Endocrine disrupters in the aquatic environment

Possible mechanisms to explain endocrine effects on reproduction and sex differentiation are presented for selected pharmaceuticals, agrochemicals, industrial chemicals and plant sterols which are known to be present in the aquatic environment. Disruptions of the hormonal coordination can be induced by xenobiotics on various levels of the hierachically organised endocrine system of vertebrates. Phthalate plasticisers, for example, may disrupt the pituitary control of gonadal functions; prenatal/larval exposure to synthetic estrogen impairs sex differentiation and neuroendocrine sexual determination of the central nervous system; phenylurea herbicides block the androgen receptor; the biotransformation of weakly estrogenic plant sterol components of paper mill wastewater (e.g. βsitosterol) may lead to androgenic compounds. The effect of hypolipidemic drugs on lipid homeostasis (peroxysom proliferation) is transmitted via a receptor protein that seems to be closely related to the endocrine system both functionally as well as phylogenetically; possible interferences with the neuroendocrine control of sex differentiation are also discussed. In invertebrates, tributyltin is known to effect the biosynthesis of steroidal sexual hormones. PCBs are suspected to be competitive inhibitors of the steroid catabolism. In order to identify potential risks caused by chemicals to the reproductive capacities of aquatic animals and to the quality of drinking water, methods should be established to detect endocrine disrupters at the various levels of the endocrine system.     

Benzotriazoles, Alkylphenols and Bisphenol A in Municipal Wastewaters and in the Glatt River, Switzerland

- DOI: http://dx.doi.org/10.1065/espr2006.01.295 Background Many organic micropollutants occur at trace concentrations in municipal wastewater effluents and in the aquatic environment. Some of these xenobiotic chemicals can be considered as 'emerging' contaminants and some are suspect to have endocrine disrupting effects. Among the latter are nonylphenol (NP), octylphenol (OP) and bisphenol A (BPA), which deserve special attention due to their ubiquitous occurrence in the aquatic environment. The complexing agents benzotriazole (BT) and tolyltriazole (TT) are applied as anticorrosive agents (e.g. in cooling and hydraulic fluids, in antifreezing fluids, in aircraft deicing fluids, in dish washing liquids for silver protection), as antifogging agents and as intermediates for the synthesis of various chemicals. The environmental occurrence of NP and OP is caused by the fact that they are intermediate products (metabolites) in the biodegradation of alkylphenol polyethoxylate surfactants. BPA is globally used for the production of polycarbonate and epoxy resins. Methods BT, TT, NP, OP and BPA were quantitatively determined in municipal wastewater effluents in Switzerland and in the Glatt River. The analytes were enriched by solid-phase enrichment. BT and TT were determined underivatized by electrospray LC/tandem MS. Reversed-phase LC was performed on octylsilica columns with isocratic water/methanol elution. Multiple reaction monitoring of the positive ions provided selective and sensitive detection for reliable quantifications. NP, OP and BPA were determined by GC/MS after derivatization with N-methyl-N-(trimethylsilyl)-trifluoroacetamide. Results and Discussion BT and TT concentrations in primary and secondary effluents of municipal wastewater treatment plants varied from below 10 to 100 μg/L. The ranges of the concentrations in the Glatt River in ng/L were 636–3,690 for BT, 122–628 for TT, 68–326 for NP, 6–22 for OP and 9–76 for BPA. The corresponding mass flows in g/d were 93–1,870 for BT, 18–360 for TT, 24–183 for NP, 1–16 for OP and 2–72 for BPA. The concentrations and mass flows of NP in the River Glatt were drastically lower than the analogous values found 15 years ago. Thus, a substantially decreased environmental exposure can be observed due to the reduction of the use of alkyphenol polyethoxylate surfactants in Switzerland. The current concentrations of NP, OP and BPA are within the ranges reported for weakly impacted surface waters. Conclusion The investigated contaminants occur at quantitatively measurable but varying concentrations in municipal wastewaters and in the Glatt River reflecting their ubiquitous input into wastewaters and their different behaviour during biological wastewater treatment.     

