Oxidative stress is an important mechanism for cell damage and generally occures when organisms are exposed to environmental stressors, such as food scarcity, unusual environmental conditions, parasite infections, or of course intoxication. What happens then is the formation of reactive oxygen species (ROS), like for instance hydroxy radicals. These molecules chemically react with any component of the cells, be it proteins, nucleic acids, lipopolysaccharides of membranes, etc. The reaction is not targeted but highly random, leading to damage and eventually cell death. ROS induction is not necessarily by accident. The mechanism of programmed cell death, so called apoptosis, includes the effectiveness of ROS.
The good thing about ROS is that they appear very early in stressed cells, and clearly before cytotoxicity is measurable. Hence, ROS induction is a sensitive early alert marker also for chemical exposure. No surprise that assays were already described to determine ROS induction in a variety of different test organisms. The method is rather simple: a fluorescent dye that gets activated by reaction with ROS is added to the experiment, and subsequently the fluorescence is being measured. For cells and cellular organisms like algae this can be done quantitatively by means of a multiwell photometer.
However, for zebrafish larvae only a semi-quatitative procedure was developed so far, by colleagues from Spain. This involves inspection of the zebrafish using a fluorescence microscope and a subsequent categorisation of the fluorescence intensity - and thus the level of ROS induction.
We now took their protocol and combined it with a method for fluorescence measurement in a multiwell photometer that determines endocrine activity in a transgenic fish strain. We adapted the whole test procedure to the requirements of automatic fluorescence readout, and came up with a test system that initially allows detection of ROS induction in zebrafish larvae by a control substance.
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| ROS induction in zebrafish, semi-quantitative assessment by means of microscopy |
In the next steps we will testdrive the method with a several single substances and created mixtures. We will further finetune the assay where necessary, and finally will verify suitability for detecting the biological activity of complex environmental samples, such as water samples and extracts.
Our hope is that the assay will help to better understand and assess other effects determined using the zebrafish model. It will be included in our upcoming integrated zebrafish test battery that measures a multitude of biomarkers and endpoints in just one experiment.
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