estrogenic effects of TBC via the estrogen receptor α (ERα)-mediated signaling pathway. Within a dose range between 1 × 10- 9 and 1 × 10- 7 M, TBC significantly inhibited 17ß-estradiol (E2)-induced cell proliferation in a breast cancer cell line. The luciferase activity induced by E2 was also significantly inhibited by TBC in a dose-dependent manner. Moreover, neither TBC nor E2 affected proliferation of the ERα-negative breast cancer cell line MDA-MB-231. These experimental results confirmed that TBC has anti-estrogenic effects by affecting the ERα-mediated signaling pathway. By comparing TBC with known antagonists of ERα, we found that TBC has similar molecular structure as certain co-activator binding inhibitors. Therefore, using molecular docking and molecular dynamics simulations, TBC was further predicted to competitively occupy the surface site of ERα rather than the canonical E2-binding pocket of ERα, thus disrupt subsequent co-activator recruitment and transcription activation. Our findings elucidate the anti-estrogenic mechanism of TBC at the atomic level and highlight the biological importance of surface sites of nuclear receptors for a risk assessment of potential environmental pollutants.

Molecular mechanisms of 3,3'4,4',5-pentachlorobiphenyl-induced epithelial-mesenchymal transition in human hepatocellular carcinoma cells.

Polychlorinated biphenyls (PCBs) are classic persistent organic pollutants (POPs). Many studies have found a positive association between the progression of hepatocellular carcinoma (HCC) and PCBs exposure. However, the influence of PCBs on epithelial-mesenchymal transition (EMT) of HCC remains to be unclear. In this study, we explored the effect of PCB126 on EMT in HCC cells and its underlying mechanisms. The data showed that PCB126, exposing both Bel-7402 and SMMC-7721 cells for 48h, promoted EMT that was demonstrated by E-cadherin repression, up-regulation of N-cadherin and vimentin, and morphological alteration. We found that signal transducer and activator of transcription 3 (STAT3)/Snail1 signaling was activated after PCB126 exposure, and the addition of STAT3 inhibitor WP1066 blocked PCB126-induced down-regulation of E-cadherin as well as up-regulation of N-cadherin and vimentin. Moreover, PCB126 exposure increased pyruvate kinase M2 (PKM2) expression and its nuclear translocation, whereas treatment with PKM2 shRNA suppressed the activation of STAT3/Snail1 signaling and the alternation of EMT-related molecules (E-cadherin, N-cadherin and vimentin). Furthermore, this study indicated estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) were involved in PCB126-induced effects on PKM2, STAT3/Snail1 signaling and EMT by according treatment using ER inhibitor ICI and AhR shRNA. Notably, PCB126-increased reactive oxygen species (ROS) production via AhR is associated with activation of PKM2/STAT3/Snail1 cascades and contributes to EMT. Taken together, these results indicated that PCB126 promotes EMT process of HCC cells via PKM2/STAT3/Snail1 signaling which is mediated by ER and AhR.

Phenotype anchoring in zebrafish reveals a potential role for matrix metalloproteinases (MMPs) in tamoxifen's effects on skin epithelium.

The zebrafish is a powerful alternative model used to link phenotypes with molecular effects to discover drug mode of action. Using a zebrafish embryo-larval toxicity bioassay, we evaluated the effects of tamoxifen--a widely used anti-estrogen chemotherapeutic. Zebrafish exposed to ≥ 10 µM tamoxifen exhibited a unique necrotic caudal fin phenotype that was rapidly induced regardless of developmental life-stage when treatment was applied. To define tamoxifen's bioactivity resulting in this phenotype, targeted gene expression was used to evaluate 100 transcripts involved in tissue remodeling, calcium signaling, cell cycle and cell death, growth factors, angiogenesis and hypoxia. The most robustly misregulated transcripts in the tail were matrix metalloproteinases mmp9 and mmp13a, induced 127 and 1145 fold, respectively. Expression of c-fos, c-jun, and ap1s1 were also moderately elevated (3-7 fold), consistent with AP-1 activity--a transcription factor that regulates MMP expression. Immunohistochemistry confirmed high levels of induction for MMP13a in affected caudal fin skin epithelial tissue. The necrotic caudal fin phenotype was significantly attenuated or prevented by three functionally unique MMP inhibitors: EDTA (metal chelator), GM 6001 (broad MMP inhibitor), and SR 11302 (AP-1 transcription factor inhibitor), suggesting MMP-dependence. SR 11302 also inhibited induction of mmp9, mmp13a, and a putative MMP target, igfbp1a. Overall, our studies suggest that tamoxifen's effect is the result of perturbation of the MMP system in the skin leading to ectopic expression, cytotoxicity, and the necrotic caudal fin phenotype. These studies help advance our understanding of tamoxifen's non-classical mode of action and implicate a possible role for MMPs in tissues such as skin.

