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Abstract: Chemical and toxicological profiles were assessed in surface sediments (fraction <63μm) from the southern North Sea. In extracts of freeze-dried samples, polybrominated biphenyl (PBB), Irgarol 1051 and phthalate concentrations were below the respective detection limits (except di(2-ethylhexyl)phthalate, which was between 170 and 3300μgkg−1 dry weight (dw)). Hexabromocyclododecane (HBCD) concentrations were between 0.8 and 6.9μgkg−1dw, with highest concentrations at river mouths. Polybrominated diphenylethers (PBDE) concentrations were 0.4–0.6μgkg−1dw, decabromodiphenylether (BDE209) 1–32μgkg−1dw. The ratio BDE209/PCB153 was used as a tracer for recent emissions, and pointed towards a BDE209 source in the Western Scheldt’s upper estuary. PCBs and PAHs were between 0.19–4.7 and 2.6–200μgkg−1dw respectively and generally had highest concentrations at near-shore locations and river mouths. Responses in the Microtox broad-spectrum and the Mutatox genotoxicity assays were generally low, with near-shore locations giving higher responses. The umu-C genotoxicity and the ER-CALUX assay for estrogenicity showed no response, with the exception of one near-shore location (IJmuiden outer harbour, ER-CALUX). Highest dioxin-like toxicity (DR-CALUX) was found at near-shore locations, in the outflow of the Rhine/Meuse estuary including a dumping site of harbour sludge. At the Oyster Grounds, DR-CALUX responses appeared to be linked to the occurrence of larger PAHs (4–6 rings). A new, non-destructive clean up procedure resulted in significantly higher DR-CALUX responses than the current protocol. The Dutch legislation on disposal of harbour sludge at sea, dictates the use of the conventional clean up procedure. Our results therefore indicate that probably more dioxin-like toxicity associated with harbour sludge is disposed off at sea than assumed. [Copyright &y& Elsevier]

Estrogenic and thyroid activities of 91 monohydroxylated PCBs were measured with two-hybrid assays using yeast cells containing the human estrogen receptor ERα or human thyroid receptor TRα. Estrogenic activity of 30 of the 91 compounds, including all compounds active in the yeast two-hybrid assay, were also measured by a reporter gene assay employing Chinese hamster ovary cells. The mammalian cell assay was more sensitive than the yeast assay but the rank order of estrogenicities of the compounds were in broad agreement for the two assays. Results for estrogenicity and thyroid activity were analyzed by inspection and those for estrogenicity by a theoretical treatment. Inspection indicated para-hydroxyl was more likely to be estrogenically active than meta-, which was more likely to be active than ortho-; one ortho-chlorine was important for activity but additional ortho-chlorines did not increase activity; and 2 lateral chlorines or 2,4,6-chloro- substitution of the non-phenol ring were favorable. In contrast, thyroid activity appeared not to depend strongly on the position of the hydroxyl group although ortho-hydroxyls occurred in the most active compounds. Activity was usually associated with at least one ortho-chlorine, with 2 chlorines in the phenolic ring and, importantly, two chlorines in the non-phenolic ring, and with 1 or 2 chlorines ortho to the hydroxyl group. Examination of the torsion angle between the rings, in the theoretical examination of estrogenicity, suggested that perpendicular orientation (i.e., rigidity) was not essential for activity. Intramolecular hydrogen bonding of the phenolic groups to adjacent chlorines or to the π-electron cloud of the non-phenol ring possibly decreased activity—the hydroxyl should be free of intramolecular interactions for maximum activity. It was difficult to predict the estrogenic activity of a congener from its obtained potential energy curve (PEC). In general, estrogenically active congeners had large values for the sum of the atomic charges on the carbon atoms of the hydroxylated ring, and on the oxygen atom. Hydroxyl substitution at the para-position allowed the compounds to become more polarizable in the x-axis (molecular axis), whereas OH substitution at the ortho-position made the congeners less polarizable in the same direction. However, no general statement about polarizability and estrogenic activity was possible. [ABSTRACT FROM PUBLISHER]

To evaluate the decomposition and formation characteristics of estrogenic compounds during ozonation, semibatch ozonation experiments were conducted using the prototypical estrogen, 17β-estradiol (E 2 ). Ozonation was shown to be very effective for decomposition of E 2 and decrease of estrogenicity. An E 2 equivalent concentration index was introduced based on the estrogenicity intensity measured with Estrogen Competition Binding Assay (ECB Assay). The importance of evaluation of estrogenicity by ECB Assay was shown by the difference in removal efficiencies between E 2 equivalent concentration and the E 2 itself. Through this research, ozonation was shown to be a promising method to remove E 2 without any formation of estrogenic by-products from secondary-treated sewage, which contained E 2 of ngL level and total organic carbon (TOC) of several mgL. [ABSTRACT FROM AUTHOR]

A group of heterocyclic aromatic compounds, dinaphthofurans (DNFs), recently have been identified as potentially significant contaminants in freshwater sediments. In the present study, a battery of in vitro assays was used for detection of toxic effects of DNFs that are potentially associated with endocrine disruption and tumor promotion. Dinaphthofurans were found to act as relatively potent inducers of aryl hydrocarbon receptor (AhR)--mediated activity in the chemical-activated luciferase reporter gene expression DR-CALUX assay. The relative AhR-inducing potencies of DNFs were similar or even higher than relative potencies of unsubstituted polycyclic aromatic hydrocarbons (PAHs), with dinaphtho[1,2-b;2'3'-d]furan being the most potent AhR agonist. Two compounds, dinaphtho[2,1-b;2'3'-d]furan and dinaphtho[1,2-b;1'2'-d]furan, induced estrogen receptor (ER)--mediated activity in the estrogen receptor--mediated CALUX (the ER-CALUX) assay. Two types of potential tumor-promoting effects of DNFs were investigated, using in vitro bioassays for detection of inhibition of gap-junctional intercellular communication and detection of a release from contact inhibition. Although the acute inhibition of gap-junctional intercellular communication was not observed, all six tested DNFs were able to release rat liver epithelial WB-F344 cells from contact inhibition at concentrations as low as 100 nM. In summary, the present study indicated that DNFs can exert multiple biological effects in vitro, including induction of the AhR-mediated activity, release of cells from contact inhibition, and induction of ER-mediated activity. [ABSTRACT FROM AUTHOR]

