The effect of endocrine disrupting chemicals on the vitronectin-receptor (integrin αvß3)-mediated cell adhesion of human umbilical vein endothelial cells.

Endocrine disrupting chemicals (EDCs) are associated with cancer development and progression due to their promotion of increased cell invasiveness and metastasis formation. However, the effects of EDCs on cell adhesion mediated through integrins have not been well studied to date. Their actions are implicated by binding sites for hormones on the vitronectin receptor (VTNR; or integrin αvß3), which is involved in tumor angiogenesis and metastasis. VTNR-expressing human umbilical vein endothelial cells (HUVECs) were used to determine the effects of EDCs and endogenous hormones on cell adhesion to vitronectin-coated surfaces, and on VTNR activation. Cell adhesion was significantly increased for bisphenol A, triclocarban, and triclosan (10, 100 nM; p < 0.05), with similar trends for bisphenols AF and S (10, 100 nM; p > 0.05). No changes in cell adhesion were seen for 5α-dihydrotestosterone, 17ß-estradiol, triiodothyronine, imatinib and paroxetine. These data indicate that EDC-mediated increases in HUVEC adhesion to vitronectin are not mediated through androgenic, estrogenic, or thyroid activities, nor through activation of VTNR. Although these effects of EDCs on HUVEC adhesion require further investigation of the underlying mechanism(s) of action to define their biological relevance, the low-dose effects and nonmonotonic responses revealed here define the need for further investigation of these EDCs.

A critical review on antibiotics and hormones in swine wastewater: Water pollution problems and control approaches.

Swine wastewater (SW) is an important source of antibiotics and hormones (A&H) in the environment due to their large-scale application in swine industry. A&H in SW can be released into the water environment through the direct discharge of SW, effluent from SW treatment plants, and runoff and leaching from farmland polluted by swine wastes. The presence of A&H in the water environment has become an increasing global concern considering their adverse effects to the aquatic organism and human. This review critically discusses: (i) the occurrence of A&H in global water environment and their potential risks to water organisms and human; (ii) the management and technical approaches for reducing the emission of A&H in SW to the water environment. The development of antibiotic alternatives and the enhanced implementation of vaccination and biosecurity are promising management approaches to cut down the consumption of antibiotics during swine production. Through the comparison of different biological treatment technologies for removing A&H in SW, membrane-based bioprocesses have relatively higher and more stable removal efficiencies. Whereas, the combined system of bioprocesses and AOPs is expected to be a promising technology for elimination and mineralization of A&H in swine wastewater. Further study on this system is therefore necessary.

Toxic effects of single animal hormones and their mixtures on the growth of Chlorella vulgaris and Scenedesmus armatus.

In their environments, aquatic organisms are simultaneously exposed to mixtures of several endocrine disrupting compounds, including hormones. However, most of the toxicity studies so far focused on effects of single contaminants. The available information on the potential toxicity of combined hormones on microalgae is extremely limited. For these reasons the aim of this study was to evaluate the individual and mixture effect of estrone (E1), ß-estradiol (E2), estriol (E3), 17-α-ethinylestradiol (EE2), progesterone (PRO), 5-pregnen-3ß-ol-20-one (PRE), levonorgestrel (LG) and testosterone (TST) on Chlorella vulgaris and Scenedesmus armatus. Green algae cells were exposed to different concentrations (0.1-100 mg L-1) of hormones for 14 days. Biomass in the form of dry weight and chlorophyll a was examined. The decreasing order of toxicity (based on EC50, 14d) to Chlorella vulgaris and Scenedesmus armatus was: EE2>PRO > E2>PRE > TST > E3>LG > E1 and EE2>PRO > TST > E2>PRE > LG > E1>E3, respectively. Chlorella vulgaris was more sensitive to the effects of hormones than Scenedesmus armatus. Although mixed hormones were more toxic to green algae than single hormones, in the ecosystem mixtures can pose higher ecological risk than single pollutants. Therefore, data on the toxicology of both single and mixed hormones is very valuable for assessment of the possibility of adverse ecological effects caused by these pollutants. Furthermore, these results suggest that environmental exposure to hormone mixtures may cause toxicity levels different to the sum of those of the single hormones and provides a basic understanding of their toxic effect on algae.