Assessment of estrogenic and androgenic activity in PM10 air samples from an urban, industrial and rural area in Flanders (Belgium) using the CALUX bioassay.

BACKGROUND: Endocrine disrupting chemicals represent a broad class of compounds, are widespread in the environment and can pose severe health effects. OBJECTIVES: The objective of this study was to investigate and compare the overall estrogen and androgen activating potential of PM10 air samples at an urban, rural and industrial location in Flanders, using a human in vitro cell bioassay. METHODS: PM10 samples were collected on glass fiber filters every six days between April 2013 and January 2014 using a high-volume sampler. Extraction was executed with a hexane/acetone mixture before analysis using a recombinant estrogen- or androgen responsive human carcinoma cell line. Results were expressed as bioanalytical equivalents (BEQs) per cubic meter of air. RESULTS: High fluctuations in estrogenic activity were observed during the entire sampling period, with median BEQs of 32.1, 35.9 and 31.1 fg E2-Eq m(-)³ in the industrial, urban and rural background area, respectively. Estrogenic activity was measured in 70% of the samples, while no androgenic activity was observed in any of the samples. The estrogenic activity in the industrial area was positively correlated with the airborne concentration of the sum of the non-carcinogenic PAHs pyrene and fluoranthene (rho=0.48; p<0.01) and the sum of the carcinogenic PAHs (rho=0.36; p=0.05). CONCLUSIONS: This study showed that no androgenic activity was present in PM10 and that although the median estrogenic activity was rather low and comparable in the three locations, high fluctuations in estrogenic response exist over time. While atmospheric PAHs contributed to the observed estrogenic response, especially in the industrial area, the chemicals responsible for the majority of estrogenic activity remain to be identified.

Inhibitory effect of cadmium on estrogen signaling in zebrafish brain and protection by zinc.

The present study was conducted to assess the effects of Cd exposure on estrogen signaling in the zebrafish brain, as well as the potential protective role of Zn against Cd-induced toxicity. For this purpose, the effects on transcriptional activation of the estrogen receptors (ERs), aromatase B (Aro-B) protein expression and molecular expression of related genes were examined in vivo using wild-type and transgenic zebrafish embryos. For in vitro studies, an ER-negative glial cell line (U251MG) transfected with different zebrafish ER subtypes (ERα, ERß1 and ERß2) was also used. Embryos were exposed either to estradiol (E2 ), Cd, E2 +Cd or E2 +Cd+Zn for 72 h and cells were exposed to the same treatments for 30 h. Our results show that E2 treatment promoted the transcriptional activation of ERs and increased Aro-B expression, at both the protein and mRNA levels. Although exposure to Cd, does not affect the studied parameters when administered alone, it significantly abolished the E2 -stimulated transcriptional response of the reporter gene for the three ER subtypes in U251-MG cells, and clearly inhibited the E2 induction of Aro-B in radial glial cells of zebrafish embryos. These inhibitory effects were accompanied by a significant downregulation of the expression of esr1, esr2a, esr2b and cyp19a1b genes compared to the E2 -treated group used as a positive control. Zn administration during simultaneous exposure to E2 and Cd strongly stimulated zebrafish ERs transactivation and increased Aro-B protein expression, whereas mRNA levels of the three ERs as well as the cyp19a1b remained unchanged in comparison with Cd-treated embryos. In conclusion, our results clearly demonstrate that Cd acts as a potent anti-estrogen in vivo and in vitro, and that Cd-induced E2 antagonism can be reversed, at the protein level, by Zn supplement. Copyright © 2016 John Wiley & Sons, Ltd.

In silico prediction and in vivo analysis of antiestrogenic potential of 2-isopropylthioxanthone (2-ITX) in juvenile goldfish (Carassius auratus).