Recently several advisory committees (EDSTAC, ICCVAM) have recommended that stable estrogen-dependent gene expression assays be developed for screening chemicals for estrogenic activity because of the high degree of specificity of the response and potential for use in a high-throughput mode. In this paper we describe a specific, sensitive assay developed for screening chemicals for estrogenic and antiestrogenic activities. T47D human breast cancer cells, which naturally express estrogen receptor (ER) alpha and beta, were stably transfected with a triplet ERE (estrogen-responsive elements)–promoter–luciferase reporter gene construct. The transformed cells were named T47D-KBluc. These cells are sensitive to the potent estrogens, 17β-estradiol, ethynyl estradiol, and diethylstibesterol, and well-characterized weaker environmental estrogens like genistein, HPTE (an estrogenic pesticide metabolite), and 4-nonylphenol. The EC50 for estradiol was about 0.01 nM, reaching maximal induction at 0.1 nM. The antiestrogen, ICI 182,780, was able to completely inhibit the induction of luciferase expression by 0.1 nM estradiol at 10 nM, with an IC50 of 1 nM. In addition, we were able to replicate, in this in vitro assay, the observation that low concentrations of cadmium were able to induce estrogen-dependent gene expression, an effect that was completely inhibited by the potent antiestrogen ICI 182,780. The potent glucocorticoid receptor agonist, dexamethasone, was without effect as an ER agonist at concentrations up to 10 nM, whereas the potent androgen, dihydrotestosterone (DHT), showed no induction at concentration of 50 μM, but was a partial agonist at high concentrations of 0.2 mM and above. In summary, we have developed a specific, sensitive estrogen-responsive gene expression assay in a stable cell line that could possibly be adapted for high throughput screening of large numbers of chemicals for estrogenic and antiestrogenic activity. In addition, herein we also provide key protocol recommendations necessary to identify and eliminate common problems encountered in in vitro screening for estrogenicity. [ABSTRACT FROM AUTHOR]

There are 10 manufacturers who produce fossil fuel products in the Da-Hse Industrial District, Kaohsiung County, southwestern Taiwan. Before discharging the wastewater into the nearby aquatic environment, the pretreated wastewater from these manufacturers must be processed in a treatment plant which includes four major processing units: equalization, a primary clarifier, an aeration basin, and a final clarifier. In order to estimate the potential environmental risks of industrial wastewater from each manufacturer and the treatment efficiency of the powdered activated carbon treatment (PACT) system used in this wastewater treatment plant, in vitro bioassays for estrogenicity and oxidative hepatotoxicity were carried out using a stably transfected human breast cancer cell line, MVLN, and a human hepatocellular carcinoma cell line, HepG2, respectively. Estrogenic substances occurred in nine of 10 pretreated wastewaters from these manufacturers in which the relative luciferase activity ranged from 18.9% to 98.0% for 1-fold-condensed wastewaters corresponding to the concentration of the original wastewater. The estrogenicity was highest at the influent of the treatment plant and decreased through the treatment process. About 81% estrogenicity clearance was found through all processing units. On the other hand, oxidative hepatotoxic substances existed in seven of 10 pretreated wastewaters from these manufacturers in which relative TBARs activity ranged from 18.5% to 43.0% for 1-fold-condensed wastewater. The TBARs of influent samples apparently decreased through the processing units until the aeration basin, but abruptly rose in the final clarifier, which was a result of the addition of an active charcoal-retrieving agent with the molecular formula of (C2H3)nCONHCH2N(CH3)3Cl and which had high TBARs activity. No TBARs activity being found in effluent samples could be a consequence of allowing sufficient time for coagulation between the active charcoal and its retrieval agent which decreased the residual active charcoal-retrieving agent. We concluded that the industrial wastewater treatment plant using the PACT system in the Da-Hse Industrial District is suitable for removing estrogenic substances and oxidative hepatotoxic substances discharged from these industrial manufacturers. [Copyright &y& Elsevier]

We developed a DNA microarray to evaluate the estrogen activity of natural estrogens and industrial chemicals. Using MCF-7 cells, we conducted a comprehensive analysis of estrogen-responsive genes among approximately 20,000 human genes. On the basis of reproducible and reliable responses of the genes to estrogen, we selected 172 genes to be used for developing a customized DNA microarray. Using this DNA microarray, we examined estrogen activity among natural estrogens (17β-estradiol, estriol, estrone, genistein), industrial chemicals (diethylstilbestrol, bisphenol A, nonylphenol, methoxychlor), and dioxin. We obtained results identical to those for other bioassays that are used for detecting estrogen activity. On the basis of statistical correlations analysis, these bioassays have shown more sensitivity for dioxin and methoxychlor. [ABSTRACT FROM AUTHOR]

We have evaluated whether mixtures of estrogens, present in the mix at doses that are individually inactive in the immature rat uterotrophic assay, can give a uterotrophic response. Seven chemicals were evaluated: nonylphenol, bisphenol A (BPA), methoxychlor, genistein (GEN), estradiol, diethylstilbestrol, and ethinyl estradiol. Dose responses in the uterotrophic assay were constructed for each chemical. The first series of experiments involved evaluating binary mixtures of BPA and GEN at dose levels that gave moderate uterotrophic responses when tested individually. The mixtures generally showed an intermediate or reduced uterotrophic effect compared with when the components of the mixture were tested alone at the dose used in the mixture. The next series of experiments used a multicomponent (complex) mixture of all seven chemicals evaluated at doses that gave either weakly positive or inactive uterotrophic responses when tested individually in the assay. Doses that were nominally equi-uterotrophic ranged over approximately six orders of magnitude for the seven chemicals. Doses of agents that gave a weak uterotrophic response when tested individually gave a marginally enhanced positive response in the assay when tested combined as a mixture. Doses of agents that gave a negative uterotrophic response when tested individually gave a positive response when tested as a mixture. These data indicate that a variety of different estrogen receptor (ER) agonists, present individually at subeffective doses, can act simultaneously to evoke an ER-regulated response. However, translating these findings into the process of environmental hazard assessment will be difficult. The simple addition of the observed, or predicted, activities for the components of a mixture is confirmed here to be inappropriate and to overestimate the actual effect induced by the mixture. Equally, isobole analysis is only suitable for two- or three-component mixtures, and concentration addition requires access to dose-response data and EC50 values (concentration giving 50% of the maximum response) for the individual components of the mixture--requirements that will rarely be fulfilled for complex environmental samples. Given these uncertainties, we conclude that it may be most expedient to select and bio- assay whole environmental mixtures of potential concern. [ABSTRACT FROM AUTHOR]

Previously, we have reported that sealants incorporating bisphenol A dimethacrylate showed estrogenicity by a reporter gene assay. This study tested the hypothesis that commercial composites, which contain various monomers and additives, exhibit estrogenic activity in vitro. The estrogenic activities of eluates obtained from 24 composites and 18 chemicals identified from the composites tested were examined with the use of the reporter gene assay. Among the 24 composites, 6 products were estrogenic, and among the 18 constituents, 1 photostabilizer, 2-hydroxy-4-methoxy-benzophenone (HMBP), 1 photoinitiator, 2,2-dimethoxy-2-phenyl-acetophenone (DMPA), and 1 inhibitor, 2,6-di-tert-butyl-p-cresol (BHT) had significant estrogenic activity. The concentration of HMBP in 4 estrogenic eluates was greater than the minimum concentration required for estrogenicity, and DMPA was found at a higher level than the minimum estrogenic concentration in the remaining 2 estrogenic specimens. These results suggest that the observed estrogenic activity of 6 composites is associated with the elution of either HMBP or DMPA. [ABSTRACT FROM AUTHOR]