The complexation of steroid hormones into cyclodextrin alters the toxic effects on the biological parameters of zebrafish (Danio rerio).

The objective of this study was to evaluate the toxic effects of progesterone (P4F) and estradiol (E2F) and the effect of these steroid hormones complexed into cyclodextrins, commercially available drugs, such as micronized progesterone (P4M) and transdermal estradiol (E2T), and evaluate them as endocrine disruptors through biological parameters of Danio rerio. An acute toxicity test was performed with hormones using D. rerio embryos according to OECD 236 guidelines. The heart rate, mortality, and teratogenic effects were evaluated. In addition, a chronic toxicity test was assayed with adult animals for evaluation of animal behavior, reproductive capacity, and electrophysiological responses of the retina. Analysis of the results of the acute toxicity test with embryos exposed to progestins and estrogens showed that free hormones caused a higher percentage of teratogenic effects such as pericardial edema, yolk sac edema, and spinal deformation. Behavioral evaluation (30-60 days) of adult animals exposed to P4M, E2F, and E2T demonstrated higher frequencies of aggressive behaviors such as Chase away, Persecution, Escape, and Attack. Analysis of reproductive capacity did not show significant differences in the number of viable eggs, and no significant changes were observed in the electrophysiological responses of the retina. According to these results, there is a higher toxicity effect of hormones in the free form when compared to the commercial forms and inclusion complexes. This indicates that complexation into cyclodextrin reduced the toxicity of the hormones according to the parameters studied.

Reproductive drugs and environmental contamination: quantum, impact assessment and control strategies.

Industrial and municipal solid wastes, noise, pesticides, fertilizers and vehicular emission are visible pollutants responsible for environmental contamination and ill-effects on health of all living systems. But, environmental contamination due to drugs or medicines used for different purposes in humans and animals goes unseen largely and can affect the health of living system severely. During the last few decades, the usage of drugs has increased drastically, resulting in increased drug load in soil and water. Contraceptive and fertility drugs are extensively and effectively used in humans as well as animals for different purposes. Usage of these reproductive drugs in humans is increased manifold to manage reproductive problems and/or for birth control with changing lifestyles. These drugs are excreted in urine and faeces as metabolite or conjugated forms, leading to contamination of water, milk and animal produce, which are consumed directly by humans as well as animals. These drugs are not eliminated even by water treatment plant. Consumption of such contaminated water, milk, meat and poultry products results in reproductive disorders such as fertility loss in men and increase risk of different types of cancers in humans. Therefore, assessment of impact of environmental contamination by these drugs on living system is of paramount importance. The purpose of this review article is to provide a comprehensive analysis of various research and review reports on different contraceptive and fertility drugs used in human and animals, their occurrence in the environment and their ill-effects on living systems. The approaches to control this invisible menace have also been proposed.

Transcriptomic and phenotypic profiling in developing zebrafish exposed to thyroid hormone receptor agonists.

There continues to be a need to develop in vivo high-throughput screening (HTS) and computational methods to screen chemicals for interaction with the estrogen, androgen, and thyroid pathways and as complements to in vitro HTS assays. This study explored the utility of an embryonic zebrafish HTS approach to identify and classify endocrine bioactivity using phenotypically-anchored transcriptome profiling. Transcriptome analysis was conducted on zebrafish embryos exposed to 25 estrogen-, androgen-, or thyroid-active chemicals at concentrations that elicited adverse malformations or mortality at 120 h post-fertilization in 80% of animals exposed. Analysis of the top 1000 significant differentially expressed transcripts and developmental toxicity profiles across all treatments identified a unique transcriptional and phenotypic signature for thyroid hormone receptor agonists. This unique signature has the potential to be used as a tiered in vivo HTS and may aid in identifying chemicals that interact with the thyroid hormone receptor.

Endocrine disruptors affect larval zebrafish behavior: Testing potential mechanisms and comparisons of behavioral sensitivity to alternative biomarkers.

