The 3,4-Quinones of Estrone and Estradiol Are the Initiators of Cancer whereas Resveratrol and N-acetylcysteine Are the Preventers.

This article reviews evidence suggesting that a common mechanism of initiation leads to the development of many prevalent types of cancer. Endogenous estrogens, in the form of catechol estrogen-3,4-quinones, play a central role in this pathway of cancer initiation. The catechol estrogen-3,4-quinones react with specific purine bases in DNA to form depurinating estrogen-DNA adducts that generate apurinic sites. The apurinic sites can then lead to cancer-causing mutations. The process of cancer initiation has been demonstrated using results from test tube reactions, cultured mammalian cells, and human subjects. Increased amounts of estrogen-DNA adducts are found not only in people with several different types of cancer but also in women at high risk for breast cancer, indicating that the formation of adducts is on the pathway to cancer initiation. Two compounds, resveratrol, and N-acetylcysteine, are particularly good at preventing the formation of estrogen-DNA adducts in humans and are, thus, potential cancer-prevention compounds.

Evaluation of the oestrogenic potential of oestrone and bisphenol-A on the reproduction of Astyanax bimaculatus males after subacute exposure.

In the last decades, oestrogenic compounds have often been reported in environmentally relevant concentrations in aquatic environments around the world. Most laboratory studies of oestrogens try to understand the effects of a single contaminant, but in natural environments, the effects may be quite different due to interactions with other compounds. The present study aimed to compare the action of oestrone (E1) and bisphenol-A (BPA), acting singularly and in combination, on the spermatogenesis of Astyanax bimaculatus. After exposure to 100 ng/L of E1, BPA and a mixture of the two for 15 days, our results showed that E1 and the E1 + BPA mixture significantly altered the number of spermatogenic cells. BPA presented high cytotoxicity when compared to other treatments. Analysis of the two oestrogenic compounds suggests that the E1 + BPA mixture has no additive or synergistic effects. Together, the results of the present study indicate that endocrine-disrupting chemicals (EDCs) analysed alone may behave differently than when administered with other substances.

Opposite estrogen effects of estrone and 2-hydroxyestrone on MCF-7 sensitivity to the cytotoxic action of cell growth, oxidative stress and inflammation activity.

There are many kinds of estrogens, and endogenous estrogens produce a variety of estrogen metabolites with similar structure but with different physiological effects after metabolism in vivo. Studies have shown that estrone (E1) widely occurs in the environment and animal-derived food. Because of its estrogen effect, E1 can have adverse effects on the human body as an endocrine disruptor. In this study, we found that E1 and 2-hydroxyestrone (2-OH-E1), the hydroxylation metabolite of estrogen, have opposite proliferative effects on breast cancer cells (MCF-7) through cell proliferation experiments and comparison of their effects by molecular docking and detection of ROS, Ca2+, and cell pathway proteins. The effects of 2-methoxyestrone (2-MeO-E1) and 16α-hydroxyestrone (16α-OH-E1) on the biochemical and protein levels of MCF-7 were further studied to compare the effects of metabolic sites and modes on estrogen effects. Hydroxylation of E1 at the C2 site weakened the estrogen effect, down-regulated the expression of the mammalian target of rapamycin (mTOR) and protein kinase B (Akt) pathway proteins, inhibited the proliferation of cancer cells, and enhanced anti-oxidative stress and anti-inflammation. Methoxylation at the C2 position also inhibited the expression of inflammatory and oxidative stress pathway proteins but did not greatly affect the estrogen effects. However, hydroxylation on C16 had no significant effect on the biological effects of estrogen. Therefore, the structural changes of estrogen on C2 are important reasons for the different physiological effects of estrogen and its metabolites. Thus, by regulating the gene Cytochrome P450 1B1(CYP1B1), which affects the hydroxylation metabolism of estrogen, and promoting the hydroxylation of estrone at the C2 position, the estrogen effect of estrone can be effectively reduced, thus reducing the harm its poses in food and the environment.

Synthesis, and anti-proliferative, Pim-1 kinase inhibitors and molecular docking of thiophenes derived from estrone.