An integrated procedure using mass spectrometry and molecular biology for determination of estrogenicity in natural waters and sediments is reported. Solid-phase extraction (SPE) and pressurized-liquid extraction (PLE), respectively, were used for isolation of endocrine-disrupting compounds (EDC) from surface waters and sediments, followed by liquid chromatography–mass spectrometry using an electrospray interface (LC–ESI-MS). Twenty seven EDC were determined: non-ionic surfactants (nonylphenol ethoxylate), alkylphenols (e.g. nonylphenol and octylphenol), bisphenol A, phthalates, and natural and synthetic steroid sex hormones. Limits of detection varied from 0.02 to 0.22 μg L-1 and from 1 to 10 μg kg-1 in water and sediments, respectively. Recoveries ranged from 65 to 125% and 73 to 97% for waters and sediments, respectively. In addition to LC–ESI-MS determination, extracts obtained by SPE and PLE were analyzed by the recombinant yeast assay (RYA) to assess total estrogenic activity. This bioassay detects natural estrogens and xenoestrogens, producing a quantitative measurement of EDC irrespective of the identity of the chemical responsible for the activity. As a novelty, a relative estrogenicity factor was determined for 19 analytes with EC50 values ranging from 10-10 to 10-9 mol L-1 for synthetic estrogens, from 10-7 to 10-5 mol L-1 for alkylphenol derivatives, and from 10-5 to 10-4 mol L-1 for phthalates and benzothiazoles. By use of this integrated chemical–ecotoxicological approach good correlation was usually established between chemical composition and estrogenic effects for surface water and sediment samples from Portugal. Estrogenic activity observed was mainly attributed to the presence of nonylphenolic compounds (with concentrations of NP ranging from 0.1 up to 44 μg L-1 in waters and up to 1172 μg kg-1 in sediments), and to the sporadic presence of estrogens, detected at ng L-1 levels. [ABSTRACT FROM AUTHOR]

In the present study, the yeast estrogen screen (YES) was used to estimate the estrogenic potential of solid phase-extracted water samples from the effluents of two municipal sewage treatment plants (STPs 1 + 2) and from four lanes (left to right) of the river Rhine at Worms, Germany, i.e. downstream the STPs. Estrogenic activities of extracted water samples were expressed as 17β-estradiol equivalents (E2-EQs). Estrogenic activity was detected in the effluents of both STPs with values of 0.242±0.038 nm (65.96±10.4 ng/l) and 0.125±0.026 nM E2-EQs (34.1±7.18 ng/l) at STP 1 and 2, respectively. In river Rhine water, estrogenic activity was lower, however, displaying significant differences between the left and right bank of the river (0.044±0.003 nm E2-EQs [11.97±0.7 ng/l] for lanes 1–3; 0.071±0.01 nm E2-EQs [19.42±2.8 ng/l] for lane 4). Chemical analysis of corresponding water samples resulted in a potential estrogenic response in the YES, expressed as E2-EQs for the known estrogens and phytoestrogens in the STP effluents with values up to 0.0662 nm E2-EQs (18.04 ng/l). In Rhine water from lane 4, however, total estrogenic activity of steroidal estrogens was equal to 0.014 nM E2-EQs (3.8 ng/l). Furthermore, total concentrations of flavonoids, fecal- and phytosteroids and resorcyclic lactones were about 1.2 μg/l at STP 1, 0.62 μg/l at STP 2 and 0.25 μg/l at the river Rhine, lane 4. Results indicate that estrogenic activity can clearly be measured in SPT effluents as well as in river Rhine water using the YES in combination with chemical analysis. Results from the bioassay, however, indicated a higher estrogenic potential (expressed as E2-EQs) than that obtained by chemical analysis. [Copyright &y& Elsevier]

To evaluate possible estrogenic effects of bisphenol A (BPA) in an amphibian model, Xenopus laevis tadpoles were exposed to BPA and 17β-estradiol (E2) during larval development. After metamorphosis, the gonadal phenotype was determined by gross morphology, and testes were further examined histologically to validate the results. BPA treatment altered the normal sex ratio toward females depending on the BPA concentrations added. Chemical analysis showed a time-dependent decline of BPA during semistatic exposure, indicating that BPA is taken up and metabolized to some extent by tadpoles. In addition, tadpoles were exposed to BPA and E2 for 2 weeks during sensitive stages of sexual differentiation. Afterward, the expression of an estrogenic biomarker, estrogen receptor (ER) mRNA, was assessed by semiquantitative RT-PCR. Both BPA and E2 up-regulated ER mRNA significantly. In conclusion, these results show clear evidence that BPA induces feminization in X. laevis tadpoles, revealing an estrogenic potency of BPA that influences sexual development in amphibians. [Copyright &y& Elsevier]

We have employed an estrogen receptor dependent transcriptional expression assay and E-Screen assay systems to evaluate the estrogenicity of various heavy metals and their species. Using the former, the following estrogenicity ranking was measured: bis(tri-n-butyltin)>cadmium chloride>antimony chloride>barium chloride=chromium chloride>lithium hydroxide>sodium selenate=lead acetate>stannous chloride. Using the latter, the following estrogenicity ranking was measured: bis(tri-n-butyltin)>cadmium chloride>antimony chloride>lithium hydroxide>barium chloride>sodium selenate>chromium chloride. Especially, bis(tri-n-butyltin), cadmium chloride, antimony chloride, lithium hydroxide, barium chloride, and chromium chloride showed estrogenicity in both assay systems. Recent studies suggesting that bis(tri-n-butyltin), cadmium chloride, and lithium hydroxide have estrogenicities are compatible with the present findings. Furthermore, our studies are the first to suggest that antimony, barium, chromium may be estrogenic. A range of estrogenicity was observed for different species of the same heavy metal. The results demonstrate that an estrogen receptor dependent transcriptional expression assay and the E-Screen assay systems could serve as a useful method to assess the estrogenicity of heavy metals. [Copyright &y& Elsevier]

