Intercalibration exercise using a stickleback endocrine disrupter screening assay (original) (raw)

The 21-day androgenised female stickleback endocrine screening assay

2009

1 2 Executive summary 1. The current OECD test guidelines for screening chemicals with potential endocrine 3 disrupting activity (TG 229 and TG 230) cannot clearly identify androgen antagonists due 4 to the lack of a suitable end-point in the 3 core species involved, the fathead minnow, the 5 medaka and the zebrafish. 6 2. The reported antiandrogenic activity in the aquatic environment based on the Yeast 7 Androgen Screen (YAS), a suitable in vitro test, is substantial. The evidence of high levels 8 of antiandrogens in the environment emphasises the need to develop a suitable assay for 9 chemical screening. 10 3. The 3-spined stickleback posses a unique trait, the presence of an androgen regulated 11 protein in their kidney, spiggin, which can be deployed in the detection of environmental 12 androgens and antiandrogens. 13 4. There are two in vivo tests using the stickleback that have the potential to identify 14 compounds with antiandrogenic activity, the stickleback-breeding test and the 15 androgenised female stickleback screen. 16 5. The design of the androgenised female stickleback screen (AFSS) is better suited as an 17 endocrine screen due to its simplicity and reproducibility in any laboratory. The fish are 18 simultaneously treated with a model androgen (dihydro-testosterone, DHT) at 5g/L and a 19 range of concentrations of the putative antiandrogen. Any antiandrogenic activity is 20 detected by the degree of reduction/inhibition of spiggin induction by the DHT treatment. 21 6. This report also addresses a retrospective validation of a large dataset using the AFSS 22 collaboration with Japanese co-workers (Nagae et al, 2007) but more methods are coming 87 to light (Hogan et al, 2009). 88 15. The bulk of the existing data were produced during the course of the EU-funded project 89 Endocrine disrupters: exploring novel endpoints, exposure, low-dose-and mixture-effects in 90 humans, aquatic wildlife and laboratory animals (EDEN), that was particularly focusing on 91 the effects of chemical mixtures. A sub-set of the data for exposures to single chemicals 92 rather than mixtures has been analysed in this report. 93 16. The second milestone was to undertake independent statistical analysis of the existing 94 data to assess whether or not the main end-point of the assay (kidney spiggin levels) has the 95 3 17. In support of the third milestone we generated of a small set of data anticipated as 99 essential for the validation process under the OECD guidelines. These data were focused 100 on two separate items: 101  To investigate the effect of carrier solvent on the end-point employed by the assay. The 102 OECD guideline for the fish screen explicitly states that the use of solvent should be avoided all together and if it absolutely necessary to employ a solvent for the in vivo exposures then the level of solvent should not exceed 0.01% at the fish aquaria. However all existing data employed methanol as a carrier solvent for administering the chemicals to the experimental aquaria reaching a final concentration of 0.1%. 103 104 105 106 107 109 110 111 119 120  Investigate the effect of a non-endocrine active substance on the end-point employed by 108 the assay. Currently the OECD guideline suggests that either potassium permanganate and/or n-Octanol are suitable negative control substances. We investigated the suitability of potassium permanganate (PP) for this purpose.

Comparison of Short-Term Estrogenicity Tests for Identification of Hormone-Disrupting Chemicals

Environmental Health Perspectives, 1999

The aim of this study was to compare results obtained by eight different short-term assays of estrogenlike actions of chemicals conducted in 10 different laboratories in five countries. Twenty chemicals were selected to represent direct-acting estrogens, compounds with estrogenic metabolites, estrogenic antagonists, and a known cytotoxic agent. Also included in the test panel were 1 7,Bestradiol as a positive control and ethanol as solvent control. The test compounds were coded before distribution. Test methods included direct binding to the estrogen receptor (ER), proliferation of MCF-7 cells, transient reporter gene expression in MCF-7 cells, reporter gene expression in yeast strains stably transfected with the human ER and an estrogen-responsive reporter gene, and vitellogenin production in juvenile rainbow trout. 17p-Estradiol, 177a-ethynyl estradiol, and diethylstilbestrol induced a strong estrogenic response in all test systems. Colchicine caused cytotoxicity only. Bisphenol A induced an estrogenic response in all assays. The results obtained for the remaining test compounds-tamoxifen, ICI 182.780, testosterone, bisphenol A dimethacrylate, 4-n-octylphenol, 4-n-nonylphenol, nonylphenol dodecylethoxylate, butylbenzylphthalate, dibutylphthalate, methoxychlor, o,p'-DDT, p,p'-DDE, endosulfan, chlomequat chloride, and ethanol-varied among the assays. The results demonstrate that careful standardization is necessary to obtain a reasonable degree of reproducibility. Also, similar methods vary in their sensitivity to estrogenic compounds. Thus, short-term tests are useful for screening purposes, but the methods must be further validated by additional interlaboratory and interassay comparisons to document the reliability of the methods.

