Effect-directed analysis for estrogenic compounds in a fluvial sediment sample using transgenic cyp19a1b-GFP zebrafish embryos

Abstract

Xenoestrogens may persist in the environment by binding to sediments or suspended particulate matter serving as long-term reservoir and source of exposure, particularly for organisms living in or in contact with sediments. In this study, we present for the first time an effect-directed analysis (EDA) for identifying estrogenic compounds in a sediment sample using embryos of a transgenic reporter fish strain. In the tg(cyp19a1b-GFP) transgenic zebrafish strain, the expression of GFP (green fluorescent protein) in the brain is driven by an oestrogen responsive element in the promoter of the cyp19a1b (aromatase) gene. The selected sediment sample of the Czech river Bilina had already been analysed in a previous EDA using the yeast oestrogen screening assay and had revealed fractions containing estrogenic compounds. When normal phase HPLC (high performance liquid chromatography) fractionation was used for the separation of the sediment sample, the biotest with transgenic fish embryos revealed two estrogenic fractions. Chemical analysis of candidate compounds in these sediment fractions suggested alkylphenols and estrone as candidate compounds responsible for the observed estrogenic effect. Alkylphenol concentrations could partially explain the estrogenicity of the fractions. However, xenoestrogens below the analytical detection limit or non-targeted estrogenic compounds have probably also contributed to the sample's estrogenic potency. The results indicated the suitability of the tg(cyp19a1b-GFP) fish embryo for an integrated chemical-biological analysis of estrogenic effects.

The value of zebrafish as an integrative model in effect-directed analysis - a review

“…When instead the aim of multiple methods was to analyse different aspects of the same bioactivity or toxicity mechanism, there was the prevalence of tiered approach biotesting [19,39,45]. When applied in screening phase, zebrafish bioassays aimed to identify active fractions by organism-level endpoints, which were later further investigated by additional methods with zebrafish [52] or with other experimental models [19,45]. As an example, zebrafish bioassays were applied to screen extracts and fractions for anti-angiogenic effects, followed by further investigations on human cells and transgenic zebrafish embryos [19].…”

“…[36,53,[55][56][57]) or a combination of methods (e.g. [44,52]) to evaluate endpoints in zebrafish. Other studies applied methods with additional experimental models, mostly cell-based (e.g.…”

“…Natural product studies aimed to identify bioactive compounds for pharmacological applications, investigating mostly plant extracts [19,[34][35][36][37][38][39][40][41][42][43][44][45] but also extracts of bacteria [46], cyanobacteria and algae [47], seaweed [48] and marine organisms [49]. Environmental toxicology studies aimed to identify the toxic compounds in various environmental samples, including marine and fluvial sediments [50][51][52], soil [53], cyanobacteria and algae [54,55], industrial effluent [33], rubber tyre leachates [32], oil sand process waters [56,57] and river pore water [58]. Finally, fish skin extracts were investigated in a behavioural sciences study [59].…”

Evaluation of estrogenic activity in the Pearl River by using effect-directed analysis

“…Previous studies found most estrogenic activity in polar fractions of log K ow 2-4 of effluent, river water, and sediment extracts (Creusot et al 2013;Grung et al 2011;Houtman et al 2004;Smital et al 2011). Fetter et al (2014 further suggested alkylphenols and estrone as Water F22 F16 F15 F14 F12 F10 F9 F8 F7 F5 F3 F2 S1 S2 S5 S8 S9 S10 S11 The present study did demonstrate that polar and mid-polar compounds such as E1, E2, and BPA are widely present in both water and sediment samples.…”

Soil Contamination and Human Health: A Major Challenge for Global Soil Security

“…In addition to the collection of legacy data on historical uses of sites, one of the solutions would be, to intensify the coupling between soil biomonitoring and 12 analytical tools and methods, where health effects are observed on soil fauna and flora. This can be done for instance with Effect Directed Analysis which aims to identify the compounds causing these effects after reducing sample complexity by combining biotests with fractionations (Fetter et al, 2014). More generally, further (eco)toxicological research would be necessary to better assess human health risk particularly on chemical forms of contaminants and their toxicity related to environmental and biological conditions.…”