In pursuit of novel steroid sulfatase (STS) inhibitors devoid of estrogenicity, several D-ring modified steroid derivatives were synthesised. In vitro evaluation of the compounds identified two highly potent inhibitors, 4a and 4b, which were 18 times more active than estrone-3-O-sulfamate (EMATE), both having IC50 values of ca. 1 nM. These 16,17-seco-estra-1,3,5(10)-triene-16,17-imide derivatives were synthesised from estrone, via the intermediate 1, which was easily alkylated, deprotected and sulfamoylated affording the final compounds in high yields. In order to assess their biological profile, the selected inhibitors were tested for their in vivo inhibitory potency and estrogenicity in ovariectomised rats. After an oral dose of 10 mg/kg per day for 5 days, 4a and 4b were found to inhibit rat liver steroid sulfatase by 99%. They were also devoid of estrogenic activity in the uterine weight gain assay, indicating that these two leads have therapeutic potential for the treatment of hormone-dependent breast cancer. [Copyright &y& Elsevier]

It has been suggested that the estrogenicity of PAHs could contribute to their carcinogenic effects via increased tissue-specific cell proliferation. Both benzo[a]pyrene (BaP) and benz[a]anthracene (BaA) are known to weakly activate estrogen receptor (ER)-dependent reporter constructs. In this study, several other PAHs, including fluorene, fluoranthene, pyrene, chrysene, phenanthrene and anthracene, were found to act as very weak inducers of ER-mediated activity in the MCF-7 cell line stably transfected with a luciferase reporter gene. The effects of PAHs were time-dependent and they were not completely inhibited by antiestrogen ICI 182,780. In addition, BaP and BaA, as well as weakly estrogenic fluoranthene, significantly potentiated the maximum ER-mediated activity of 17β-estradiol. Therefore, the effects of inhibitors of several types of protein kinases known to activate ERα in a ligand-independent manner were investigated. However, neither inhibitors nor inducers of extracellular signal-regulated kinases 1 and 2 (ERK1/2), phosphatidylinositol-3 kinase, protein kinase C, c-Src, or protein kinase A modified ER-mediated activity in this model. Neither estradiol nor BaA activated ERK1/2, two kinases suggested to play significant roles in ER signaling, suggesting that another kinase is involved in the observed phosphorylation of ERα. Similar to 17β-estradiol, BaA stimulated G0/G1–S-phase transition in MCF-7 cells, which was fully suppressed by ICI 182,780. In conclusion, some PAHs can potentiate 17β-estradiol-induced ER activation and stimulate cell cycle entry in vitro. However, their exact mode(s) of action and whether this phenomenon is of in vivo relevance remains to be elucidated. [ABSTRACT FROM PUBLISHER]

The application of organic, conventional and biotechnology techniques can alter the intrinsic levels of natural toxicants in crop foods and methods are needed to screen for unexpected changes in toxicant levels. We evaluated crude, aqueous preparations of 37 foods purchased from a local market in a battery of four in vitro mammalian toxicity screens. The foods were evaluated in one or more of the following tests: (1) cytotoxicity (37 foods) and (2) chromosomal aberration test (nine foods), both in Chinese hamster ovary cells, (3) limb bud micromass assay (nine foods) using 11-day old CD-1 mouse embryos and (4) estrogenicity (MCF-7 cells transfected with estrogen receptor and lucerifase reporter constructs, 12 foods). IC50s for cellular proliferation ranged from <1% (v/v, garlic) to >10% (v/v, 18 foods), the maximal concentration tested. Five of nine preparations (soybeans, broccoli, garlic, snow peas and corn) were clastogenic and two (soybeans and snow peas) inhibited chrondrogenesis in the limb bud micromass assay. Five of nine preparations (soybeans, snow peas, cumin, asparagus and bean sprouts) produced significant estrogenic responses. Overall, the 12 foods evaluated in two or more of the tests showed different patterns of response. These preliminary data indicate that screening for potential toxicants is possible with fast, relatively inexpensive in vitro tests. These in vitro tests, while potentially useful to detect unexpected toxicants in plants that may signal the need for further evaluation, are not directly useful to predict human or animal risk from eating these plants. [Copyright &y& Elsevier]

Propolis obtained from honeybee hives has been used in Oriental folk medicine as an anti-inflammatory, anti-carcinogenic, or immunomodulatory agent. The potential estrogenic activity of propolis was investigated in vitro using the MCF-7 human breast cancer cell proliferation, human estrogen receptor (hER) binding and yeast-based steroid receptor transcription, and in vivo using the immature rat uterotrophic effect. Treatments with ethanol extract of propolis (EEP) and ether extract of propolis (REP) enhanced MCF-7 cell proliferation in concentrations ranging from 0.8 to 4 μg/ml. Both EEP and REP competed for binding of [3H]17β-estradiol to the hER with IC50 values of 9.14 and 9.72 μg/ml, respectively. In yeast estrogen receptor transcription assay, both EEP and REP were found to be estrogenic with EC50 values of 9.48, and 8.55 μg/ml, respectively. Animals treated with EEP or REP for 4 days (500–1000 mg/kg per day, s.c.) exhibited significant dose-dependent increases in uterine wet weight. However, in the yeast androgen and progesterone receptor transcription assays, either EEP or REP was found not to be active. The results suggest that propolis produces estrogenic effects through activation of estrogen receptors. [Copyright &y& Elsevier]

In order to assess in fish the maternal transfer of alkylphenolic compounds to the progeny, the identification and quantification of the labelled compounds present in oocytes and embryos was conducted after dietary exposure of mature female mosquitofish to 14C-4n-nonylphenol during vitellogenesis and embryogenesis respectively. Radioactivity found in bile and liver extracts accounted for 0.9–0.6 and 0.2–0.1% of ingested radioactivity for females exposed during vitellogenesis and embryogenesis respectively. The amount of extractable radioactivity present in oocytes and embryos was 0.19 and 0.07% of the ingested dose respectively. The radio-HPLC profiles obtained from bile, liver, oocyte and embryo extracts were similar. They showed the presence of 4n-NP-glucuronide as the major metabolite and traces of unchanged 4n-NP. The other metabolites corresponded to 8-hydroxynonylphenol, 9-(4-hydroxyphenyl)-nonanoic acid and para-hydroxybenzoic acid which is the final product of the alkyl chain oxidation. Our results indicate that exposure of ovoviviparous female fish to 4-NP during vitellogenesis and embryogenesis leads to the contamination of the progeny by 4-NP and its metabolites. [Copyright &y& Elsevier]