Comparison of shor-term estrogenicity tests for identification of hormone-disrupting chemicals

1999

The E-screen assay as a tool to identify estrogens: An update on estrogenic environmental pollutants

Environmental Health Perspectives, 1995

Estrogens are defined by their ability to induce the proliferation of cells of the female genital tract. The wide chemical diversity of estrogenic compounds precludes an accurate prediction of estrogenic activity on the basis of chemical structure. Rodent bioassays are not suited for the large-scale screening of chemicals before their release into the environment because of their cost, complexity, and ethical concerns. The E-SCREEN assay was developed to assess the estrogenicity of environmental chemicals using the proliferative effect of estrogens on their target cells as an end point. This quantitative assay compares the cell number achieved by similar inocula of MCF-7 cells in the absence of estrogens (negative control) and in the presence of 1 7,B-estradiol (positive control) and a range of concentrations of chemicals suspected to be estrogenic. Among the compounds tested, several "new" estrogens were found; alkylphenols, phthalates, some PCB congeners and hydroxylated PCBs, and the insecticides dieldrin, endosulfan, and toxaphene were estrogenic by the E-SCREEN assay. In addition, these compounds competed with estradiol for binding to the estrogen receptor and increased the levels of progesterone receptor and pS2 in MCF-7 cells, as expected from estrogen mimics. Recombinant human growth factors (bFGF, EGF, IGF-1) and insulin did not increase cell yields. The aims of the work summarized in this paper were a) to validate the E-SCREEN assay; b) to screen a variety of chemicals present in the environment to identify those that may be causing reproductive effects in wildlife and humans; c) to assess whether environmental estrogens may act cumulatively; and finally d) to discuss the reliability of this and other assays to screen chemicals for their estrogenicity before they are released into the environment. -Environ Health Perspect 1 03(Suppl 7): 113-122 (1995)

Comparative responsiveness to natural and synthetic estrogens of fish species commonly used in the laboratory and field monitoring

Aquatic Toxicology, 2012

Exposure to estrogenic chemicals discharged into the aquatic environment has been shown to induce feminization in wild freshwater fish and although fish species have been reported to differ in their susceptibility for these effects, empirical studies that directly address this hypothesis are lacking. In this study, in vitro ER␣ activation assays were applied in a range of fish species used widely in chemical testing (including, zebrafish, fathead minnow, medaka) and/or as environmental monitoring species (including, roach, stickleback, carp) to assess their comparative responsiveness to natural (estrone, estradiol, estriol) and synthetic (17␣-ethinylestradiol (EE2), diethylstilbestrol (DES)) estrogens. In vivo exposures to EE2 via the water (nominal 2 and 10 ng/L for 7 days) were also conducted for seven fish species to compare their responsiveness for hepatic vitellogenin (VTG) mRNA induction (an ER mediated response). Of the fish species tested, zebrafish ER␣ was found to be the most responsive and carp and stickleback ER␣ the least responsive to natural steroid estrogens. This was also the case for exposure to EE2 with an ER␣mediated response sensitivity order of zebrafish > medaka > roach > fathead minnow > carp > stickleback. For VTG mRNA induction in vivo, the order of species responsiveness was: rainbow trout (not tested in the ER␣ activation assays) > zebrafish > fathead minnow > medaka > roach > stickleback > carp. Overall, the responses to steroid estrogens in vitro via ER␣ compared well with those seen in vivo (VTG induction for exposure to EE2) showing in vitro screening of chemicals using fish ER␣-mediated responses indicative of estrogenic responses (VTG induction) in vivo.

An exposure:activity profiling method for interpreting high-throughput screening data for estrogenic activity—Proof of concept

Regulatory Toxicology and Pharmacology, 2015

Rapid high throughput in vitro screening (HTS) assays are now available for characterizing dose-respons- 35 es in assays that have been selected for their sensitivity in detecting estrogen-related endpoints. For 36 example, EPA's ToxCast™ program recently released endocrine assay results for more than 1800 sub-37 stances and the interagency Tox21 consortium is in the process of releasing data for approximately 38 10,000 chemicals. But such activity measurements alone fall short for the purposes of priority setting 39 or screening because the relevant exposure context is not considered. Here, we extend the method of 40 exposure:activity profiling by calculating the exposure:activity ratios (EARs) using human exposure esti-41 mates and AC50 values for a range of chemicals tested in a suite of seven estrogenic assays in ToxCast™ 42 and Tox21. To provide additional context, relative estrogenic exposure:activity quotients (REEAQ) were 43 derived by comparing chemical-specific EARs to the EAR of the ubiquitous dietary phytoestrogen, genis-44 tein (GEN). Although the activity of a substance in HTS-endocrine assays is not a measure of health haz-45 ard or risk, understanding how such a dose compares to human exposures provides a valuable additional 46 metric that can be used in decision-making; substances with small EARs and REEAQs would indicate low 47 priority for further endocrine screening or testing.

In Vitro Models in Endocrine Disruptor Screening

The public and scientific concern that chemicals present in the human diet and the environment and their ability to disrupt the normal hormonal milieu in humans and wildlife have become a high-profile international issue. In 1998, the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) convened by the Environmental Protection Agency (EPA) recommended a tiered testing approach for the evaluation of estrogen, androgen, and thyroid-related effects of some 87,000 commercial chemicals and environmental contaminants. The function of this committee concluded with its final report, and the further implementation of the recommended testing strategy has now been carried forward with the assistance of the Endocrine Disruptor Methods Validation Subcommittee. The function of this body is to provide advice to the EPA on scientific and technical issues related specifically to the conduct of studies required for the validation of assays proposed by the EDSTAC as part of the tiered screening program. The EDSTAC recommended and alternative screening batteries encompass four in vitro mammalian assays. The current methodologies and validation status of the proposed in vitro EDSTAC assays are discussed and consist of estrogen/ androgen receptor binding, estrogen/androgen gene transactivation, and minced testis, and one alternate (placental aromatase) in vitro screening assay.

Intercalibration exercise using a stickleback endocrine disrupter screening assay (original) (raw)

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