Heterocyclization of steroids were reported to give biologically active products where ring D modification occured. Estrone (1) was used as a template to develop new heterocyclic compounds. Ring D modification of 1 through its reaction with cyanoacetylhydrazine and elemental sulfur gave the thiophene derivative 3. The latter compound reacted with acetophenone derivatives 4a-c to give the hydrazide-hydrazone derivatives 5a-c, respectively. In addition, compound 3 formed thiazole derivatives through its first reaction with phenylisothiocyanate to give the thiourea derivative 9 followed by the reaction of the later with α-halocarbonyl compounds. In the present work a series of novel estrone derivatives were designed, synthesized and evaluated for their in vitro biological activities against c-Met kinase, and six typical cancer cell lines (A549, H460, HT-29, MKN-45, U87MG and SMMC-7721). The most promising compounds 5b, 5c, 11a, 13c, 15b, 15c, 15d, 17a and 17b were further investigated against the five tyrosine kinases c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR. Compounds 5b, 15d, 17a and 17b were selected to examine their Pim-1 kinase inhibition activity where compounds 15d and 17b showed high activities. Molecular docking of some of the most potent compounds was demonstrated.

Occurrence and ecological risk assessment of 22 emerging contaminants in the Jilin Songhua River (Northeast China).

Rivers may receive pharmaceuticals, personal care products, and environment estrogens, which are emerging concerns, from various sources. Understanding the fate of these emerging contaminants (ECs) from the sources to their receiving river is important for assessing their ecosystem risk. Here, the occurrence, seasonal variation, spatial distribution, and ecological risk of 22 ECs in water and sediments from the Jilin Songhua River, as well as in the effluents from the riverside Jilin wastewater treatment plant (WWTP) were investigated. Results indicated that estriol with the highest median concentration of 21.5 ng L-1 in the river water and with the highest median concentration of 481.5 ng g-1 in the sediments, and methylparaben with the highest concentration of 29.6 ± 2.9 ng L-1 in the WWTP effluents were the predominant contaminants. The total concentration of ECs in the river water in the dry season was about 1.5 times higher than that in the wet season. The concentrations of these ECs close to the contaminated tributary and the WWTP were relatively high. Risk assessment showed that the maximum risk quotient value of estrone was 1.07 in the river water and estriol was 2.10 in the effluents. In addition, erythromycin posed generally medium risk in the river water and WWTP effluents. It should be paid attention to the prior control of the three contaminants in the river region.

Modeling the exposure of wild fish to endocrine active chemicals: Potential linkages of total estrogenicity to field-observed intersex.

Decades of studies on endocrine disruption have suggested the need to manage the release of key estrogens from municipal wastewater treatment plants (WWTP). However, the proposed thresholds are below the detection limits of most routine chemical analysis, thereby restricting the ability of watershed managers to assess the environmental exposure appropriately. In this study, we demonstrated the utility of a mechanistic model to address the data gaps on estrogen exposure. Concentrations of the prominent estrogenic contaminants in wastewaters (estrone, estradiol, and ethinylestradiol) were simulated in the Grand River in southern Ontario (Canada) for nine years, including a period when major WWTP upgrades occurred. The predicted concentrations expressed as total estrogenicity (E2 equivalent concentrations) were contrasted to a key estrogenic response (i.e., intersex) in rainbow darter (Etheostoma caeruleum), a wild sentinel fish species. A predicted total estrogenicity in the river of ≥10 ng/L E2 equivalents was associated with high intersex incidence and severity, whereas concentrations <0.1 ng/L E2 equivalents were associated with minimal intersex expression. Exposure to a predicted river concentration of 0.4 ng/L E2 equivalents, the environmental quality standard (EQS) proposed by the European Union for estradiol, was associated with 34% (95% CI:30-38) intersex incidence and a very low severity score of 0.6 (95% CI:0.5-0.7). This exposure is not predicted to cause adverse effects in rainbow darter. The analyses completed in this study were only based on the predicted presence of three major estrogens (E1, E2, EE2), so caution must be exercised when interpreting the results. Nevertheless, this study illustrates the use of models for exposure assessment, especially when measured data are not available.

Estrogenic activity, selected plasticizers and potential health risks associated with bottled water in South Africa.

