Novel procedures for whole organism detection and quantification of fluorescence as a measurement for oxidative stress in zebrafish (Danio rerio) larvae

Abstract

The modes of action of pollutants are diverse, and a common consequences to pollutant exposure is oxidative stress. This phenomenon is caused by an imbalance or disurption in the control of Reactive Oxygen Species (ROS) resulting in an accumulation of free radicals. Oxidative stress may cause damages to the DNA, phospholipids and proteins, and lead to cell death. Due to the possible contribution of oxidative stress to pollutant toxicity, it is valuable to assess its occurrence, role and mechanism. Detection of oxidative stress at low concentrations soon after the onset of exposure can be a sensitive, general marker for contamination. This study aimed at developing and benchmarking a set of novel fluorescence-based procedures to assess the occurrence of oxidative stress in zebrafish larvae (96 hpf) by measuring the antioxidant glutathione (GSH) and general ROS. Zebrafish larvae were exposed to tert-butyl hydroperoxide (t-BHP). ROS and GSH were made visible by means of specific fluorescent molecular probes in different experimental scenarios. The induction was qualified using microscopy and quantified through photometric measurement. For quantitative assessment, an approach based on homogenized larvae and a non-invasive plate assay were developed. The novel procedures proved suitable for oxidative stress detection. Comparisons of qualitative to quantitative data showed that the orientation of the larvae in the well can influence fluorescence data evaluation. The non-invasive quantitative assay proved robust against any influence of the orientation of the larvae. The developed protocols promise to be useful tools for the detection of oxidative stress in zebrafish larvae.

Keywords: Danio rerio; Fluorescence probes; GSH; Oxidative stress; ROS; Zebrafish larvae.