Larval zebrafish (Danio rerio) are a tool for assessing endocrine disruption during early development. Here, we investigated the extent to which a simple light/dark behavioral test at five days post fertilization could compliment current methods within the field. We exposed fertilized embryos to hormones (17ß-estradiol, testosterone, dihydrotestosterone, 11-ketotestosterone, thyroxine, triiodothyronine, progesterone, and hydrocortisone) and other relevant compounds (17α ethinylestradiol, bisphenol A, bisphenol S, nonylphenol, flutamide, nilutamide, linuron, drospirenone, potassium perchlorate, mifepristone, and fadrozole) to screen for behavioral effects between 96 and 118h post fertilization (hpf). With the exception of progesterone, all the hormones tested resulted in altered behaviors. However, some inconsistencies were observed regarding the age of the larvae at testing. For example, the xenoestrogens 17α- ethinylestradiol and nonylphenol had behavioral effects at 96hpf, but not at 118hpf. Furthermore, although thyroxine exposure had pronounced effects on behavior, the thyroid disruptor potassium perchlorate did not. Finally, we were unable to demonstrate a role of nuclear receptors following testosterone and 17α- ethinylestradiol exposure, as neither the androgen receptor antagonist flutamide nor the general estrogen receptor inhibitor fulvestrant (ICI) could rescue the observed behavioral effects, respectively. Similarly, molecular markers for androgen and estrogen disruption were upregulated at concentrations below which behavioral effects were observed. These results demonstrate hormones and endocrine disruptors can alter the behavior of larval zebrafish, but the mechanistic pathways remain unclear.

Loss of ATF3 promotes hormone-induced prostate carcinogenesis and the emergence of CK5(+)CK8(+) epithelial cells.

Steroid sex hormones can induce prostate carcinogenesis, and are thought to contribute to the development of prostate cancer during aging. However, the mechanism for hormone-induced prostate carcinogenesis remains elusive. Here, we report that activating transcription factor 3 (ATF3)-a broad stress sensor-suppressed hormone-induced prostate carcinogenesis in mice. Although implantation of testosterone and estradiol (T+E2) pellets for 2 months in wild-type mice rarely induced prostatic intraepithelial neoplasia (PIN) in dorsal prostates (one out of eight mice), the loss of ATF3 led to the appearance of not only PIN but also invasive lesions in almost all examined animals. The enhanced carcinogenic effects of hormones on ATF3-deficient prostates did not appear to be caused by a change in estrogen signaling, but were more likely a consequence of elevated androgen signaling that stimulated differentiation of prostatic basal cells into transformation-preferable luminal cells. Indeed, we found that hormone-induced lesions in ATF3-knockout mice often contained cells with both basal and luminal characteristics, such as p63(+) cells (a basal-cell marker) showing luminal-like morphology, or cells double-stained with basal (CK5(+)) and luminal (CK8(+)) markers. Consistent with these findings, low ATF3 expression was found to be a poor prognostic marker for prostate cancer in a cohort of 245 patients. Our results thus support that ATF3 is a tumor suppressor in prostate cancer.

Abnormal error processing in depressive states: a translational examination in humans and rats.

Depression has been associated with poor performance following errors, but the clinical implications, response to treatment and neurobiological mechanisms of this post-error behavioral adjustment abnormality remain unclear. To fill this gap in knowledge, we tested depressed patients in a partial hospital setting before and after treatment (cognitive behavior therapy combined with medication) using a flanker task. To evaluate the translational relevance of this metric in rodents, we performed a secondary analysis on existing data from rats tested in the 5-choice serial reaction time task after treatment with corticotropin-releasing factor (CRF), a stress peptide that produces depressive-like signs in rodent models relevant to depression. In addition, to examine the effect of treatment on post-error behavior in rodents, we examined a second cohort of rodents treated with JDTic, a kappa-opioid receptor antagonist that produces antidepressant-like effects in laboratory animals. In depressed patients, baseline post-error accuracy was lower than post-correct accuracy, and, as expected, post-error accuracy improved with treatment. Moreover, baseline post-error accuracy predicted attentional control and rumination (but not depressive symptoms) after treatment. In rats, CRF significantly degraded post-error accuracy, but not post-correct accuracy, and this effect was attenuated by JDTic. Our findings demonstrate deficits in post-error accuracy in depressed patients, as well as a rodent model relevant to depression. These deficits respond to intervention in both species. Although post-error behavior predicted treatment-related changes in attentional control and rumination, a relationship to depressive symptoms remains to be demonstrated.

