Naslov
An assay for quantitative detection of reactive toxicity in whole zebrafish embryos - case of two pesticides

Autori
Seiler, Thomas-Benjamin ; Lackmann, Carina ; Hollert, Henner ; Rainieri, Sandra ; Martinez Santos, Monica, Spirhanzlova, Petra ; Velki, Mirna

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
7th SETAC World Congress and SETAC North America 37th Annual Meeting: Abstracts / - , 2016

Skup
7th SETAC World Congress and SETAC North America 37th Annual Meeting

Mjesto i datum
Orlando (FL), Sjedinjene Američke Države, 06.11.2016. - 10.11.2016

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
reactive toxicity; zebrafish embryos; fluorescent dye; GSH; ROS

Sažetak
Reactive toxicity is a first line response of cells upon exposure to toxic substances and part of the process that leads to cytotoxicity of contaminants. Detection of reactive toxicity in complex organisms can help to identify cell stress way before effects become visible on an organ or organism level. This allows for an early and sensitive identification of adverse effects. A zebrafish embryo assay that uses a fluorescent dye for microscopic detection of glutathion was adapted to a 96-well plate format to allow for quantification of reactive toxicity through fluorescence measurement with a multiwell plate reader. The assay was initially developed using tert-butyl hydroperoxide (t-BHP) as a reference substance, and then benchmark tested against the two pesticides diazinon and diuron. Long and short- term exposure scenarios and different protocols for the fluorescent staining were used. Hatched larvae (96 hpf) were then anesthetized, transferred to 96-well plates with conical wells, and fluorescence measured using a tailored detection method for a multiwell plate reader. Embryos were also inspected under a fluorescence microscope to qualitatively record locations and intensities of reactive toxicity in the individual specimen. Prior to the assay, lethal and sublethal effects of the pesticides on zebrafish embryos had been determined in a common FET test. We found that the assay is capable of detecting reactive toxicity of diazinon and diuron at concentrations as low as 0.1 mg/L and 0.02 mg/L, respectively, using the long-term exposure scenario. For the short-term exposure LOECs were 2 mg/L for both pesticides. For the reader measurement we could not observe any difference in the data of repeated measurements after thorough shaking, while the position of the embryo in the well turned out to be crucial for microscopic inspection. The assay allows to detect effects on zebrafish embryos at an early stage and at a low level of exposure clearly before deleterious consequences on an organ or organism level become visible. Since the embryos are kept alive, subsequent investigations of, e.g., EROD or micronucleus induction or further enzymatic biomarkers are possible. We will use the assay to investigate the impact of reactive toxicity on mechanism-specific biomarkers in zebrafish embryos. This would allow to verify whether biomarker results are representing a specific mode of action, or are rather the consequence of decreasing cell fitness.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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