Potential endocrine disrupting chemicals (EDCs) are present in bottled water from various countries. In South Africa (SA), increased bottled water consumption and concomitant increases in plastic packaging create important consequences for public health. This study aimed to screen SA bottled water for estrogenic activity, selected target chemicals and assessing potential health risks. Ten bottled water brands were exposed to 20 °C and 40 °C over 10 days. Estrogenic activity was assessed using the recombinant yeast estrogen screen (YES) and the T47D-KBluc reporter gene assay. Solid phase extracts of samples were analyzed for bis(2-ethylhexyl) adipate (DEHA), selected phthalates, bisphenol-A (BPA), 4-nonylphenol (4-NP), 17ß-estradiol (E2), estrone (E1), and ethynylestradiol (EE2) using gas chromatography-mass spectrophotometry. Using a scenario-based health risk assessment, human health risks associated with bottled water consumption were evaluated. Estrogenic activity was detected at 20 °C (n = 2) and at 40 °C (n = 8). Estradiol equivalent (EEq) values ranged from 0.001 to 0.003 ng/L. BPA concentrations ranged from 0.9 ng/L to 10.06 ng/L. Although EEqs and BPA concentrations were higher in bottled water stored at 40 °C compared to 20 °C, samples posed an acceptable risk for a lifetime of exposure. Irrespective of temperature, bottled water from SA contained chemicals with acceptable health risks.

Divergent responsiveness of two isoforms of the estrogen receptor to mixtures of contaminants of emerging concern in four vertebrates.

Contaminants of emerging concern (CECs) are ubiquitous in aquatic environments with well-established endocrine-disrupting effects. A data matrix of 559 water samples was queried to identify two commonly occurring CECs mixtures in Great Lakes tributaries. One mixture consisted of eight agricultural CECs (AG), while another contained 11 urban CECs (UB). The known estrogenic compounds bisphenol A, estrone and nonylphenol were present in both mixtures. According to the EPA Tox21 in ToxCast database, AG and UB mixture at an environmentally relevant concentration were estimated to account for 6.5% and 3.4% estrogenicity of the model endocrine disruptor estradiol-17ß, respectively. Two isoforms of the estrogen receptor (Esr1 and -2, former Erα and Erß) cloned from fathead minnow, bluegill sunfish, American alligator and human, responded differently to AG and UB mixtures. Human and bluegill Esr1 were the most sensitive to AG and UB mixtures, respectively. Fathead minnow Esr1 and Esr2b were the least sensitive to 10× AG and UB in estrogen dose equivalents, respectively. Even at environmentally documented concentrations, UB significantly activated bluegill Esr1. Moreover, 100× concentrated UB hyperstimulated fathead minnow Esr1 beyond the maximum induction of estradiol-17ß. These results indicate that efficacious receptors and species differ in their response to CEC mixtures. Furthermore, estrogenicity may be present in some CECs not previously considered estrogenic, or, alternatively, estrogenicity of a mixture may be enhanced through chemical interactions. Our study highlights the need for further studies of CECs utilizing a variety of receptors cloned from diverse species.

Vulnerability of marsh frog Pelophylax ridibundus to the typical wastewater effluents ibuprofen, triclosan and estrone, detected by multi-biomarker approach.

Pharmaceutical and personal care products (PPCPs) are the environmental pollutants of growing concern. The aim of this study was to indicate the effects of typical PPCPs on the marsh frog Pelophylax ridibundus. We treated male frogs with waterborne ibuprofen (IBU, 250ng·L-1), triclosan (TCS, 500ng·L-1), or estrone (E1, 100ng·L-1) for 14days. Common vulnerability of the frogs was detected from dramatic decrease of Zn, total and metalated metallothionein (MT) concentrations, Zn/Cu ratio, the elevation of activity of glutathione-S-transferase, cathepsin D and DNA instability in the liver, the depletion of cholinesterase in the brain and cortisol in the blood plasma in all exposures. Nevertheless, lipofuscin concentration in the liver was always decreased. The groups were best distinguished by cytochrome P450 (CYP450) activity determined by ELISA. The exposure to IBU caused lesser damage, but elevated the levels of oxyradicals and glutathione (GSH and GSSG) and lysosomal membrane instability. Exposures to TCS and E1 provoked the endocrine disturbance (increased levels of vitellogenin and thyrotropin in blood plasma), decreased lactate dehydrogenase activity and increased level of pyruvate in the liver. TCS caused the increase of GSSG by 7.3 times and lactate levels. Only E1 lead to decrease of deiodinase activity in the liver, activation of CYP450 and caspase-3 and efflux of cathepsin D from lysosomes. Spectrophotometric and ELISA assays of MTs and CYP450 gave distinct results in E1-group. Broad disruption of the hormonal pathways caused by E1 could be of concern for the health status of frogs in their habitats.

