High-Throughput Effect-Directed Analysis Using Downscaled in Vitro Reporter Gene Assays To Identify Endocrine Disruptors in Surface Water

Effect-directed analysis (EDA) is a commonly used approach for effect-based identification of endocrine disruptive chemicals in complex (environmental) mixtures. However, for routine toxicity assessment of, for example, water samples, current EDA approaches are considered time-consuming and laborious. We achieved faster EDA and identification by downscaling of sensitive cell-based hormone reporter gene assays and increasing fractionation resolution to allow testing of smaller fractions with reduced complexity. The high-resolution EDA approach is demonstrated by analysis of four environmental passive sampler extracts. Downscaling of the assays to a 384-well format allowed analysis of 64 fractions in triplicate (or 192 fractions without technical replicates) without affecting sensitivity compared to the standard 96-well format. Through a parallel exposure method, agonistic and antagonistic androgen and estrogen receptor activity could be measured in a single experiment following a single fractionation. From 16 selected candidate compounds, identified through nontargeted analysis, 13 could be confirmed chemically and 10 were found to be biologically active, of which the most potent nonsteroidal estrogens were identified as oxybenzone and piperine. The increased fractionation resolution and the higher throughput that downscaling provides allow for future application in routine high-resolution screening of large numbers of samples in order to accelerate identification of (emerging) endocrine disruptors

High-Throughput Effect-Directed Analysis Using Downscaled in Vitro Reporter Gene Assays To Identify Endocrine Disruptors in Surface Water

Effect-directed analysis (EDA) is a commonly used approach for effect-based identification of endocrine disruptive chemicals in complex (environmental) mixtures. However, for routine toxicity assessment of, for example, water samples, current EDA approaches are considered time-consuming and laborious. We achieved faster EDA and identification by downscaling of sensitive cell-based hormone reporter gene assays and increasing fractionation resolution to allow testing of smaller fractions with reduced complexity. The high-resolution EDA approach is demonstrated by analysis of four environmental passive sampler extracts. Downscaling of the assays to a 384-well format allowed analysis of 64 fractions in triplicate (or 192 fractions without technical replicates) without affecting sensitivity compared to the standard 96-well format. Through a parallel exposure method, agonistic and antagonistic androgen and estrogen receptor activity could be measured in a single experiment following a single fractionation. From 16 selected candidate compounds, identified through nontargeted analysis, 13 could be confirmed chemically and 10 were found to be biologically active, of which the most potent nonsteroidal estrogens were identified as oxybenzone and piperine. The increased fractionation resolution and the higher throughput that downscaling provides allow for future application in routine high-resolution screening of large numbers of samples in order to accelerate identification of (emerging) endocrine disruptors

Identification of Photosynthesis Inhibitors of Pelagic Marine Algae Using 96-Well Plate Microfractionation for Enhanced Throughput in Effect-Directed Analysis

Because of large-scale production and use of an increasing diversity of chemicals in modern society, estuarine and coastal waters may be contaminated with numerous substances. Some of these compounds have the potential to affect microalgae at the base of the pelagic food chain. Therefore, we identified the main chemical stressors that negatively affect the effective photosystem II efficiency (ϕPSII) in marine microalgae of the Dutch estuarine and coastal waters. An enhanced effect-directed analysis (EDA) was carried out by combining reversed-phase ultra performance liquid chromatography fractionation of extracts from passive samplers, followed by effect assessment using the pulse amplitude modulation fluorometry assay and chemical analysis of biologically active fractions using high-resolution mass spectrometry. This study focuses on a novel microfractionation technique using 96-well plates to enhance throughput in EDA, structure elucidation, and the analytical and effect confirmation of the compounds that are identified. Although there are numerous unknown compounds present in estuarine and coastal waters, our EDA study shows that atrazine, diuron, irgarol, isoproturon, terbutryn, and terbutylazine are the main contributors to the observed effect on the ϕPSII of marine microalgae

Development of an Online Cell-Based Bioactivity Screening Method by Coupling Liquid Chromatography to Flow Cytometry with Parallel Mass Spectrometry

This study describes a new platform for the fast and efficient functional screening for bioactive compounds in complex natural mixtures using a cell-based assay. The platform combines reversed-phase liquid chromatography (LC) with online flow cytometry (FC) and mass spectrometry (MS). As a model (an example or proof-of-concept study) we have used a functional calcium-flux assay in human neuroblastoma SH-SY5Y cells stably overexpressing the α-7 nicotinic acetylcholine receptor (α7-nAChR), a potential therapeutic target for central nervous system (CNS) related diseases. We have designed the coupled LC–FC system employing the neuroblastoma cells followed by analytical and pharmacological evaluation of the hyphenated setup in agonist and mixed antagonist–agonist assay modes. Using standard receptor ligands we have validated pharmacological responses and standardized good assay quality parameters. The applicability of the screening system was evaluated by analysis of various types of natural samples, such as a tobacco plant extract (in agonist assay mode) and snake venoms (in mixed antagonist–agonist assay mode). The bioactivity responses were correlated directly to the respective accurate masses of the compounds. Using simultaneous functional agonist and antagonist responses nicotine and known neurotoxins were detected from tobacco extract and snake venoms, respectively. Thus, the developed analytical screening technique represents a new tool for rapid measurement of functional cell-based responses and parallel separation and identification of compounds in complex mixtures targeting the α7-nAChR. It is anticipated that other fast-response cell-based assays (e.g., other ion flux assays) can be incorporated in this analytical setup

Comparison of morphological data and quantitative data for three venoms that have varying effects on the cells.

