Deliverables
Identification of the harmful effects of pharmaceuticals and cosmetics is essential, although often requires costly and time-consuming animal testing. In the last decade the effects of chemicals on the endocrine system have been identified as a potential cause for concern and have promoted considerable political debate and research activity. It is still difficult to identify definitively endocrine disrupters, and alternatives to animal tests are sought.
One such alternative is the use of computational modelling. Techniques such as quantitative structure-activity relationships (QSARs) attempt to relate the toxicity of chemicals to their physico-chemical and structural properties. In order to develop QSARs successfully many high quality data are required. The purpose of this study was to collate data relating to endocrine disruption into a consistent database.
A comprehensive database has been compiled including information about the name, CAS, 2D-structure in the form of SMILES strings, and all available endpoints of suspected endocrine disrupting, as well as being non-active, for over 500 chemicals. The data on the chemicals have been collected from the literature. The chemicals are grouped according to the structural type they belong to. This database should become a reliable and publicly available source for an effective screening and testing of endocrine disrupters. In addition to the biological data a large number of pertinent physico-chemical properties and structural descriptors were calculated and included in the database.
The full database (approximate size 25 MB) is available on request.
There is a significant requirement to predict biological activity from chemical structure. There are various methods to achieve this, all loosely defined as quantitative structure-activity relationships (QSARs). Such tools have proven successful in priority setting for risk assessment and are likely to gain some form of acceptance in the regulatory community. QSAR modelling employs statistical approaches to correlate or rationalise variations in the biological activity of a series of chemicals with variations in molecular structure.
In this study a large database of quantitative and qualitative data have been collated for oestrogenicity and two different QSAR approaches were applied to enable prediction from structure. The first approach is introduction of simple molecular features as rejection filters to eliminate chemicals that are unlikely to be oestrogenic. The rejection filters should not generate any false negatives and be able to reduce significantly the number of chemicals for further evaluation.
The dataset used in this study consisted of over 500 chemicals for which at least one oestrogenicity endpoint was available. Following molecular features were studied: molecular weight range, lack of a ring motif, log P range, number of hydrogen bond donor and acceptor groups and number of flexible bonds. It was founds that molecular weight range together with the lack of a ring motif were successful criteria, eliminating larger number of inactive chemicals with no false negatives. The second QSAR approach involved a linear correlation between oestrogen receptor binding affinity of 131 chemicals and the structural descriptors. The resulting QSAR was able to predict log RBA values of chemicals in the test set with a good accuracy.
In order to study oestrogenic activity more thoroughly, three-dimensional (3D) quantitative structure-activity relationship (QSAR) and structure-activity relationship (SAR) analyses were applied concurrently to a data set of highly selective oestrogen receptor (ER) alpha and beta agonists.
The data set consisted of diphenolic azoles characterized by similar structural skeletons but with different binding modes to the oestrogen receptor site. Models were developed separately with respect to the relative binding affinities (RBAs) to ER alpha and ER beta. Steric and electrostatic fields were calculated for a training set of 72 compounds using comparative molecular field analysis (CoMFA). The model developed for ER alpha RBA yielded a squared correlation coefficient of 0.91 and a cross-validated squared correlation coefficient of 0.60. The model developed for ER beta RBA yielded a squared correlation coefficient of 0.95 and a cross-validated squared correlation coefficient of 0.40. Both models were validated successfully using an external test set of 32 compounds.
A new concept of test set evaluation based on the variability of the biological response due to the variability of the living organism was been introduced. The CoMFA analysis was supported by a SAR study. In addition to the most favourable steric and electrostatic regions identified by CoMFA, a number of structural descriptors were identified as being important for binding.
Quantitative activity-activity relationships were also investigated to determine the relationship between biological assays. The measurements of oestrogen disruptor chemicals resulting from the [3H]-estradiol radioligand binding assay, reporter gene assay, and E-screen assay for determination of the estrogenicity were evaluated with respect to the correlation between the endpoint values and the concordance between the assays. The correlation between the data points was determined using regression analysis. The concordance between the assays was determined by the estimation of the statistical equality of two populations using the Kolmogorov-Smirnov test.
Experiments were carried out for the following cases:
- For endpoints of compounds for which identical or different assays were performed in different laboratories and
- For endpoints of compounds for which all assays were performed in the same laboratory.
The results demonstrate that for a small number of measurements (17-21 datapoints), the endpoint pKi shows high correlation to the RBA (derived from IC50 or EC50), RA and PC50 endpoints and agonism. The Kolmogorov-Smirnov test resulted in no evidence of an existing difference between the assays applied to the endpoint pKi and the endpoints RBA, RA and PC50, respectively for the same number of data points.
The significance of the QSAR and QAAR analysis is that they provide methods to ascertain the hazard associated with a chemical without resort to costly and time-consuming animal testing.
The induction of vitellogenin (Vtg) in fish is an estrogen dependent process, which normally active in maturing females, can also be activated, in immature females and males when exposed to estrogens and their mimics. Paralleling what happens in plasma, Vtg can be detected in skin mucus and enhanced by estrogens. This observation has supported the proposal of using skin mucus Vtg as a non destructive biomarker of exposure to estrogens.