Previous studies have shown both anti-estrogenic and anti-androgenic activities of 2-isopropylthioxanthone (2-ITX), a well known food contaminant, in in vitro assays. However, no data are available on the anti-estrogenic potentials and risks of 2-ITX in aquatic organisms. This work evaluated the potential endocrine disrupting effects of 2-ITX at the level of estrogen receptor (ER) signaling cascade using juvenile goldfish (Carassius auratus) as model. Firstly, we investigated the ligand binding efficiency of 2-ITX to the ligand binding domains (LBD) of goldfish ER subtypes using a molecular docking approach. Secondly, we assessed the effects of 2-ITX on E2-induced hepatic expression of ERα1, ERß1, ERß2, and vitellogenin (VTG) in vivo. Crosstalk between ER-VTG and aryl hydrocarbon receptor 2 (AhR2)-cytochrome P4501A (CYP1A) was also investigated. Fish were injected with increasing doses of 2-ITX ranging from 2 to 10µg/g BW, and results were compared to the effect of tamoxifen, a well-known ER modulator. We observed that compared to ERß, the interaction potentials of 2-ITX to goldfish ERα1 LBD was more stable in the inactive receptor conformation. The in silico docking simulation analysis also revealed that 2-ITX acted as agonist for the goldfish AhR2 LBDs suggesting the ability of this compound to activate the cross-talk between the ERα- and AhR-signaling pathways. In vivo experiments confirm in silico simulation predictions demonstrating that 2-ITX reduced the estrogenicity of E2 at both transcriptional and post-transcriptional levels, indicating a clear anti-estrogenic effect. Co-exposure of E2 and 2-ITX also resulted in a significant decrease of CYP1A gene expression with respect to 2-ITX alone. Results from these studies collectively revealed that the antiestrogenic property of 2-ITX can be ascribed to a combination of effects on multiple signaling pathways suggesting the potential for this environmental contaminant to affect the hormonal control of reproductive processes in fish.

Developmental toxicity, oxidative stress, and related gene expression induced by dioxin-like PCB 126 in zebrafish (Danio rerio).

3,3',4,4',5-Pentachlorobiphenyl (PCB126) cause multiple adverse effects in organisms including animals and humans. Although PCB toxicities are linked to oxidative damage in rodents, the mechanism in early life stages of zebrafish is not clear. To explore the developmental toxicity mechanism of PCB126, three paradigms (toxicological phenotypes, biochemical changes, and molecular changes) were studied in 3-h postfertilization (hpf) zebrafish (Danio rerio) embryos exposed to different PCB126 concentrations (0, 16, 32, 64, and 128 µg/L) until 168 hpf. Developmental malformations, including pericardial and yolk sac edema, impaired lower jaw growth, spinal curvature, head edema and failure to inflate the swim bladder were observed, some as early as 72 hpf. Mortality was not apparent in early stages but significantly increased in a dose-dependent manner from 144 hpf onward. A dose-dependent significant increase in malformation rate was observed from 72 hpf onward with up to 100% at 132 hpf in embryos exposed to 128 µg/L of PCB126. Higher doses of PCB126 significantly decreased the copper-zinc superoxide dismutase (CuZn-Sod), catalase (Cat), and glutathione peroxidase (Gpx) enzyme activities at 96, 132 hpf, but markedly declined from thereafter. PCB126 at 128 µg/L significantly increased the malondialdehyde content at 72, 96, and 132 hpf. The transcriptional gene expression of antioxidant enzymes Cat and Gpx was upregulated in embryos exposed to 64 µg/L of PCB126 at 24 and 96 hpf. Sod1 messenger RNA (mRNA) was low in embryos exposed to 32 µg/L at 72 and 96 hpf but was induced in embryos exposed to 64 and 128 µg/L doses at 132 hpf. Collectively, the results suggest oxidative stress as a major factor in the induction of multiple developmental abnormalities in early life stages of zebrafish exposed to PCB126. However, the relationship between the antioxidant enzyme activity and the mRNA expression was not clear and the potential reasons for this are discussed.

Combined action of estrogen receptor agonists and antagonists in two-hybrid recombinant yeast in vitro.

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 17ß-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.

MEK1/2 inhibition suppresses tamoxifen toxicity on CNS glial progenitor cells.