AhR-mediated and antiestrogenic activity of humic substances

Humic substances (HS) were for decades regarded as inert in the ecosystems with respect to their possible toxicity. However, HS have been recently shown to elicit various adverse effects generally attributed to xenobiotics. In our study, we used MVLN and H4IIE-luc cell lines stably transfected with luciferase gene under control of estrogen receptor (ER) and Ah receptor (AhR; receptor connected with so-called dioxin-like toxicity) for assessment of anti/estrogenic and AhR-mediated effects of 12 commercially available humic substances. Out of those, five humic acids were shown to induce AhR-mediated activity with relative potencies related to TCDD 2.6 x 10(-8)-7.4 x 10(-8). Organic extracts of HS solutions also elicited high activities what means that lipophilic molecules are responsible for a great part of effect. However, relatively high activity remaining in extracted solution suggests also presence of polar AhR-agonists. Contribution of persistent organic compounds to the observed effects was ruled out by H(2)SO(4) treatment. Eight out of twelve HS elicited significant antiestrogenic effects with IC(50) ranging from 40 to 164 mg l(-1). The possible explanations of the antiestrogenic effect include sorption of 17-beta-estradiol (E2) on HS, changes in membrane permeability for E2 or another specific mechanism.     

Low dose effects and non-monotonic dose responses for endocrine active chemicals: Science to practice workshop: Workshop summary

A workshop was held in Berlin September 12–14th 2012 to assess the state of the science of the data supporting low dose effects and non-monotonic dose responses (“low dose hypothesis”) for chemicals with endocrine activity (endocrine disrupting chemicals or EDCs). This workshop consisted of lectures to present the current state of the science of EDC action and also the risk assessment process. These lectures were followed by breakout sessions to integrate scientists from various backgrounds to discuss in an open and unbiased manner the data supporting the “low dose hypothesis”. While no consensus was reached the robust discussions were helpful to inform both basic scientists and risk assessors on all the issues. There were a number of important ideas developed to help continue the discussion and improve communication over the next few years.     

不同尾水输送方案对尾水水质的影响研究

通过管道实验和明渠实验模拟尾水输送过程,研究不同输送方案对尾水中COD、BOD5、SS、TN、氨氮、TP、DO、雌酮(E1)、辛基酚(OP)、壬基酚(4-n-NP)等指标的去除规律,为徐州市截污导流工程尾水输送方案的选取提供理论依据.结果表明:(1)除DO、TP和SS外,管道输送方案对其他指标的控制能力均优于明渠;管道输送过程中,除SS和TN外,污染物的浓度变化主要发生在0～48 h.(2)在选择管道输送方案的基础上,可以考虑在实际输送时提高流速,缩短尾水在管道中的停留时间.     

Screening of high production volume chemicals for estrogen receptor binding activity (II) by the MultiCASE expert system

A structurally and functionally diverse and cross-validated quantitative structure-activity knowledge database generated by the MultiCASE expert system was used to screen 2526 high production volume chemicals (HPVCs) for their estrogen receptor binding activity. 73 HPVCs were found to contain structural features or biophores that have been documented as having the ability to bind to the estrogen receptor. Potential chemicals were ranked according to their quantitatively predicted ER binding potential and the details of the biophores found in them are discussed.     

4-Alkylphenols and related chemicals show similar effect on the function of human and rat estrogen receptor alpha in reporter gene assay