The necessary premise to a routine application of this biomarker was the development of a simple assay, which can rapidly detect Vtg in the mucus of large numbers of fish. The method of choice was immunological and the dipstick assay developed for carp Vtg was applied both to fish exposed in laboratory and to the many individuals captured in seven areas of the River Po basin, with different levels of contamination. The point of strength of this tool is that it is non destructive and harmless to fish. Moreover, although still a prototype, the dipstick resulted to be very rapid to perform and of easy applicability, particularly in field. Also the evaluation of results is quite simple, since a coloured test line appears when Vtg is present in the sample.
Comparison of Vtg levels in plasma and mucus of the same fish using a quantitative sandwich ELISA previously established (Nilsen et al., 2004) indicated a positive correlation in almost all groups of carp investigated. Interestingly, in some of the fish groups, no correlation or a negative correlation was observed. This was most pronounced in samples from the field and from the tamoxifen (TAM) exposure groups. The lack of correlation between plasma and mucus Vtg indicates different turnover of Vtg in these sample types. In the field sampling, there was a general lack of Vtg in the mucus, although varying levels of Vtg was found in plasma. It is possible that fish of various age groups show different Vtg kinetics, and that fish displaying correlations that were more consistent and significant, like the EE2, flutamide, methyldihydrotestosterone, and mixed exposure, were from more homogeneous populations.
Therefore, care should be taken when using the non-invasive strategy in the field. Apparently, exposure to antiestrogens may disturb the Vtg turnover such that mucus and plasma Vtg are no longer in equilibrium. Possibly, a blood sample should be taken, which still could be analysed by the LFIA in a few minutes in the field. From larger specimens, blood sampling could also be a non-destructive strategy. Further studies should be performed to elucidate this.
The impact of endocrine disrupting chemicals (EDC) on sex steroids, biomarkers and histology in Xenopus laevis were investigated. This combined approach of different endpoints for endocrine disruption should deliver information about the sensitivity of examined endpoints for the disruption of normal endocrine function.
For this reason, X. laevis were exposed in vivo to an estrogenic compound, ethinylestradiol (EE2), an antiestrogenic compound, tamoxifen (TAM), an androgenic compound, methyldihydrotestosterone (MDHT), an antiandrogenic compound, flutamide (FLU) at a concentration of 10-8M and to Lambro river water for four weeks. Biomarkers (RBP, TTR and TF as described in detail at Nr. 1 of main results) and sex steroids (estradiol and testosterone) were changed by (anti)estrogenic but not by (anti)androgenic model compounds.
In contrary, severe changes in the histology of gonads of X. laevis were found by all treatments including the exposure to Lambro river water.
Summarising, histology of gonads was a suitable endpoint for endocrine disruption indicating (anti)estrogenic and (anti)androgenic mode of actions. The mRNA expression of examined genes and sex steroids showed endocrine disruption by (anti)estrogenic mode of action. Biomarkers for (anti)androgenic mode of actions have still to be developed.
The effects of endocrine disrupting compounds (EDC) on the hypothalamus-pituitary-gonad axis, regulating reproduction, were investigated in Xenopus laevis by determining their potential impact on gene expression of luteinizing hormone β-subunit (LHβ) and follicle-stimulating hormone β-subunit (FSHβ) in brain and pituitary using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).
Lambro river water and four model compounds, ethinylestradiol (EE2), tamoxifen (TAM), methyldihydrotestosterone (MDHT), flutamide (FLU), corresponding to (anti)estrogenic and (anti)androgenic modes of action were used at 10-8 M during a four weeks exposure of adults of both sexes. EE2 and MDHT treatment decreased LHβ mRNA expression in the brain of male X. laevis, while only EE2 but not MDHT reduced LHβ mRNA in females indicating classical negative feed-back mechanisms on hypophyseal gonadotropin expression. TAM increased LHβ mRNA and FSHβ mRNA expression in female X. laevis while none of the other treatments showed an effect on FSHβ mRNA expression. Exposure of X. laevis to Lambro river water had no significant effect on any of the genes examined.
It is reported for the first time in amphibians that gonadotropin mRNA expression is differentially regulated by (anti)estrogenic and (anti)androgenic EDC indicating disturbance of reproductive processes at higher regulatory centres.
Results are concerned with the development and application of new analytical methods in GC-MS and LC-MS/MS for the identification and quantification of selected endocrine disrupting compounds in environmental and biological samples: water, sediment, macroinvertebrates, tissue from fishes plasma, bile, liver, muscle.
The analytical work has been concentrated on a restricted number of potentially effective chemicals confirmed both to be endocrine disrupting substances and, in the mean time, to be really present in relevant way in the areas utilised for in field applicability of novel testing methods. The selected analytes have been estrone, estradiol, ethynilestradiol, estrone-gluoronide, estradiol-3gluronide, estradiol-17glucoronide, octylphenol, nonylphenol mix, bisphenol A, tertoctylphenol.