Background. Recent studies have indicated that the risk of thromboembolic disease (VTE) in users of combined oral contraceptive pills (COCs) varies not only with estrogen dose, but also with the progestogen in pills with the same estrogen dose. The aim of this article is to discuss sex hormone binding globulin (SHBG) as a marker of estrogenicity and as a surrogate indicator for the potential risk of VTE in users of COC. Material and methods. Using data from the literature, we investigated the relationship between the risk of VTE with various COCs and their effects on SHBG. We also collected data on the effects on SHBG by some combined preparations, where there are no VTE data. Results. There appears to be a relationship between the risk of VTE and the effect on SHBG. Monophasic preparations containing levonorgestrel, having the lowest risk of VTE, cause an average SHBG increase of around 50%. COCs containing desogestrel or gestodene cause an average SHBG increase of 200–300%. A preparation with cyproterone acetate, carrying a higher risk of VTE than desogestrel and gestodene, cause a 300–400% SHBG increase. With the recently developed combined preparations, there is a 150% SHBG increase with norgestimate and a 250–300% increase with drosperinone and dienogest. Conclusions. We propose that the change in SHBG with a COC could be interpreted as a measure of total estrogenicity and used as a predictor of the risk of VTE. Preparations containing drosperinone, dienogest, cyproterone acetate and norgestimate are equally or more estrogenic than the more thoroughly studied COCs, containing desogestrel or gestodene and should not be considered a safer substitute. [ABSTRACT FROM AUTHOR]

In the past decade the list of chemicals in the environment that are able to mimic the natural hormone estrogen, thereby disrupting endocrine function, has grown rapidly. These chemicals are able to bind to estrogen receptors (ERs) and influence estrogen signalling pathways, although several of them have structures that differ substantially from the endogenous hormone 17β-estradiol. In this study, six extensively used ultraviolet (UV) filters were assessed for transcriptional activation of estrogen receptors. Because of their high lipophilicity, these UV filters tend to bioaccumulate in the environment. They have been found in surface waters, fish, and in human milk fat. Using a sensitive in vitro reporter gene assay, we found that all six compounds induce estrogenic activity towards ERα, while four out of six compounds induced transcriptional activity of ERβ. Zebrafish, in which an estrogen responsive luciferase reporter gene has been stably introduced, were used for in vivo testing. In this transgenic zebrafish assay none of the compounds showed estrogenic activity. Our findings suggest that one should be aware of over-interpretation when predicting in vivo effects from weak in vitro data. However, it can not be ruled out that these UV filters have long-term effects in the environment. [ABSTRACT FROM AUTHOR]

Groups of five male and female Wistar rats were treated by gavage with 0, 0.01, 0.05 or 0.2 mg/kg body weight of the known synthetic estrogen ethinylestradiol for 28–32 days according to a modified enhanced OECD Test Guideline no. 407 in order to investigate which of the current and/or additional parameters would detect effects on the endocrine system reliably and sensitively and to provide data on intra-laboratory variability. Two identical studies (A and B) were run concurrently. The modified enhanced protocol requests the additional determination of triiodothyronine, thyroxine and thyroid-stimulating hormone (TSH), of the stage of the estrous cycle to ensure necropsy of all females in diestrus, of the number and morphology of cauda epididymal spermatozoa, and of additional organ weights (ovaries, uterus, thyroid, and male accessory reproductive organs), and histopathology of additional organs (pituitary, epididymides, coagulation glands, pancreas, and vagina). There were no treatment-related mortalities, clinical signs or changes in behavioral parameters. In male rats, 0.2 mg/kg was the maximum tolerated dose (MTD) resulting in reduced body weight gain. The only treatment-related alteration in hematological parameter was prolonged blood clotting time in high-dose females of both studies. Changes in clinical chemistry observed in study A were elevated alkaline phosphatase activity (high-dose females) and triglyceride levels (mid- and high-dose females and high-dose males). Changes in thyroid hormones and TSH of treated animals showed high variability with no clear dose-dependency, and could not be clearly related to estrogenic activity. In accordance to a suppression of the hypothalamic-pituitary-gonadal axis, decreased relative organ weights of the male accessory reproductive organs were obtained in both studies at the high dose. Corresponding histological changes were degeneration of the testicular germinal epithelium and atrophy of Leydig cells and of all accessory sex glands. Atrophy of the coagulating gland (study A) and seminal vesicles (study B) was also seen at 0.05 mg/kg. A marked increase in relative adrenal weight in male rats, accompanied by decreased vacuolization of zona fasciculata cells observed in both studies at the high dose seems to reflect an activation of the hypothalamic-pituitary-adrenal axis. The male mammary gland was sensitively affected. Increased numbers of small basophilic over large acidophilic cells indicated an estrogen-mediated feminisation and were detected at the low (study A) or mid dose (study B). Co-mitogenic properties of estrogens in rat liver were reflected by increased relative liver weights in females at the mid and high dose of study A and also at the high dose in study B. No treatment-related changes in endocrine organ weights were observed in treated females. Histological changes in the ovaries were increased numbers of apoptotic corpora lutea (from mid dose, study B) and of early stage follicles at the high dose in both studies. Classical direct estrogenic effects on the uterus, i.e. an increased height of luminal and glandular epithelium and increased granulocytic infiltration of the endometrium, were observed even at the low dose in both studies. Uterine findings occurring with a greater variability were dilation, squamous metaplasia of glands and thickened walls. Although females were necropsied in diestrus, as diagnosed by vaginal cytology, typical signs of estrogenic action in the vagina such as keratinization (indicative of estrus in normally cycling rats), mucification (indicative of proestrus), or thickened epithelia were observed in both studies even at the lowest dose. This unexpected discrepancy between vaginal cytology and vaginal and uterine morphology of treated females was considered to be treatment-related as it was not observed in the controls. Studies on liver enzymes that were performed outside the scope of the enhanced protocol showed that ethinylestradiol at 0.2 mg/kg decreased the activity of the sex-specific testosterone-dependent liver enzyme CYP2C11 in male rats. A simulation of doubling group size (to ten animals) by combining both studies did not increase the sensitivity of detection of endocrine-mediated effects above the level already obtained by histopathological examination of groups containing five animals. Only some of the enhancements to the current OECD Test Guideline no. 407 evaluated in this study (additional organs weights and additional histopathological investigations) were helpful in detecting the endocrine-mediated effects of ethinylestradiol, while other enhancements did not contribute towards this aim. Spermatology was completely insensitive at the MTD and measurement of thyroid hormones and TSH did not contribute to increased sensitivity. Vaginal cytology appeared to be an unreliable procedure for estrous cycle staging in estrogen-treated animals. Ongoing investigations, according to the modified version of the enhanced OECD Test Guideline no. 407 protocol, into the interference of ten compounds with the endocrine system by different mechanisms will result in the identification of the most appropriate enhancements. [ABSTRACT FROM AUTHOR]