Alkylphenols (APs) are widely used as important industrial materials and have attracted lots of attention because of their potential estrogenic activities. In this study, we developed human estrogen receptor alpha (hERalpha) and rat estrogen receptor alpha (rERalpha) mediated reporter gene assays and compared the estrogenic activity of APs and related chemicals based on the two ERalpha. Human breast cancer cell line MCF-7 was co-transfected with Gal4-fused hERalpha and corresponding reporter plasmid; African green monkey kidney cell line CV-1 was co-transfected with rERalpha and reporter gene. Both assays showed acceptable response to natural estrogen 17beta-estradiol (E2) with EC50 of 0.16 nM and 4.7 nM. Then the estrogenic activity of 4-APs, 4-phenylphenol and bisphenol-A were evaluated and compared with the effects of E2. The data suggested that test APs and related chemicals possessed weakly estrogenic activity and the activity of test APs increased with the increase of substituent size. This structure-activity relationship helped to infer the activity of chemicals with similar feature. Furthermore, test APs showed similar effect on the function of hERalpha and rERalpha. This consistency helped to extrapolate in vivo rodent data to human being when performing risk assessment of endocrine disruptors.     

The constitutively active Ah receptor (CA-AhR) mouse as a model for dioxin exposure – Effects in reproductive organs

The dioxin/aryl hydrocarbon receptor (AhR) mediates most toxic effects of dioxins. In utero/lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) impairs fetal/neonatal development and the developing male reproductive tract are among the most sensitive tissues. TCDD causes antiestrogenic responses in rodent mammary gland and uterus and in human breast cancer cell lines in the presence of estrogen. Also, more recently an estrogen-like effect of TCDD/AhR has been suggested in the absence of estrogen. A transgenic mouse expressing a constitutively active AhR (CA-AhR) was developed as a model mimicking a situation of constant exposure to AhR agonists. Male and female reproductive tissues of CA-AhR mice were characterized for some of the effects commonly seen after dioxin exposure. Sexually mature CA-AhR female mice showed decreased uterus weight, while an uterotrophic assay in immature CA-AhR mice resulted in increased uterus weight. In immature mice, both TCDD-exposure and CA-AhR increased the expression of the estrogen receptor target gene Cathepsin D. When co-treated with 17β-estradiol no increase in Cathepsin D levels occurred in either TCDD-exposed or CA-AhR mice. In sexually mature male CA-AhR mice the weights of testis and ventral prostate were decreased and the epididymal sperm reserve was reduced. The results of the present study are in accordance with previous studies on dioxin-exposed rodents in that an activated AhR (here CA-AhR) leads to antiestrogenic effects in the presence of estrogen, but to estrogenic effects in the absence of estrogen. These results suggest the CA-AhR mouse model as a useful tool for studies of continuous low activity of the AhR from early development, resembling the human exposure situation.     

The constitutively active Ah receptor (CA-AhR) mouse as a model for dioxin exposure - effects in reproductive organs

The dioxin/aryl hydrocarbon receptor (AhR) mediates most toxic effects of dioxins. In utero/lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) impairs fetal/neonatal development and the developing male reproductive tract are among the most sensitive tissues. TCDD causes antiestrogenic responses in rodent mammary gland and uterus and in human breast cancer cell lines in the presence of estrogen. Also, more recently an estrogen-like effect of TCDD/AhR has been suggested in the absence of estrogen. A transgenic mouse expressing a constitutively active AhR (CA-AhR) was developed as a model mimicking a situation of constant exposure to AhR agonists. Male and female reproductive tissues of CA-AhR mice were characterized for some of the effects commonly seen after dioxin exposure. Sexually mature CA-AhR female mice showed decreased uterus weight, while an uterotrophic assay in immature CA-AhR mice resulted in increased uterus weight. In immature mice, both TCDD-exposure and CA-AhR increased the expression of the estrogen receptor target gene Cathepsin D. When co-treated with 17β-estradiol no increase in Cathepsin D levels occurred in either TCDD-exposed or CA-AhR mice. In sexually mature male CA-AhR mice the weights of testis and ventral prostate were decreased and the epididymal sperm reserve was reduced. The results of the present study are in accordance with previous studies on dioxin-exposed rodents in that an activated AhR (here CA-AhR) leads to antiestrogenic effects in the presence of estrogen, but to estrogenic effects in the absence of estrogen. These results suggest the CA-AhR mouse model as a useful tool for studies of continuous low activity of the AhR from early development, resembling the human exposure situation.     