(A) Morphological data represented by immunofluorescent microscopy images showing morphology of RPTEC/TERT1cells after 24 hours exposure. Images were captured using confocal microscopy with 10X magnification. H342 staining is shown in blue, and PI in orange. 0.1% Triton T-100 was used as a positive control. Yellow stars represent wells in which activity was observed. Scale bar represents 200 μm. (B) Quantitative data of four cellular bioassays shown as bar graphs, with the activity of the venom represented relative to negative control (0 μg/mL). Live cell count (orange); cell surface area (grey); resazurin reduction activity (blue); ATP level (black). Increasing venom concentrations on the X-axes (in μg/mL) and percentage relative to negative control on the Y-axes. Measurements are presented as the mean of three individual experiments (N = 3), error bars depict SD; ‘*’ represents a statistically significant difference when compared to negative control, two-tailed test, p < 0.05 (Bonferroni-corrected).</p

DataSheet1_Optimizing drug discovery for snakebite envenoming via a high-throughput phospholipase A2 screening platform.docx

Snakebite envenoming is a neglected tropical disease that causes as many as 1.8 million envenomings and 140,000 deaths annually. To address treatment limitations that exist with current antivenoms, the search for small molecule drug-based inhibitors that can be administered as early interventions has recently gained traction. Snake venoms are complex mixtures of proteins, peptides and small molecules and their composition varies substantially between and within snake species. The phospholipases A2 (PLA2) are one of the main pathogenic toxin classes found in medically important viper and elapid snake venoms, yet varespladib, a drug originally developed for the treatment of acute coronary syndrome, remains the only PLA2 inhibitor shown to effectively neutralise venom toxicity in vitro and in vivo, resulting in an extremely limited drug portfolio. Here, we describe a high-throughput drug screen to identify novel PLA2 inhibitors for repurposing as snakebite treatments. We present method optimisation of a 384-well plate, colorimetric, high-throughput screening assay that allowed for a throughput of ∼2,800 drugs per day, and report on the screening of a ∼3,500 post-phase I repurposed drug library against the venom of the Russell’s viper, Daboia russelii. We further explore the broad-spectrum inhibitory potential and efficacy of the resulting top hits against a range of medically important snake venoms and demonstrate the utility of our method in determining drug EC50s. Collectively, our findings support the future application of this method to fully explore the chemical space to discover novel PLA2-inhibiting drugs of value for preventing severe pathology caused by snakebite envenoming.</p

Identification of bioactive fractions of <i>N</i>. <i>mossambica</i> venom (1 mg/mL) by correlating bioactivity data with proteomics data.

i: bioactivity chromatograms obtained by plotting the results of three bioassays: cell surface area (grey); resazurin reduction (blue); live cell count (orange). The peaks with negative minima indicate the presence of bioactive fractions; ii: Graphs representing the protein score chromatograms (PSCs), showing the individual venom proteins found with Mascot database searching of the digested well fractions. iii: UV traces of the snake venoms at 220 nm obtained by RP-HPLC. The vertical outer lines mark the bioactivity window, which includes the main activity peaks and their corresponding PSC peaks and RP-HPLC-UV chromatogram peaks. Measurements are presented as the mean of three individual experiments (N = 3), error bars depict SD.</p

Brightfield microscopy images of RPTEC/TERT1 cells in response to three different venoms, which were used to quantify the cell surface area.

Conditions shown for three different venoms: cytotoxic viper, cytotoxic elapid and non-cytotoxic elapid. Top row represents the ‘training’ of the software to differentiate cell populations (red dots) from the ‘empty well area’ (green dots) using a simple learn-by-example approach. The lower set of images provides the calculated cell surface area (depicted in red) and the ‘empty well area’ (in green). The scale bar represents 200 μm. (EPS)</p

Brightfield and immunofluorescent microscopy images showing the difference in morphology of RPTEC/TERT1 cells in presence of two venoms with contrasting effects.

(A) Cells exposed to 100 μg/mL E. ocellatus venom, with various channels (brightfield, Hoechst & PI) at 10X, 20X and 63X magnification. (B) Cells exposed to 100 μg/mL N. mossambica venom, with various channels (brightfield, Hoechst & PI) at 10X, 20X and 63X magnification. (C). Time series (0–150 min) of the effects of E. ocellatus venom (100 μg/mL), with a higher-magnification section (63X) clearly showing the monolayer detachment (60–70 min). (D). Time series (0–15 min) of the effects of N. mossambica venom (100 μg/mL) on cells, with a higher-magnification section (63X) showing the PI entering the cell (0–15 min). Scale bars lengths represented in the images.</p

All raw data files, including raw bioassay data and proteomics data.

All raw data files, including raw bioassay data and proteomics data.</p