Pharmaceuticals and personal care products (PPCPs): a review on environmental contamination in China.

Pharmaceuticals and personal care products (PPCPs) which contain diverse organic groups, such as antibiotics, hormones, antimicrobial agents, synthetic musks, etc., have raised significant concerns in recently years for their persistent input and potential threat to ecological environment and human health. China is a large country with high production and consumption of PPCPs for its economic development and population growth in recent years. This may result in PPCP contamination in different environmental media of China. This review summarizes the current contamination status of different environment media, including sewage, surface water, sludge, sediments, soil, and wild animals, in China by PPCPs. The human body burden and adverse effects derived from PPCPs are also evaluated. Based on this review, it has been concluded that more contamination information of aquatic environment and wildlife as well as human body burden of PPCPs in different areas of China is urgent. Studies about their environmental behavior and control technologies need to be conducted, and acute and chronic toxicities of different PPCP groups should be investigated for assessing their potential ecological and health risks.

Predicting the concentration range of unmonitored chemicals in wastewater-dominated streams and in run-off from biosolids-amended soils.

Organic compounds such as sterols and hormones have been detected in surface waters at ecologically relevant concentrations with sources including effluent discharged from publicly owned treatment works (POTWs) as well as leachate and runoff from land amended with municipal sludge (biosolids). Greater than 20% of regulated effluents discharged into U.S. surface waters experience in-stream dilution of <10-fold and potential impacts are particularly likely in receiving waters dominated by POTW effluents. The increasing use of biosolids on agricultural land exerts additional stress, thereby necessitating environmental monitoring for potential ecological and human health effects. Alternatively or in addition to monitoring efforts, screening for potentially hazardous chemicals can be performed using empirical models that are scalable and can deliver results rapidly. The present study makes use of data from U.S. EPA's Targeted National Sewage Sludge Survey (TNSSS) to predict the aqueous-phase concentrations and removal efficiencies of 10 sterols (campesterol, ß-sitosterol, stigmasterol, ß-stigmastanol, cholesterol, desmosterol, cholestanol, coprostanol, epicoprostanol, and ergosterol) as well as the putative toxicity posed by four specific hormones based on their reported biosolids concentrations using published empirical models. Model predictions indicate that removal efficiencies for sterols are uniformly high (~99%) and closely match removal rates calculated from chemical monitoring at POTWs (paired t-test; p=0.01). Results from toxicity modeling indicate that the hormones estrone, estradiol and estriol had the highest leaching potentials amongst the compounds considered here and that 17 ß-ethinylestradiol was found to pose a potentially significant threat to fathead minnows (Pimephales promelas) via run-off or leaching from biosolids-amended fields. This study exemplifies the use of in silico analysis to (i) identify potentially problematic organic compounds in biosolids, (ii) predict influent and effluent levels for hydrophobic organic compounds (HOCs) of emerging concern, and (iii) provide initial estimates of runoff concentrations, in this case for four prominent hormones known to act as endocrine disruptors.

Freshwater mudsnail (Potamopyrgus antipodarum) estrogen receptor: identification and expression analysis under exposure to (xeno-)hormones.

Molluscs are raising attention as ecotoxicological test organisms due to their high diversity and ecological importance. The ovoviviparous prosobranch gastropod Potamopyrgus antipodarum (freshwater mudsnail) responds very sensitively to xenobiotics and has therefore been proposed as OECD standard test organism. Endocrine disrupting chemicals influence the reproduction of P. antipodarum, which can be assessed by embryo numbers in the brood pouch. However, the knowledge about the endocrine system of P. antipodarum is rather limited. The aim of this study was to identify an estrogen receptor in the endocrine system of P. antipodarum and to investigate if this receptor is differentially expressed under exposure to (xeno-)hormones (17α-ethinylestradiol, bisphenol A and 17α-methyltestosterone). The DNA-binding domain of the identified ER-like transcript has an amino acid identity of 92 percent compared to the ER of the gastropod Nucella lapillus (84 percent to human ERα) and 83 percent in the ligand binding domain (38 percent to human ERα). Furthermore, the P. antipodarum ER is transcriptionally regulated as shown by quantitative real-time PCRs of (xeno-)hormone exposed snails. 17α-ethinylestradiol and bisphenol A exposure resulted in a transitory ER-mRNA increase while17α-methyltestosterone caused a transitory reduction of ER-mRNA. In addition the solvent dimethyl sulfoxide had also a modulating effect on the receptor.