Bioluminescent yeast assays for detecting estrogenic and androgenic activity in different matrices

In this paper we describe the construction and use of a set of bioluminescent yeast strains for the detection of compounds that can affect androgen or estrogen receptor mediated hormonal signalling. The set includes Saccharomyces cerevisiae strains expressing human androgen receptor (AR), estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta), along with firefly luciferase controlled by a respective hormone responsive promoter. A constitutively luminescent strain was included in the set for determining the cytotoxicity of the sample. Yeast cells were incubated with pure chemicals or complex samples for 2.5 h, after which the signal could be detected from the cell-sample mixture after simply adding the D-luciferin substrate. The assays could be completed in one day and they required no cell lysis or centrifugation steps, which makes them suitable for high-throughput analysis of samples. Due to a short incubation time the assays are directly applicable to different sample matrices, requiring no pretreatment of the samples. The assays were used to assess the hormonal activity in moisturizing lotions as an example of a complex sample matrix known to contain endocrine disrupting chemicals. Six out of eight tested moisturisers showed high estrogenic activity, whereas no androgenic activity was observed in the samples.     

Atropisomers of 2,2′,3,3′,6,6′-hexachlorobiphenyl (PCB 136) exhibit stereoselective effects on activation of nuclear receptors in vitro

PCB 136 is an environmentally relevant chiral PCB congener, which has been found in vivo to be present in form of rotational isomers (atropisomers). Its atropselective biotransformation or neurotoxic effects linked with sensitization of ryanodine receptor suggest that it might interact also with other intracellular receptors in a stereospecific manner. However, possible atropselective effects of PCB 136 on nuclear receptor transactivation remain unknown. Therefore, in this study, atropselective effects of PCB 136 on nuclear receptors controlling endocrine signaling and/or expression of xenobiotic and steroid hormone catabolism were investigated. PCB136 atropisomers were found to exert differential effects on estrogen receptor (ER) activation; (+)-PCB 136 was estrogenic, while (?)-PCB 136 was antiestrogenic. In contrast, inhibition of androgen receptor (AR) activity was not stereospecific. Both PCB136 stereoisomers induced the constitutive androgen receptor (CAR)-dependent gene expression; however, no significant stereospecificity of PCB 136 atropisomers was observed. PCB136 was a partial inducer of the pregnane X receptor (PXR)-dependent gene expression. Here, (?)-PCB 136 was a significantly more potent inducer of PXR activity than (+)-PCB 136. Taken together, the present results indicate that at least two nuclear receptors participating in endocrine regulation or metabolism, ER and PXR, could be regulated in an atropselective manner by chiral PCB 136. The enantioselective enrichment of PCB atropisomers in animal and human tissues may thus have significant consequences for endocrine-disrupting effects of chiral ortho-substituted PCB congeners.     

Evaluation of the fish short term reproduction assay for detecting endocrine disrupters

In a fish testing strategy, positive results of the fish short term reproduction assay (FSTR), often trigger a definitive test like the fish sexual development test (FSDT) or the fish full life cycle test (FFLC), entailing ethical and economic problems. This study analysed 137 studies encompassing 35 chemicals with different modes of actions (MOAs). Variability is quantified for MOA endpoints vitellogenin (VTG) and secondary sex characteristics (SSCs) as well as for apical endpoints. Two MOA endpoints could indicate estrogenic, anti-estrogenic, androgenic, anti-androgenic and steroidogenesis activities. Great variability, however, has been observed for chemicals with anti-androgenic and steroidogenesis activities, suggesting that TG229/230 may not be sensitive enough to detect these types of chemicals and may produce false negatives. Changes in apical endpoints like fecundity are not limited to endocrine disrupting chemicals (EDCs). Non-EDCs could induce the similar effects on these apical endpoints. If elucidating MOA is needed, targeted in vitro MOA tests are suggested. Positive in vitro MOA results trigger a definitive test, which could be used for confirmation of the MOA in vivo and for deriving a no observed effect concentration (NOEC). Based on positive MOA results of TG229, a definitive test such as the FSDT or the FFLC is still needed, because the current TG229 has limitation on the derivation of a NOEC. An extended TG229 with more power to detect reproduction effects, as recently proposed in the OECD test guideline program, would improve the possibility to derive a NOEC and increase its usefulness in risk assessment.     