It is increasingly apparent that treatment with a variety of anticancer agents often is associated with adverse neurological consequences. Clinical studies indicate that exposure even to tamoxifen (TMX), a putatively benign antihormonal agent widely used in breast cancer treatment, causes cognitive dysfunction and changes in CNS metabolism, hippocampal volume, and brain structure. We found that TMX is toxic for a variety of CNS cell populations in vitro and also increased cell death in the corpus callosum and reduced cell division in the mouse subventricular zone, the hippocampal dentate gyrus, and the corpus callosum. We further discovered that MEK1/2 inhibition selectively rescued primary glial progenitors from TMX toxicity in vitro while enhancing TMX effects on MCF7 luminal human breast cancer cells. In vivo, MEK1/2 inhibition prevented TMX-induced cell death in systemically treated mice. Our results demonstrate unexpected cytotoxicity of this putatively benign antihormonal agent and offer a potential strategy for rescuing CNS cells from adverse effects of TMX.

Co-occurrence of estrogenic and antiestrogenic activities in wastewater: quantitative evaluation of balance by in vitro ERα reporter gene assay and chemical analysis.

Endocrine-disrupting chemicals are exogenous substances that alter the function of the endocrine system, with adverse health effects on organisms or their progeny. In vitro estrogen receptor (ER) reporter gene assays have long been used to measure estrogenic activity in wastewater. Nevertheless, there is still uncertainty about their usefulness in environmental monitoring on account of a discrepancy between the estrogenic response of the in vitro assay and concentrations of estrogenic compounds determined by chemical analysis. Here, we measured estrogenic and antiestrogenic activities in wastewater by ERα reporter gene assay. All samples were simultaneously analyzed for estrone, 17ß-estradiol, estriol, and 17α-ethynylestradiol, and the concentrations were used to predict estrogenic activity. All samples in which measured estrogenic activity was significantly lower than predicted showed strong antiestrogenic activity. In addition, we confirmed that the fraction that did not have antiestrogenic activity showed stronger estrogenic activity than the unfractionated wastewater extract. These results indicate that antiestrogenic compounds in wastewater suppress the activity of natural estrogens, and the reporter gene assay represents the net activity.

Potential estrogenic effects of phosphorus-containing flame retardants.

As the substitute of polybrominated diphenyl ethers (PBDEs), further assessments about the potential ecological safety and health risks of phosphorus-containing flame retardants (PFRs) are required because the worldwide demand for PFRs has been increasing every year. In this study, we examined the agonistic/antagonistic activity of a group of PFRs by three in vitro models (luciferase reporter gene assay, yeast two-hybrid assay, and E-screen assay). Molecule docking was used to further explain the interactions between ERα and PFRs. Data from luciferase reporter gene analysis showed three members of the nine tested PFRs significantly induced estrogenic effects, with the order of TPP > TCP > TDCPP, while TCEP and TEHP have remarkable antiestrogenic properties with calculated REC20 and RIC20 values of 10(-6) M or lower. Results from the luciferase reporter gene method are generally consistent with results obtained from the yeast two-hybrid assay and E-screen, except for the positive estrogenic activity of TBP in E-screen testing. Docking results showed that binding between ligands and ERα was stabilized by hydrophobic interactions. As a proposed alternative for brominated flame retardant, PFRs may have anti/estrogenic activity via ERα at the low dose typical of residue in environmental matrix or animals. PFRs with a short chain, halogen, and benzene ring in the substituent group tend to be estrogenic. Our research suggests that comprehensive evaluations, including health and ecological assessments, are required in determining whether PFRs are preferable as an emerging industrial substitute.

The in vitro estrogenic activities of triclosan and triclocarban.

Triclosan (TCS) and triclocarban (TCC), as broad spectrum antibacterial agents, are distributed widely in the environment and humans. Most studies have focused on their distribution and biodegradation, but the endocrine-disrupting effects of these chemicals, especially their estrogenic effects, are still unclear. In the present study, we investigated the estrogenic effects of TCS and TCC using a series of in vitro assays, including the ER reporter gene assay in the CV-1 cells, E-screen assay and evaluation of estrogen-responsive genes in the MCF-7 cells. The tested concentrations of TCS and TCC were both from 1 × 10(-9) to 1 × 10(-6) M. Results showed that TCS and TCC exerted estrogenic activities by inducing luciferase activities in an ER reporter gene assay, promoting the proliferation of the MCF-7 cells, up-regulating the expression of pS2 and down-regulating ERα expression at both the mRNA and protein levels in the MCF-7 cells. We further found that TCS and TCC could alter the expression of multiple microRNAs (mir-22, mir-206 and mir-193b) in the MCF-7 cells, which would help understand the mechanisms of their estrogenic effects on regulating the expression of ERα. In brief, our results demonstrated the potential estrogenic effects and profiled in vitro data for further risk assessment of TCS and TCC.