A study of the effects of dietary genistein on trout and sturgeon in vivo showed that sturgeon was sensitive to 20 ppm of genistein, whereas trout was not. To analyze the origin of this interspecies difference in sensitivity, a cell culture technique was developed with hepatocytes from sturgeon and compared to results obtained with hepatocytes from trout in the same system. The hepatocyte culture proved to be useful as bioassay for estrogenicity. Vitellogenin (VTG), assayed by a specific enzyme-linked immunosorbent assay, was used as a biomarker of the estrogenic activity. 17β-Estradiol, its glucuronide and sulfate derivatives, and estradiol analogues (ethynylestradiol and diethylstilbestrol) were tested. Nonestrogenic compounds such as androgens, progesterone, and cortisol were tested as negative controls. VTG production was monitored at doses ranging from 1 nM to 10 μM estradiol. Phytoestrogens, from the isoflavone family, were tested individually at increasing doses exhibiting dose response curves for concentrations from 500 nM to 10 μM. With tamoxifen, an antagonist of estrogen receptors, the estrogenic effect was partially reduced. The effect was the same with ICI182,780 in sturgeon, whereas the effect was the opposite in trout. The estrogenic potency of the isoflavones ranged differently between the two species in the following order: biochanin A < daidzein = formononetin < genistein < equol in trout and biochanin A < genistein < daidzein < formononetin < equol in sturgeon. Further, in sturgeon, formononetin was the most potent phytoestrogen in vitro, whereas its activity was weakest in vivo. These data suggest that one must reconsider the relevance of heterologous estrogenic tests and of homologous in vitro tests for estrogenic potency of chemicals. [Copyright &y& Elsevier]

Reproductive dysfunction in wildlife populations can be a result of environmental contaminants binding to aryl hydrocarbon receptor (AhR) or estrogenic receptors. Signaling by both types of receptors can be affected by polycyclic aromatic hydrocarbons (PAHs), which are potential endocrine disruptors. However, our knowledge regarding the effects of oxygenated (oxy)-PAHs and azaarenes on AhR-mediated and estrogenic activities is incomplete. In the present study, we have identified 9-fluorenone, anthrone, anthraquinone, benzanthrone, benz[a]anthracene-7,12-dione, benz[c]acridine, and dibenz[a,h]acridine as prevalent oxy-PAHs and azaarenes found in river sediments. Their concentrations in sediment samples ranged from 2.1 to 165.2 ng g-1 for oxy-PAHs and up to 27.3 ng g-1 for azaarenes. Their relative AhR-inducing and estrogenic potencies were quantified in vitro using two cell lines that were stably transfected with a luciferase reporter gene system and expressed as induction equivalency factors (IEFs). The only oxy-PAHs with detectable levels of in vitro AhR-mediated activity were benzanthrone and benz[a]anthracene-7,12-dione. However, their IEFs were approximately three to four orders of magnitude lower than those of benzo[a]pyrene. On the other hand, azaarenes showed a strong AhR-mediated activity, with dibenzo[a,h]acridine being a far more potent inducer of activity than benzo[a]pyrene. Benzanthrone, benz[a]anthracene-7,12-dione, anthraquinone, and benz[a]acridine were weak inducers of in vitro estrogenic activity, with IEFs similar to that of benzo[a]pyrene. Based on concentrations and relative potencies, our results suggest that dibenzo[a,h]acridine can significantly contribute to the overall AhR-mediated activity in river sediments, whereas the remaining compounds do not. No studied compound was found to contribute significantly to estrogen receptor-mediated activity in vitro. [ABSTRACT FROM AUTHOR]

It is controversial whether the dental resinous materials containing 2,2-bis[4-(2-hydroxy-3- methacryloyloxypropoxy)phenyl]propane (Bis- GMA), which is synthesized from the estrogenic compound bisphenol A (BPA), include unreacted BPA and/or can mimic the effects of natural steroid hormones. In the present study, the estrogenic activities of 3 fissure sealants and 5 adhesive resins, which were all unpolymerized, were determined by means of a reporter gene assay, and the relevance of the components to the estrogenicity was investigated. Two commercially available sealants were confirmed to have estrogenic activity, although none of the tested materials contained BPA. In contrast, hydrophobic monomer bisphenol A dimethacrylate (BPA-DMA), which is also estrogenic, was found to be included in these estrogenic sealants in an amount greater than the minimum concentration to show estrogenicity. This suggests that the estrogenicity of the two proprietary sealants was associated with BPA-DMA rather than with BPA. [ABSTRACT FROM AUTHOR]

Soy as several other vegetables is rich in phytoestrogens and we demonstrated that these compounds were both estrogenic and endocrine disrupters, in sturgeon and trout, in vivo and in vitro. In our laboratory, we developed several in vitro tests in these two species to screen the activity of estrogenic compounds. This paper reports hepatocytes, which synthesise VTG in response to estrogenic stimulation, and in vitro binding tests on hepatic estrogen receptors. These tests showed different reactivities in the two species suggesting a difference of sensitivity, which is also observed in vivo. Finally, comparing the two species the in vitro affinity of phytoestrogens for estrogen receptors did not reflect the VTG synthesis in hepatocyte cultures. All these results, together with the tyrosine kinase inhibitory effect of certain phytoestrogens demonstrated by other authors, suggest that the stimulation of the VTG gene transcription may not be explained exclusively by a simple ligand-receptor-DNA interaction. [ABSTRACT FROM AUTHOR]

In the present study, a battery of biological tests undertaken in vitro and in vivo was used to evaluate the toxic potential of an organophosphorus insecticide, namely Fenitrothion. The cytotoxic effect of pesticide was evaluated with the MTT assay against two human cancer cell lines: Hep-2 and MDA-MB-231. Genotoxicity was also studied using the bacterial VITOTOX® assay. The estrogenic effect was tested using the recombinant yeasts (YES) assay. Likewise, bioluminescence assays using V. fischeri and D. magna immobilization were performed. The results showed that Fenitrothion exhibits a variable cytotoxic effect depending on the dose as well as the studied cell lines, and no genotoxicity was observed in the tested sample. However, an estrogenic effect was recorded when investigating Fenitrothion using the recombinant yeasts (YES) assay. Analogously, acute toxicity was observed for both organisms and at all tested concentrations of Fenitrothion. Overall, these results underline the crucial importance of in vitro and in vivo bioassays in monitoring toxicity of pesticides. [ABSTRACT FROM AUTHOR]

Zearalenone and its cis -isomer, cis -zearalenone, are nonsteroidal mycotoxins that elicit an estrogenic response upon binding to the estrogen receptor. This study compares the estrogenicity of eleven congeners including novel metabolites as 15-OH-zearalenone, zearalenone-14-sulfate, α- cis -zearalenol and β- cis -zearalenol using the E-Screen assay. Overall, a change in the configuration from trans to cis retains significant estrogenic activity. In contrast, alterations of the aromatic moiety including hydroxylation and sulfation showed a markedly decreased estrogenicity when compared to zearalenone. [ABSTRACT FROM AUTHOR]