环境内分泌干扰物诱发小鼠睾丸细胞DNA损伤的研究

应用单细胞凝胶电泳技术技术，研究了烷基酚类化合物对离体小鼠睾丸细胞DNA的损伤作用。实验结果表明，9种烷基酚类化合物对小鼠睾丸细胞均能引起不同程度的DNA损伤，并呈现剂量效应关系，其高剂量组与对照组相比，均有极显著性差异（P＜0．01）。实验结果亦表明，其DNA的损伤程序与化学结构有一定的关系。     

The Precautionary Principle and Chemicals Regulation: Past Achievements and Future Possibilities (8 pages)

Background, Aim and Scope

The paper investigates the development of the institutional basis of the present modes of chemicals regulation and management, with special attention to interrelations with the precautionary principle. Main Features: The paper elucidates on how the precautionary principle has been shaped in relation to chemicals regulation and management since Carson's Silent Spring (years before the principle was confirmed as a policy-principle in German and European legislation. Furthermore, it is examined how the precautionary principle interacted with the development of the present chemicals regulatory regime, in a complex interplay within the OECD and Member Countries. The present modes of precaution in the new EU chemical legislation – REACH – are investigated with respect to the precautionary principle, and tested against two contemporary problems; brominated flame retardants and endocrine disrupting substances. Results: The analysis demonstrates the changing character of the integration of the precautionary principle. The main tendencies are from implicit to more explicit precaution and from a closed expert-orientation towards a more deliberative approach to scientific knowledge and uncertainty. The results demonstrate that the precautionary principle is manifest in both the design of the testing strategy and in policy provisions. In particular, the substitution of hazardous substances with less hazardous is important. Discussion: Despite explicit attention to the precautionary principle, is the present reformulation of the European Chemicals policy in danger of falling into loop-holes that equal problems related to the present regulation of existing chemicals? 'Precaution' has been reduced virtually to an abstract concept that is more or less devoid of practical meaning in the regulatory process. Conclusions: It is concluded that the role of the precautionary principle in chemicals regulation will require continued scrutiny in the future shaping of the REACH strategy. Perspectives: Continued development of robust and precaution-based chemicals regulation will have to involve both new data-generation strategies and new forms of political decision-making, with special attention given to transparency and deliberative policymaking.     

再生水中内分泌干扰物质的存在及处理现状

随着污水回用大力开展,人们与再生水的接触机会增加,必须保障再生水的水质安全.资料调研发现,作为再生水水源的污水处理厂出水中含有多种内分泌干扰物质,活性炭吸附、超滤、高级氧化技术及膜技术可有效去除再生水中的内分泌干扰物质,但鉴于经济和管理上的困难,目前仍不能全面推广.中国常用的澄清(混凝沉淀)/过滤工艺不能有效去除污水处理厂二级出水中的内分泌十扰物质,这对接触人群的健康产生潜在威胁,对生物种群也有一定的影响.生物处理方法未来有可能成为去除内分泌干扰物质很有前途的方法.     