Morphological changes in Daphnia galeata induced by a crustacean terpenoid hormone and its analog.

Terpenoid hormones in insects (i.e., juvenile hormones) have various effects on physiology, morphology, and behavior, producing a wide range of phenotypic variation. Recent studies have shown that sex determination in cladoceran crustaceans is under the strong control of a major terpenoid hormone of crustaceans, methyl farnesoatote (MF). It can be easily conceived that MF is also a major determinant of other traits in cladocerans. In the present study, morphological changes known as antipredatory responses in a cladoceran Daphnia galeata in response to exposure to MF and a juvenile hormone-mimicking pesticide, fenoxycarb, were investigated. Morphological change was studied using neonates less than 24 h old, exposed either to MF at the concentrations from 1.9 to 30 µg/L, or fenoxycarb at the concentrations from 13 to 200 ng/L, for 6 d. Animals developed a longer helmet at 1.9 µg/L of MF and 25 ng/L of fenoxycarb, and showed a concentration-dependent elongation. However, the tail spine was reduced in size in a concentration-dependent manner. Results of the present study not only give new insight into the mechanisms of inducible defenses in cladocerans, but also provide invaluable information to understand ecological and evolutionary consequences of endocrine disruption through the shift in biological interaction between predator and prey.

Characterization of additional sewage treatment technologies: ecotoxicological effects and levels of selected pharmaceuticals, hormones and endocrine disruptors.

In the present study, two conventional (with and without sand filter) and four additional (moving bed biofilm reactor, ozone, moving bed biofilm reactor combined with ozone and a membrane bio reactor) treatment technologies were operated in small-scale at Hammarby Sjöstad sewage treatment plant, Stockholm, Sweden. The effluents were tested with five short-term (≤ 7 days exposure) ecotoxicological tests, and analyzed for a number of target analytes, comprising pharmaceuticals, natural hormones and industrial chemicals. Overall, the tested effluents generated few adverse effects at lower concentrations (< 50% sewage effluent), and no major differences were observed between any of the treatments. The effluent treated with the moving bed biofilm reactor resulted in the lowest effects in the ecotoxicological tests. The most efficient treatment technology with regard to the pharmaceutical residues was the ozone treatment, which however caused negative effects in some of the ecotoxicological tests.

Degradation of fifteen emerging contaminants at microg L(-1) initial concentrations by mild solar photo-Fenton in MWTP effluents.

The degradation of 15 emerging contaminants (ECs) at low concentrations in simulated and real effluent of municipal wastewater treatment plant with photo-Fenton at unchanged pH and Fe=5 mg L(-1) in a pilot-scale solar CPC reactor was studied. The degradation of those 15 compounds (Acetaminophen, Antipyrine, Atrazine, Caffeine, Carbamazepine, Diclofenac, Flumequine, Hydroxybiphenyl, Ibuprofen, Isoproturon, Ketorolac, Ofloxacin, Progesterone, Sulfamethoxazole and Triclosan), each with an initial concentration of 100 microg L(-1), was found to depend on the presence of CO(3)(2-) and HCO(3)(-) (hydroxyl radicals scavengers) and on the type of water (simulated water, simulated effluent wastewater and real effluent wastewater), but is relatively independent of pH, the type of acid used for release of hydroxyl radicals scavengers and the initial H(2)O(2) concentration used. Toxicity tests with Vibrio fisheri showed that degradation of the compounds in real effluent wastewater led to toxicity increase.

17alpha-ethinylestradiol alters reproductive behaviors, circulating hormones, and sexual morphology in male fathead minnows (Pimephales promelas).