Mycotoxins are toxic metabolites of filamentous fungi. Previous studies demonstrated the co-occurrence of Fusarium and Alternaria toxins, including zearalenone (ZEN), ZEN metabolites, and alternariol (AOH). These xenoestrogenic mycotoxins appear in soy-based meals and dietary supplements, resulting in the co-exposure to ZEN and AOH with the phytoestrogen genistein (GEN). In this study, the cytotoxic and estrogenic effects of ZEN, reduced ZEN metabolites, AOH, and GEN are examined to evaluate their individual and combined impacts. Our results demonstrate that reduced ZEN metabolites, AOH, and GEN can aggravate ZEN-induced toxicity; in addition, the compounds tested exerted mostly synergism or additive combined effects regarding cytotoxicity and/or estrogenicity. Therefore, these observations underline the importance and the considerable risk of mycotoxin co-exposure and the combined effects of mycoestrogens with phytoestrogens. [ABSTRACT FROM AUTHOR]

A number of chemicals released into the environment have the potential to interfere with physiological and developmental processes by disrupting endocrine pathways. Among the best known of these endocrine disruptors are compounds that mimic the action of the steroid hormone 17β-estradiol. These xenobiotic estrogens are believed to pose health risks to both humans and wildlife. Our laboratories are designing in vivo bioassays for xenobiotic estrogens based on induction of the egg-yolk precursor protein vitellogenin. Vitellogenin is normally produced by the liver of adult female nonmammalian vertebrates under estrogen stimulation. In immature or male animals, which have low levels of endogenous estrogens, vitellogenin can serve as a reliable biomarker for exposure to xenobiotic estrogens. Our model system used the African clawed frog, Xenopus laevis, an ideal species for laboratory screening of endocrine disruptors. Xenopus laevis vitellogenin was purified by diethylaminoethyl (DEAE) chromatography and used to generate polyclonal antibodies in rabbits. The resulting antiserum was used to develop an enzyme-linked immunosorbent assay (ELISA) for measurement of serum vitellogenin. Frogs were exposed to compounds by immersion in order to mimic environmental exposure to aquatic contaminants. Initially, frogs were immersed in the potent estrogenic agent diethylstilbestrol (DES) at a concentration of 1 ppm for 11 d to test the efficacy of the immersion protocol. Diethylstilbestrol exposed animals showed substantial induction of serum vitellogenin, indicating that the frogs are capable of responding to estrogenic agents present in their aquatic environment. Vitellogenin induction was then investigated for chlordane, dieldrin, endosulfan, and toxaphene, compounds that have been shown through in vitro assays to be weakly estrogenic when administered individually but more strongly estrogenic in combination. Adult male frogs were immersed in water containing the compoun... [ABSTRACT FROM AUTHOR]

Dienogest (DIE) and drospirenone (DRO) are two fourth-generation synthetic progestins widely used as oral contraceptives. Despite their increasing detection in wastewaters and surface waters, their fate during biological wastewater treatment is unclear. Here, we investigated DIE and DRO biotransformation with representative activated sludge batch incubations and identified relevant transformation products (TPs) using high-resolution mass spectrometry. DIE exhibited slow biotransformation (16-30 h half-life) and proceeded through a quantitative aromatic dehydrogenation to form TP 309 (molar yields of ∼55%), an aromatic TP ∼30% estrogenic as 17β-estradiol. DRO experienced more rapid biotransformation (<0.5 h half-life), and 1,2-dehydrogenation formed the major TP 364 (molar yields of ∼40%), an antimineralocorticoid drug candidate named as spirorenone. Lactone ring hydrolysis was another important biotransformation pathway for DRO (molar yields of ∼20%) and generated pharmacologically inactive TP 384. Other minor pathways for DIE and DRO included hydroxylation, methoxylation, and 3-keto and C4(5) double-bond hydrogenation; distinct bioactivities are plausible for such TPs, including antigestagenic activity, antigonadotropic activity, and pregnancy inhibition effects. Thus, biotransformation products of DIE and DRO during wastewater treatment should be considered in environmental assessments of synthetic progestins, especially certain TPs such as the estrogenic TP 309 of DIE and the antimineralocorticoid spirorenone (TP 364) of DRO.

Major rivers in India are subject to ongoing impacts from urban drain discharges, most of which contain high levels of domestic and industrial wastewater and stormwater. The aim of the present study was to determine the levels of bioactive organic micropollutants at the discharge points of major urban drains in comparison to upstream and downstream sites. To achieve this, we employed a panel of in vitro bioanalytical tools to quantify estrogenic, androgenic, progestogenic, glucocorticoid and peroxisome proliferator-like activity in water extracts collected from two Indian cities in the Ganga Basin. Cytotoxicity of the water extracts in a human-derived cell line and the potential to cause oxidative stress in a fish cell line were also investigated. We found high levels of activity for all endpoints in samples directly receiving urban drain discharge and low levels at sites upstream from drain discharges. Estrogenicity was detected at levels equivalent to 10 ng/L 17β-estradiol, representing a high likelihood of biomarker effects in fish. Sites located downstream from drain discharges exhibited low to intermediate activity in all assays. This study demonstrates the importance of managing urban drain discharges and the utility of applying bioanalytical tools to assess water quality. ga1 ● Water extracts from Ganga River were analysed using a suite of bioanalytical tools. ● Urban drain discharge samples were collected pre- and post-monsoon season. ● Sewage was the dominant source of organic micropollutants in urban drain effluents. ● Estrogenicity was detected at levels that could cause reproductive effects in fish. ● Effect-based trigger values help prioritise endocrine activity in the Ganga River. [ABSTRACT FROM AUTHOR]

The Advanced Oxidation System (AOS) is a novel electrochemical advanced oxidation process that effectively removes bacterial and organic contaminants from wastewater. However, potential formation of secondary oxidative species may pose additional hazards to aquatic organisms living in the receiving water affected by the post-treatment effluent. The effect of exposure to AOS treated water, especially the potential long-term effects on aquatic organisms, requires further investigation to demonstrate both efficacy and safety of this process. To examine the potential adverse effects of AOS treated water, three aquatic species, including daphnia, zebrafish, and rainbow trout, were exposed to treated and untreated municipal wastewater effluent (MWE) spiked with one of two model organic contaminants, benzo[a]pyrene (BaP) and 17β-estradiol (E2). The results indicated AOS treatment significantly reduced the adverse effects caused by exposure to MWE and model organic contaminants to baseline levels in daphnia (reduced fecundity), zebrafish embryo (elevated EROD activity), and rainbow trout (elevated plasma vitellogenin). The Ames test was also conducted to confirm the removal efficacy of carcinogenicity of BaP spiked in MWE. Overall, this study demonstrated that AOS treatment is a promising and environmentally friendly technology for wastewater treatment, remediation, and management. Image 1 • AOS treatment greatly attenuates the bioactivity of benzo[a]pyrene. • AOS treatment removes bioactivity of 17β-estradiol. • No additional hazards in the post-treatment effluent were detected. • AOS treatment is a promising and environmentally friendly method for wastewater remediation. [ABSTRACT FROM AUTHOR]