Biological screening of cucurbitacin inspired estrone analogs targeting mitogen-activated protein kinase (MAPK) pathway.

Assembly of cucurbitacin inspired estrone analogs has been previously synthesized and screened against melanoma cell lines. Further synthetic optimization was executed via installation of Azide polar functional moiety across 23, 24 α, ß-unsaturated ketone side chain using Michael addition reaction. This was followed by biological screening against melanoma cell lines employing MTT assay, in-cell-based ELISA assay, and Western blot analysis to monitor the potential of the synthesized analogs to inhibit the phosphorylated ERK levels. This resulted in evolution of MH-4 possessing IC50 of 3.59 µm with significant decrease in the p-ERK and targeting MAPK pathway.

Experimental paradigm for in-laboratory proxy aquatic studies under conditions of static, non-flow-through chemical exposures.

Endocrine-disrupting chemicals (EDCs) such as 17α-ethynylestradiol, 17ß-estradiol, estrone, and para-nonylphenol have been measured in wastewater-treatment plant effluents, surface waters, sediments, and sludge and have been shown to induce liver-specific vitellogenin (vtg) messenger RNA in male fathead minnows (Pimephales promelas). The purpose of the present study was to establish minimal concentrations of select EDCs necessary to induce transcription of vtg in 48-h static renewal exposures, as measured by quantitative real-time thermal cycle amplification. Adult males were exposed to 17α-ethynylestradiol, 17ß-estradiol, estrone, and para-nonylphenol. Dose-dependent increases in vtg expression were significant with all chemicals tested. The lowest concentrations of these chemicals to induce measurable vtg expression, with significant difference from respective controls, were 17α-ethynylestradiol, 2.2 ng L(-1); para-nonylphenol, 13.9 µg L(-1); 17ß-estradiol, 42.7 ng L(-1); and estrone, 46.7 ng L(-1), measured as 48-h average concentrations. The present experiments were designed to frame a commonly acceptable approach for investigators who conduct static, in-laboratory proxy environmental aquatic exposures. The present study highlights the need for investigators to report in peer-reviewed submissions the observed concentration values for minimal induction levels when measuring molecular responses to chemical exposures by means of real-time polymerase chain reaction, quantitative polymerase chain reaction, or other "omic" technologies.

Mechanisms of estrogen carcinogenesis: The role of E2/E1-quinone metabolites suggests new approaches to preventive intervention--A review.

Studies in hamsters, mice and rats have demonstrated that estradiol (E2), its interconvertible metabolite estrone (E1) and their catechol metabolites, in particular 4-hydroxy E2/E1, are carcinogenic in the kidney, uterus and mammary gland. Observational studies and clinical trials consistently show that sustained exposure to E2/E1 is associated with the development of sporadic breast cancer. The weight of evidence supports the contribution of two complementary pathways in the initiation, promotion and progression of breast cancer. One pathway involves activation of nuclear and cytoplasmic signaling pathways through the binding of estrogen to nuclear and membrane-bound estrogen receptors leading to increased cell proliferation. The other pathway involves the oxidative metabolism of E2/E1 to catechols and then reactive quinones that can contribute to oxidative DNA damage and form specific, mutagenic depurinating adducts with adenine and guanine which then in turn can serve as biomarkers for the occurrence of these processes. Both pathways can serve as portals to preventive intervention. Antiestrogens are used clinically to block receptor-mediated signaling to block tumor growth. Various chemopreventive agents such as sulforaphane (SFN) and resveratrol have been shown in cell culture to block oxidative metabolism of E2/E1 and thus prevent DNA damage. Pretreatment of MCF-7 and MCF-10F cells with and inhibitor of catechol-O-methyltransferase (COMT) followed by treatment with E2 or 4-OH E2 caused increased oxidative DNA damage (8-oxo-dG) and depurinating DNA adducts showing the importance of E2-catechol O-methylation by COMT as a protective pathway. E2 treatment of MCF-10A cells with E2 or 4-OH E2 caused an increase in E2-adenine and guanine adducts. Treatment with sulforaphane increased NAD(P)H: quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase A1 (GSTA1) expression without affecting expression of catechol-O-methyltransferase (COMT) or cytochrome P450 1B1. Pretreatment with SFN decreased depurinating DNA adducts while increasing levels of 4-OCH3E1/2 and 4-OHE1/2-glutathione conjugates. Treatment of MCF-10F cells with E2 or 4-OH-E2 also caused increased depurinating DNA adducts and neoplastic transformation while pretreatment with resveratrol caused a reduction in adduct levels and neoplastic transformation. Increased levels of estrogen-quinone conjugates and DNA adducts have also been detected in urine of women at increased risk for and with breast cancer. These observations support the notion that targeting the estrogen/estrone metabolism pathway may be another way to reduce breast cancer risk.