Ecologically relevant indicators of endocrine disruption in fish must be linked with measures of reproductive success. The ability of male fathead minnows (Pimephales promelas) to compete for, maintain, and defend a spawning substrate is paramount to reproductive success. The present study quantified alterations in male fathead minnow reproductive behaviors after exposure to environmentally relevant concentrations (0, 10, 20, or 40 ng/L) of 17alpha-ethinylestradiol (EE2) for 21 d. A video-based behavioral quantification system examined changes in male-male competitive behaviors (chasing and head-butting) and ability of males to maintain spawning substrates (nibbling and scrubbing). Behaviors analyzed included time under the spawning substrate, frequency of substrate cleaning, and conspecific aggression. Plasma hormone levels (11-ketotestosterone [11-KT], testosterone, and estradiol [E2]), vitellogenin (VTG), secondary male characteristics (tubercle count and dorsal nape pad rank), gonadosomatic index (GSI), and gonad histology also were evaluated. Exposure to 40 ng/L of EE2 decreased the ability of exposed males to compete with control males for spawning substrates (p = 0.09). Furthermore, exposed males displayed reduced frequency of substrate cleaning activities as well as chasing male competitors (p < or = 0.05). 11-Ketotestosterone, testosterone, and E2 were lower, and VTG was notably higher, in EE2-exposed males compared with control males (p < or = 0.03). 17alpha-Ethinylestradiol exposure in males also was associated with reductions in tubercles; lower GSI, gonadal maturity ranks, and number of resorbed tubercles; and presence of an ovipositor (p < or = 0.001). These data reveal alterations in male reproductive behavior that coincide with decreased hormone levels and secondary sex characteristics. Behavioral endpoints to discern potential ecological consequences in fish exposed to low concentrations of endocrine-disrupting chemicals may provide sensitive and functional indices of effect.

Screening health risk assessment of micropullutants for indirect potable reuse schemes: a three-tiered approach.

Indirect potable reuse schemes are an important consideration in the sustainable management of scarce water resources. However, communities still hold real concerns about the potential health risks of micropullutants in recycled water entering their potable water supply. Microfiltration or ultrafiltration followed by reverse osmosis is currently the standard treatment technology for potable use of recycled water. Nevertheless, membranes are not 100% efficient in the removal of trace organic contaminants and the potential health risks of these micropullutants need to be assessed. The aim of this paper is to present a three-tiered approach for the preliminary assessment of micropullutants in recycled water. A risk quotient is calculated by comparing measured concentrations against benchmark values. Tier 1 corresponds to regulated chemicals; the maximum contaminant level in drinking water is used as benchmark value. Tier 2 corresponds to unregulated chemicals with toxicity information; slope factors or risk specific doses are used to calculate benchmark values. Tier 3 corresponds to unregulated chemicals without toxicity information. The "Threshold of Toxicological Concern" concept is used to calculate benchmark values. The characterization of chemicals of concern following reverse osmosis in a water reclamation plant and the application of the three-tiered approach for the evaluation of the potential health risks is presented.

Workgroup report: National Toxicology Program workshop on Hormonally Induced Reproductive Tumors - Relevance of Rodent Bioassays.

The National Toxicology Program (NTP) is currently reviewing its research portfolio as part of its efforts to implement the NTP Roadmap to achieve the NTP Vision for the 21st century. This review includes a recent workshop, "Hormonally Induced Reproductive Tumors-Relevance of Rodent Bioassays," held 22-24 May 2006, that was organized to determine the adequacy and relevance to human disease outcome of rodent models currently used in the 2-year bioassay for four types of hormonally induced reproductive tumors (ovary, mammary gland, prostate, and testis). In brief, none of the workshop's breakout groups felt the currently used models are sufficient. For some types of tumors such as prostate, no adequate animal models exist, and for others such as ovary, the predominant tumors in humans are of different cellular origins than those induced by chemicals in rodents. This inadequacy of current models also applies to the testis, although our more complete understanding of the responses of Leydig cells to hormonal changes in rats may prove predictive for effects in humans other than cancer. All breakout groups recommended that the NTP consider modifying its testing protocols (i.e., age at exposure, additional end points, etc.) and/or using alternative models (i.e., genetically engineered models, in vitro systems, etc.) to improve sensitivity. In this article we briefly review the workshop's outcome and outline some next steps forward in pursuing the workshop's recommendations. Breakout group reports and additional information on the workshop, including participants, presentations, public comments and background materials, are posted on the NTP website.