Label-Free Biosensor Detection of Endocrine Disrupting Compounds Using Engineered Estrogen Receptors.

Endocrine Disrupting Compounds (EDCs) are chemical substances shown to interfere with endogenous hormones affecting the endocrine, immune and nervous systems of mammals. EDCs are the causative agents of diseases including reproductive disorders and cancers. This highlights the urgency to develop fast and sensitive methods to detect EDCs, which are detrimental even at very low concentrations. In this work, we propose a label-free surface plasmon resonance (SPR) biosensor method to detect specific EDCs (17 ß-estradiol (E2), ethinyl-estradiol, 4-nonylphenol, tamoxifen) through their binding to estrogen receptor alpha (ERα). We show that the use of rationally designed ERα (as bio-recognition element) in combination with conformation-sensitive peptides (as amplification agent, resulting in increased responses) enables the detection of low parts per billion (ppb) levels of E2. As a proof of concept, this bioassay was used to detect E2 in (spiked) real water samples from fish farms, rivers and the sea at low ppb levels after concentration by solid phase extraction. In addition, the present SPR assay that combines a conformation-sensitive peptide with an array of ERα mutants is very promising for the assessment of the risk of potential estrogenic activity for chemical substances.

Mixtures of chemical pollutants at European legislation safety concentrations: how safe are they?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations.

Receptor-based high-throughput screening and identification of estrogens in dietary supplements using bioaffinity liquid-chromatography ion mobility mass spectrometry.

A high-throughput bioaffinity liquid chromatography-mass spectrometry (BioMS) approach was developed and applied for the screening and identification of recombinant human estrogen receptor α (ERα) ligands in dietary supplements. For screening, a semi-automated mass spectrometric ligand binding assay was developed applying (13)C2, (15) N-tamoxifen as non-radioactive label and fast ultra-high-performance-liquid chromatography-electrospray ionisation-triple-quadrupole-MS (UPLC-QqQ-MS), operated in the single reaction monitoring mode, as a readout system. Binding of the label to ERα-coated paramagnetic microbeads was inhibited by competing estrogens in the sample extract yielding decreased levels of the label in UPLC-QqQ-MS. The label showed high ionisation efficiency in positive electrospray ionisation (ESI) mode, so the developed BioMS approach is able to screen for estrogens in dietary supplements despite their poor ionisation efficiency in both positive and negative ESI modes. The assay was performed in a 96-well plate, and all these wells could be measured within 3 h. Estrogens in suspect extracts were identified by full-scan accurate mass and collision-cross section (CCS) values from a UPLC-ion mobility-Q-time-of-flight-MS (UPLC-IM-Q-ToF-MS) equipped with a novel atmospheric pressure ionisation source. Thanks to the novel ion source, this instrument provided picogram sensitivity for estrogens in the negative ion mode and an additional identification point (experimental CCS values) next to retention time, accurate mass and tandem mass spectrometry data. The developed combination of bioaffinity screening with UPLC-QqQ-MS and identification with UPLC-IM-Q-ToF-MS provides an extremely powerful analytical tool for early warning of ERα bioactive compounds in dietary supplements as demonstrated by analysis of selected dietary supplements in which different estrogens were identified.

Fluorescence detection of ligand binding to labeled cytochrome P450 BM3.

The cytochrome P450 superfamily of monoxygenases are highly relevant for pharmaceutical, environmental and biocatalytical applications. The binding of a substrate to their catalytic site is usually detectable by UV-vis spectroscopy as a low-to-high spin state transition of the heme iron. However, the discovery of potential new substrates is limited by the fact that some compounds do not cause the typical spin-shift even if they are oxidised by P450 enzymes. Here we report a fluorescence-based method able to detect the binding of such substrates to the heme domain of cytochrome P450 BM3 from Bacillus megaterium. The protein was labeled with the fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-Yl)-ethylenediamine (IANBD). Arachidonic and lauric acids are substrates of P450 BM3 and were used to validate the method, as their binding can be detected both by a spin-shift of the Soret peak from 419 to 397 nm and by the fluorescence change of the labelled protein. The fluorescence emission of the probe linked to the protein increased by a value corresponding to 121 ± 9% and 52 ± 5% with respect to the initial one, upon titration with arachidonic or lauric acids respectively. The dissociation constants were calculated by both UV-vis and fluorescence spectroscopy. Three drugs, propranolol, chlorzoxazone and nifedipine, known to be oxidized by P450 BM3 and that bind without causing spin-shift, were also tested and the fluorescence emission of IANBD was found to decrease by 29 ± 5%, 21 ± 2% and 23 ± 3%, respectively, allowing the measurement of their dissociation constants.

Direct spectroscopic evidence for binding of anastrozole to the iron heme of human aromatase. Peering into the mechanism of aromatase inhibition.

Aromatase (CYP19A1), is a microsomal cytochrome P450 catalysing the conversion of androgens to estrogens. Non-steroidal inhibitors, such as anastrozole, are important drugs in breast cancer therapy. Using hyperfine sublevel correlation (HYSCORE) spectroscopy we provide the first experimental evidence of the binding of anastrozole to the iron heme of human aromatase.

Protein and electrode engineering for the covalent immobilization of P450 BMP on gold.

Site-directed mutagenesis and functionalization of gold surfaces have been combined to obtain a stable immobilization of the heme domain of cytochrome P450 BM3 from Bacillus megaterium. Immobilization experiments were carried out using the wild type protein bearing the surface C62 and C156 and the site-directed mutants C62S, the C156S, and the double mutant C62S/C156S (no exposed cysteines). The gold surface was functionalized using two different spacers: cystamine- N-succinimidyl 3-maleimidopropionate and dithio-bismaleimidoethane, both leading to the formation of maleimide-terminated monolayers capable of covalent linkage to cysteine. Tapping mode atomic force microscopy experiments carried out on cystamine- N-succinimidyl 3-maleimidopropionate derivatized gold led to good images with expected molecular heights (5.5-6.0 nm) for the wild type and the C156S mutant. These samples also gave measurable electrochemical signals with midpoint potentials of -48 and -58 mV for the wild type and C156S, respectively. On the other hand, the dithio-bismaleimidoethane spacer led to variability on the molecular heights measured by tapping mode atomic force microscopy and the electrochemical response. This is interpreted in terms of lack of homogeneous dithio-bismaleimidoethane monolayer on gold. Furthermore, results from tapping mode atomic force microscopy show that the double mutant and the C62S did not lead to stably immobilized P450 protein, confirming the necessity of the solvent exposed C62.

Solid-phase extraction of ochratoxin A from wine based on a binding hexapeptide prepared by combinatorial synthesis.

In this paper we describe the preparation of a hexapeptide library by combinatorial synthesis and the identification of a peptide with sequence Ser-Asn-Leu-His-Pro-Lys, which showed good affinity (K(eq)=3.4 x 10(4) M(-1)) towards the mycotoxin ochratoxin A (OTA). An immunoaffinity-like stationary phase supporting such a hexapeptide was used to develop a solid-phase extraction method for the quantification of OTA in wine samples at concentration levels down to 0.10 microg l(-1). Several different wine samples fortified with OTA at 2 and 4 microg l(-1) levels showed recovery of 94.7% and 98.4% at 2.0 and 4.0 microg l(-1), respectively, without any effect on the extraction efficiency of the matrix. The efficacy of this approach was successfully tested by comparison with an immunoaffinity extraction performed on commercial cartridges.