Effects of single and mixed estrogens on single and combined cultures of D. subspicatus and P. subcapitata.

This paper investigates the effect of estrone (E1), 17ß-estradiol (E2) and 17α-ethinylestradiol (EE2) individually and mixed at equal proportions (1:1:1) on Desmodesmus subspicatus and Pseudokirchneriella subcapitata in single and combined cultures (S+) at different exposure times based on algal growth (in vivo chlorophyll fluorescence and cell counting) and coenobium formation. EE2 and E2 were more toxic to individual and combined (S+) cultures than was E1. The frequency of coenobium formation by D. subspicatus increased significantly for all estrogens and all concentrations. After 96 h, D. subspicatus prevailed in S+. The results of the exposure to E+ suggested a less-than-additive effect on D. subspicatus and S+ and additive effect on P. subcapitata. Toxic effects occurred for both species exposed to E+ with individual estrogen concentrations below the NOEC of each species. Assays must include changes in response due to the exposure of more than one species to more than one estrogen.

Label-free detection of endocrine disrupting chemicals by integrating a competitive binding assay with a piezoelectric ceramic resonator.

A piezoelectric biosensor for detection of endocrine disrupting chemicals (EDCs) was developed by incorporating chemical/biochemical recognition elements on the ceramic resonator surface for competitive binding assays. A facile electrodeposition was employed to modify the sensor surface with Au nanoparticles, which increased the surface area and enhanced the binding capacity of the immobilized probes. Thiol-labeled long chain hydrocarbon with bisphenol A (BPA) as head group was synthesized and self-assembled on the Au nanoparticle surface as the sensing probes, which showed a linear response upon the binding of estrogen receptor (ER-α) ranging from 1 to 30 nM. Detection of estrone, 17ß-estradiol and BPA was achieved by integrating a competitive binding assay with the piezoelectric sensor. In this detection scheme, different concentrations of EDCs were incubated with 30 nM of ER-α, and the un-bounded ER-α in the solution was captured by the probes immobilized on the ceramic resonator, which resulted in the frequency changes for different EDCs. The biosensor assay exhibited a linear response to EDCs with a low detection limit of 2.4-2.9 nM (S/N=3), and required only a small volume of sample (1.5 µl) with the assay time of 2h. The performance of the biosensor assay was also evaluated for rapid and facile determination of EDCs of environmental relevant concentrations in drinking water and seawater, and the recovery rate was in the range between 94.7% and 109.8%.

In vitro assessment of thyroidal and estrogenic activities in poultry and broiler manure.

Among the many chemicals found in avian manure, endocrine disruptors (EDs), of natural or anthropogenic origin, are of special environmental concern. Nowadays, an increasing amount of estrogens is being released into the environment via the use of manure to fertilize agricultural land. While most research in this field has focused on estrogenic phenomena, little is known about alterations related to other endocrine systems, such as the thyroidal one. Here we simultaneously assessed the potential estrogenic and thyroidal activity of poultry and broiler litter manure using in vitro approaches based on estrogen receptor (Er) and thyroid receptor (Tr) transactivation assays. In addition, leaching experiments were performed to assess whether the EDs present in the manure pass through a soil column and potentially reach the groundwater. Manure from four broiler and four poultry farms was collected in two sampling campaigns carried out in two seasons (fall and spring). Extracts from broiler and poultry manure exhibited strong thyroidal activity. Only poultry manure showed estrogenic activity, which is consistent with the low levels of estrogens expected in hatchlings. Leakage experiments were performed in columns with two kinds of arable soils: sandy and loamy. No estrogenicity or thyroidal activity was detectable in soils treated with the manure or in the corresponding leachates. These results indicate that substances with estrogenic or thyroidal activity were degraded in the soil under our experimental conditions. However, the long-term effects associated with the constant and intensive application of manure to agricultural land in some regions require further research.