Perfluorinated compounds affect the function of sex hormone receptors

Perfluorinated compounds (PFCs) are a large group of chemicals used in different industrial and commercial applications. Studies have suggested the potential of some PFCs to disrupt endocrine homeostasis, increasing the risk of adverse health effects. This study aimed to elucidate mechanisms behind PFC interference with steroid hormone receptor functions. Seven PFCs [perfluorohexane sulfonate (PFHxS), perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), perfluorononanoate (PFNA), perfluorodecanoate (PFDA), perfluoroundecanoate (PFUnA), and perfluorododecanoate (PFDoA)] were analyzed in vitro for their potential to affect estrogen receptor (ER) and androgen receptor (AR) transactivity as well as aromatase enzyme activity. The PFCs were assessed as single compounds and in an equimolar mixture. PFHxS, PFOS and PFOA significantly induced the ER transactivity, whereas PFHxS, PFOS, PFOA, PFNA and PFDA significantly antagonized the AR activity in a concentration-dependent manner. Moreover, PFDA weakly decreased the aromatase activity at a high test concentration. A mixture effect more than additive was observed on AR function. We conclude that five of the seven PFCs possess the potential in vitro to interfere with the function of the ER and/or the AR. The observed mixture effect emphasizes the importance of considering the combined action of PFCs in future studies to assess related health risks.     

Antiandrogenic activity of pyrethroid pesticides and their metabolite in reporter gene assay

Many pesticides possess hormonal activity and have thus been classified as endocrine disruptors. Pyrethroids are commonly used pesticides worldwide, but little has been done to characterize their antiandrogenic activity potential. We tested three frequently encountered pyrethroids (fenvalerate, cypermethrin, permethrin) and their metabolite 3-phenoxybenzoic acid (3-PBA) for antiandrogenic and androgenic activity using a human androgen receptor (AR) mediated luciferase reporter gene assay in CV-1 African green monkey kidney cell. The assay displayed appropriate response to the known AR agonist 5alpha-dihydrotestosterone and AR antagonist nilutamide and flutamide. At 0.1mM, all the three tested pyrethroids significantly suppressed the luciferase expression. Further, their metabolite 3-PBA also showed antagonist activity. None of the test chemicals showed androgenic activity. Through the antiandrogenic pathways, exposure to certain pyrethroids may contribute to the damage of reproductive system. In conclusion, pyrethroid pesticides can act as antiandrogen in vitro, and metabolizing to 3-PBA cannot eliminate the antagonist activity. This result provides useful information for risk assessment of pyrethroid pesticides.     

Endocrine modulation, inhibition of ovarian development and hepatic alterations in rainbow trout exposed to polluted river water

Under laboratory conditions, female rainbow trout were exposed to graded concentrations of water from the River Lambro, a polluted tributary of the River Po, and to the effluent of a large wastewater treatment plant which flows into the River Lambro. In field exposures, trout were held in cages in the River Po upstream and downstream from the confluence of the River Lambro. After 10-day (laboratory) and 30-day (laboratory and field) exposures, trout were examined for several chemical, biochemical and histological endpoints. The results indicated that exposure to complex mixtures of chemicals, including estrogen receptor agonists, aryl-hydrocarbon receptor agonists, and probably antiandrogens, had occurred. Exposure altered the plasma levels of 17β-estradiol and testosterone, and some treatments also enhanced the activity of hepatic ethoxyresorufin O-deethylase. Gonadal histology showed varying levels of degenerative processes characterised by oocyte atresia, haemorrhages, melano-macrophage centres (MMCs), and oogonia proliferation. Liver histology showed less severe effects.     

Photolysis of estrone generates estrogenic photoproducts with higher activity than the parent compound

In the present study, we aimed to evaluate the effect of UV-visible irradiation on the estrogenicity of an estrone aqueous solution by using chemical analysis associated with an in vitro bioassay and in silico analysis. An estrone aqueous solution was irradiated with an UV-visible high-pressure mercury lamp. By using the MELN in vitro cellular bioassay, based on the induction of a luciferase reporter gene upon the activation of the estrogen receptor by chemicals, we showed that the estrogenic potency of the solution increased after irradiation. High-performance liquid chromatography fractionation of the photolyzed solution followed by in vitro testing of fractions allowed the quantitation of the estrogenic potency of each fraction. Nine photoproducts were detected and characterized by liquid chromatography-mass spectrometry coupling. The observed estrogenic activity is mediated by mono- and multi-hydroxylated photoproducts; it is influenced by the position of hydroxyl groups on the steroidal skeleton. In addition, a structure-activity analysis of the hydroxylated photoproducts confirmed their ability to act as estrogen receptor ligands.