Ginger (Zingiber officinale Roscoe) has been used as a food, spice, supplement and flavoring agent and in traditional medicines due to its beneficial characteristics such as pungency, aroma, nutrients and pharmacological activity. Ginger and ginger extracts were reported to have numerous effects, such as those on diabetes and metabolic syndrome, cholesterol levels and lipid metabolism, and inflammation, revealed by epidemiological studies. To understand the beneficial characteristics of ginger, especially its physiological and pharmacological activities at the molecular level, the biological effects of ginger constituents, such as monoterpenes (cineole, citral, limonene and α/β-pinenes), sesquiterpenes (β-elemene, farnesene and zerumbone), phenolics (gingerols, [6]-shogaol, [6]-paradol and zingerone) and diarylheptanoids (curcumin), and the associated signaling pathways are summarized. Ginger constituents are involved in biological activities, such as apoptosis, cell cycle/DNA damage, chromatin/epigenetic regulation, cytoskeletal regulation and adhesion, immunology and inflammation, and neuroscience, and exert their effects through specific signaling pathways associated with cell functions/mechanisms such as autophagy, cellular metabolism, mitogen-activated protein kinase and other signaling, and development/differentiation. Estrogens, such as phytoestrogens, are one of the most important bioactive materials in nature, and the molecular mechanisms of estrogen actions and the assays to detect them have been discussed. The molecular mechanisms of estrogen actions induced by ginger constituents and related applications, such as the chemoprevention of cancers, and the improvement of menopausal syndromes, osteoporosis, endometriosis, prostatic hyperplasia, polycystic ovary syndrome and Alzheimer's disease, were summarized by a comprehensive search of references to understand more about their health benefits and associated health risks. [ABSTRACT FROM AUTHOR]

• g-C 3 N 4 as visible photocatalyst promote degradation of 17α-ethinylestradiol (EE2). • EE2 (3 mg/L) degradation in real hospital wastewater was also achieved. • Proposed mechanism for the degradation of EE2 by g-C 3 N 4 under visible-light irradiation. • Estrogenicity removal was confirmed by the YES assay (<45 min, 3 mg/L). • Regeneration affirm (<10 %) loss of degradation efficiency after 5 cycle. Metal-free, chemically activated crystalline graphitic carbon nitride (g-C 3 N 4) nanorods with enhanced visible-light photoactivity demonstrated rapid photodegradation of 17α-ethinylestradiol (EE2) in water and real hospital wastewater. Pure g-C 3 N 4 and another three crystalline promoted g-C 3 N 4 photocatalysts developed by hydrothermal method were characterized by, High-Resolution Transmission Electron Microscopy (HRTEM), X-ray diffraction (XRD), Fourier-Transform Infrared spectroscopy (FTIR), Brunauer–Emmett–Teller (BET), Photoluminescence (PL), Electron spin resonance (ESR), X-ray Photoelectron Spectroscopy (XPS) and Diffuse Reflectance Spectroscopy (DRS). Hydrothermal-based chemical activation did not alter the crystal structure, functional group or surface morphology, but it enhanced the specific surface area of activated g-C 3 N 4 due to intralayer delamination and depolymerization of g-C 3 N 4. Compared to pure g-C 3 N 4 , the activated g-C 3 N 4 -3 demonstrated efficient degradation of EE2 (<30 min, 3 mg/l) by visible wavelengths of the solar spectrum. This work provides advanced insight into the construction of heterojunction visible-light photocatalysts and production of O 2 − via reduction of O 2 with photogenerated electrons. Proposed and derived mechanism for photodegradation of EE2 by g-C 3 N 4 -3 using gas chromatography-mass spectrometry (GC MS). Yeast Estrogen Screen (YES) was performed to evaluate the estrogenicity of treated water samples. Efficient removal of EE2 estrogenic activity (<45 min, 3 mg/l) was achieved using the visible light-activated g-C 3 N 4. Estrogenicity removal rate corresponded well with EE2-degradation rate. [ABSTRACT FROM AUTHOR]

Zearalenone (ZEA) is produced in cereals by different species of Fusarium, being a non-steroidal estrogenic mycotoxin. Despite having a low acute toxicity, ZEA strongly interferes with estrogen receptors. Gamma-radiation has been investigated to eliminate mycotoxins from food and feed, showing promising results. The present study aims to investigate the gamma-radiation effect on ZEA at different moisture conditions and to evaluate the cytotoxicity and estrogenicity of the irradiated ZEA. Different concentrations of dehydrated ZEA and aqueous solutions of ZEA were exposed to gamma-radiation doses ranging from 0.4 to 8.6 kGy and the mycotoxin concentration determined after exposure by high performance liquid chromatography (HPLC) with fluorescence detection. Following this, the cytotoxicity of irradiated samples was assessed in HepG2 cells, by measuring alterations of metabolic activity, plasma membrane integrity and lysosomal function, and their estrogenicity by measuring luciferase activity in HeLa 9903 cells. Gamma-radiation was found to be effective in reducing ZEA, with significant increases in degradation with increased moisture content. Furthermore, a reduction of cytotoxicity with irradiation was observed. ZEA estrogenicity was also increasingly reduced with increasing radiation doses, but mainly in aqueous solutions. These results suggest reduction of ZEA levels and of its toxicity in food and feed commodities may be achieved by irradiation. [ABSTRACT FROM AUTHOR]

Contaminants of Emerging Concern (CECs) with estrogenic or estrogenic-like activity have been increasingly detected in aquatic environments and have been an issue of global concern due to their potential negative effects on wildlife and human health. This study used the MCF-7 cell proliferation assay (E-Screen) to assess the estrogenic activity profiles in Maryland Coastal Bays (MCBs), a eutrophic system of estuaries impacted by human activities. Estrogenic activity was observed in all study sites tested. Water samples from MCBs increased MCF-7 cell proliferation above the negative control from 2.1-fold at site 8, located in Sinepuxent Bay close to the Ocean City Inlet, to 6.3-fold at site 6, located in Newport Bay. The proliferative effects of the sediment samples over the negative control ranged from 1.9-fold at the Assateague Island National Seashore site to 7.7-fold at the Public Landing site. Moreover, elevated cell proliferation ( p < 0.05) was observed when cells were co-exposed with 17ß-Estradiol (E2), while reduction in cell proliferation was observed when cells were co-exposed with the antagonist ICI 182, 780 suggesting that cell proliferative effects were primarily mediated by the estrogen receptor (ER). These results suggest the occurrence of some estrogenic or hormonal-like compounds in the MCBs and are consistent with our previous findings based on vitellogenin analyses.