Effects of estrone on the early life stages and expression of vitellogenin and estrogen receptor genes of Japanese medaka (Oryzias latipes).

The fertilized eggs of Japanese medaka (Oryzias latipes) were exposed to estrone (E1) at 5-5000 ng L(-1) for 15 d, and the hatched fry were exposed continuously to the same concentrations for the additional 15 d. Adverse effects on hatchability, time to hatching, and gross abnormalities occurred at 50 ng L(-1) or above. Then the fry were divided into a continual exposure group, and a water recovery group. When the fry were exposed to E1 for another 60 d, there was a decrease in the hepatosomatic index (HSI) of males and the influence disappeared in the water recovery group. The gonadosonatic index (GSI) of females at 500 ng L(-1) decreased significantly in another 60 d exposure. While the fry were maintained in dechlorinated tap water for 60 d, a significant decrease in female GSI was observed at 50 ng L(-1) or above. An increased GSI was found in males in both continual exposure and water recovery groups at all E1 treatments. Quantitative RT-PCR showed that vitellogenin-I (Vtg-I) gene expressions in the female liver were significantly down-regulated at 50 ng L(-1) in the continual exposure group, and at 500 ng L(-1) in the water recovery group, while male Vtg-I genes were significantly up-regulated for all E1 treatments. In addition, all E1 treatments caused sex reversal of males. These results suggest that E1 at 5 ng L(-1) or above have unrecoverable impacts on the gonadal growth and development of medaka, even if only early life stages were exposed to E1.

Assessment of hormonal activities and genotoxicity of industrial effluents using in vitro bioassays combined with chemical analysis.

Wastewaters from various industries are a main source of the contaminants in aquatic environments. The authors evaluated the hormonal activities (estrogenic/anti-estrogenic activities, androgenic/anti-androgenic activities) and genotoxicity of various effluents from textile and dyeing plants, electronic and electroplate factories, pulp and paper mills, fine chemical factories, and municipal wastewater treatment plants in the Pearl River Delta region by using in vitro bioassays (yeast estrogen screen [YES]; yeast androgen screen [YAS]; and genotoxicity assay [umu/SOS]) combined with chemical analysis. The results demonstrated the presence of estrogenic, anti-estrogenic, and anti-androgenic activity in most industrial effluents, whereas no androgenic activities were detected in all of the effluents. The measured estrogenic activities expressed as estradiol equivalent concentrations (EEQs) ranged from below detection (3 of 26 samples) to 40.7 ng/L, with a mean of 7.33 ng/L in all effluents. A good linear relationship was found between the EEQs measured by YES bioassay and the EEQs calculated from chemical concentrations. These detected estrogenic compounds, such as 4-nonylphenol and estrone, were responsible for the estrogenic activities in the effluents. The genotoxic effects expressed as benzo[a]pyrene equivalent concentrations (BaP EQs) varied between below detection and 88.2 µg/L, with a mean of 8.76 µg/L in all effluents. The target polycyclic aromatic hydrocarbons were minor contributors to the genotoxicity in the effluents, and some nontarget compounds in the effluents were responsible for the measured genotoxicity. In terms of estrogenic activities and genotoxicity, discharge of these effluents could pose high risks to aquatic organisms in the receiving environments.

Endocrine disruption due to estrogens derived from humans predicted to be low in the majority of U.S. surface waters.