Experiments have been performed in order to optimise the sample preparation steps, mainly based on solid phase (SPE) methodologies. GC-MS and LC-MS/MS procedures have been developed. Studies have been performed in order to determine the characteristics of the developed procedures in term of reproducibility, limits of detection and quantification and linearity.
Real wild-life exposure and controlled environments has been studied to characterize environmental level of EDCs and to evaluate experimentally the xenobiotic concentrations able to induce the disruptive response in the studied organisms.
Analytical methods will be published and freely available in scientific literature.
Endocrine disrupting chemicals (EDC) are known to interfere with the normal endocrine system of different animals including humans. EDC can have adverse effects on the reproduction like changed sex ratios, intersex phenomena, reproductive malformations or reduced fecundity.
The use of mRNA expression of retinol binding protein (RBP), transthyretin (TTR) and transferrin (TF) were investigated as potential biomarkers for EDC in the liver of Xenopus laevis as a model organism. For this reason, X. laevis were exposed in vivo to an estrogenic compound, ethinylestradiol (EE2), an antiestrogenic compound, tamoxifen (TAM), an androgenic compound, methyldihydrotestosterone (MDHT), an antiandrogenic compound, flutamide (FLU) at a concentration of 10-8M and to Lambro river water for four weeks. TTR mRNA expression was inhibited by estrogens in male and female frogs.
In contrary, RBP mRNA was up-regulated by estrogens in males and females. The mRNA expression of TF was down-regulated by estrogens in males and females but also upregulated by antiestrogens in females.
Summarising RBP and TTR can serve as estrogenic biomarker while TF can be used as (anti)estrogenic biomarker.
One of the aims of the EASYRING project was to identify new biomarker candidates to endocrine disrupting compounds in fish and amphibians. Carp (Cyprinus carpio) and African clawed frog (Xenopus laevis) were selected as test organism for in in vivo exposures by four model compounds, ethynylestradiol (EE2), tamoxifen (TAM), metyldihydrotestosterone (MDHT) and flutamide (FLU).
Protein patterns in of different tissues (liver, plasma and mucus) were analysed by 2DE and compared with control samples. Proteins with changed levels and/ or altered pI were excised from the gels, trypsinised and analyzed by MALDI-TOF or LC-MS/MS for identification by database searches using mass-lists or unassigned MS spectra. MALDI-TOF MS-spectra were calibrated and processed with FlexAnalysis and Biotools (Bruker Daltonics) for database-searches in NCBInr using Mascot (Matrix Science). For analyses with LC-MS/MS data merge (mgf) files of the MS/MS spectra were used for peptide searches in NCBInr using Mascot and Phenyx (GeneBio). Hypotetical proteins of unknown identity and function were subjected to Blastp searches for identification.
After 2DE analyses of carp plasma samples, we chose 3 proteins as candidate biomarker proteins for endocrine disruption in carp exposed to estrogenic compounds (64 ng/l EE2). These were: 1-antitrypsin (A1AT), transferrin (TRA) and warm-temperature-acclimation-related-65 kDa-protein (WTA-65). We have selected two antigens to each protein based on different criteria for peptide selection for antibody production. Six carp specific polyclonal antibodies have been produced ands these are being tested and validated as possible new biomarker candidates.
Xenopus laevis were exposed to EE2, TAM, MDHT, FLU (all 10-8 M) and 2-DE analysis was performed on pooled samples from each treatment group. Males and females were analyzed separately and compared to controls. Estrogen regulated protein Ep45 was found to be a new candidate biomarker protein for estrogenic exposure to Xenopus. The proteins specifically repressed by MDHT treatment were identified as enolase, creatine kinase, endodermin and pentraxin fusion protein precursor. In response to FLU treatment two isoforms of albumin (68 and 74 kDa) appeared to be specifically upregulated in both male and female samples.
Of the identified proteins in Xenopus liver after exposure to EE2, oxidative stress related proteins were upregulated. These include Hsp70, Hsp90, catalase and thioredoxin.
Endocrine disrupters (estrogens, androgens, estrogenic and androgenic pharmacological agonists and antagonists) were shown to differentially interfere with several biological features of mammalian reproduction.
Four of the six drugs we tested were shown to be deleterious.
Estrogen EE2 but not E3 exerts a clearcut inhibitory effects on in vitro oocyte maturation delays zygote cleavage, as well as the very early morphogenetic events. Bisphenol A, a widely used pharmacological estrogen-receptor agonist was shown to be inhibitory of oocyte maturation only. On the opposite, tamoxifen, an estrogen antagonist has no effect on maturation but is highly toxic on secondary oocytes and cleaving embryos.
As far as androgenic pathway is concerned, the androgen MDHT was shown to exert no effect at all on all biological phenomena we analysed while the antagonist tamoxifen is dramatically inhibitory of oocyte maturation and also delays zygote cleavage.
It should be pointed out that delaying effect on cleavage of EE2, tamoxifen and flutamide was confirmed on proliferating F9 carcinoma cells. No effect was however detected on their in vitro differentiation into parietal extraembryonic endoderm.
Finally, our assays did not allow detecting any deleterious effect of alls these compounds at low concentrations similar to the one we used in Lambro mixtures.
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