In an effort to assess the combined risk estrone (E1), 17ß-estradiol (E2), 17α-ethinyl estradiol (EE2), and estriol (E3) pose to aquatic wildlife across United States watersheds, two sets of predicted-no-effect concentrations (PNECs) for significant reproductive effects in fish were compared to predicted environmental concentrations (PECs). One set of PNECs was developed for evaluation of effects following long-term exposures. A second set was derived for short-term exposures. Both sets of PNECs are expressed as a 17ß-estradiol equivalent (E2-eq), with 2 and 5 ng/L being considered the most likely levels above which fish reproduction may be harmed following long-term and short-term exposures, respectively. A geographic information system-based water quality model, Pharmaceutical Assessment and Transport Evaluation (PhATE™), was used to compare these PNECs to mean and low flow concentrations of the steroid estrogens across 12 U.S. watersheds. These watersheds represent approximately 19% of the surface area of the 48 North American states, contain 40 million people, and include over 44,000 kilometers of rivers. This analysis determined that only 0.8% of the segments (less than 1.1% of kilometers) of these watersheds would have a mean flow E2-eq concentration exceeding the long-term PNEC of 2.0 ng/L; only 0.5% of the segments (less than 0.8% of kilometers) would have a critical low flow E2-eq exceeding the short-term PNEC of 5 ng/L. Those few river segments where the PNECs were exceeded were effluent dominated, being either headwater streams with a publicly owned treatment works (POTW), or flowing through a highly urbanized environment with one or several POTWs. These results suggest that aquatic species in most U.S. surface waters are not at risk from steroid estrogens that may be present as a result of human releases.

Predicted-no-effect concentrations for the steroid estrogens estrone, 17ß-estradiol, estriol, and 17α-ethinylestradiol.

The authors derive predicted-no-effect concentrations (PNECs) for the steroid estrogens (estrone [E1], 17ß-estradiol [E2], estriol [E3], and 17α-ethinylestradiol [EE2]) appropriate for use in risk assessment of aquatic organisms. In a previous study, they developed a PNEC of 0.35 ng/L for EE2 from a species sensitivity distribution (SSD) based on all available chronic aquatic toxicity data. The present study updates that PNEC using recently published data to derive a PNEC of 0.1 ng/L for EE2. For E2, fish were the most sensitive taxa, and chronic reproductive effects were the most sensitive endpoint. Using the SSD methodology, we derived a PNEC of 2 ng/L for E2. Insufficient data were available to construct an SSD for E1 or E3. Therefore, the authors used in vivo vitellogenin (VTG) induction studies to determine the relative potency of the steroid estrogens to induce VTG. Based on the relative differences between in vivo VTG induction, they derive PNECs of 6 and 60 ng/L for E1 and E3, respectively. Thus, for long-term exposures to steroid estrogens in surface water (i.e., >60 d), the PNECs are 6, 2, 60, and 0.1 ng/L for E1, E2, E3, and EE2, respectively. Higher PNECs are recommended for short-term (i.e., a few days or weeks) exposures.

Degradation of 17α-ethinylestradiol by ozonation--identification of the by-products and assessment of their estrogenicity and toxicity.

The presence of the synthetic estrogen 17α-ethinylestradiol (EE2) in waters at low levels is a concern due to its ability to act as an endocrine disruptor. Ozone (O(3)) is commonly used in water treatment and reacts with EE2 to form by-products having characteristics that are mostly unknown. The aim of this study was to identify the by-products of E2 and EE2 ozonation and determine their estrogenicity and toxicity relative to the parent compound. Ozonation by-products were identified via LC-MS analysis. The estrogenicity was measured using the YES assay, and toxicity was determined by monitoring effects on histology of fetal rat testes and testosterone secretion by these tissues. Two EE2 by-products were identified with open phenolic ring structures (masses 302 and 344 u). The Yeast Estrogen Screening (YES) assay showed a decreased but incomplete removal of estrogenicity after ozonation of EE2. Histological analysis of fetal testes revealed that neither E2 nor EE2, with or without ozonation, had any effect on seminiferous cord formation; however, a remarkable negative effect on testosterone secretion was observed, with EE2 by-products after ozonation showing the most rapid and extensive inhibition. These results show that the removal of EE2 via reaction with O(3) resulted in the formation of by-products that are less estrogenic (as demonstrated by the YES assay), but have a greater negative impact on testosterone secretion. Thus, the disappearance of the parent compound is not a sufficient endpoint, as the by-products created may be more toxic. Care should be taken when implementing oxidation applications such as ozone during waste water treatment.