Thursday, February 18, 2016

Quantitative assessment of oxidative stress in zebrafish

We initially developed and established an assay to quantitatively determine oxidative stress in exposed zebrafish larvae.

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.
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.

Zebrafish-directed identification of culprits

The zebrafish has become a standard test species in ecotoxicology, and a versatile tool for effect measurement and assessment. Being a complex vertebrate organism it allows for a multitude of biomarker investigations on various sublethal endpoints. Since such effects are determined after exposure of a whole animal, environmental relevance is higher than for microscale cellbased assays.

Environmental relevance is also the focus of the integrated approach of effect-directed analysis (EDA). This recursive process of sample testing and fractionation helps to seperate the key toxicity drivers for final chemical identification. Thus, the real culprits for toxicity in environmental samples can be revealed and distinct measures taken for mitigation or remediation.

It sounds hence logical to combine zebrafish testing and EDA for environmental risk assessment. For our EDA Emerge project we reviewed the utility of zebrafish in EDA studies to date, described advantages and shortconings as well as further requirenents for method development.

Saturday, February 13, 2016

Is one of the best materials known to mankind toxic?

Graphene is a two-dimensional carbon layer and in many aspects the best material known to mankind. It is highly robust, has very good conductive properties, and due to its stucture an immense surface to mass ratio.

Graphene exists as nanoplatelets. Like all nanoparticles it is subject to environmental and toxicological investigations. Nanoparticles are suspected to enter cells and cause damage, harm cell membranes, and act as transport vehicles for contaminats due to their sorptive capacity for unpolar molecules.

http://polpix.sueddeutsche.com/bild/1.1008406.1355787097/640x360/physiknobelpreis.jpg
It is this possible sorption of putative pollutants that makes graphene also very interesting in terms of soil and sediment remediation. The vast surface of only a little amount of graphene platelets promises a high efficiency in capturing toxic organic molecules and thus making them unavailable for uptake by organisms.

However, prior to application in remediation measures two crucial questions need to be answered:
1. How strong is the sorption? Will the particles be a reliable sink?
2. Are graphene platelets toxic, and if yes, at which concentration? Could they be still successfully applied in concentrations where they do no harm?

We investigated these questions within two bachelor's theses in collaboration with colleagues from the Polytechnical University of Bucharest (UPB), Romania, and the RWTH Aachen University Institute of Plant Physiology. Objects of study were not only pure graphene platelets but also differently oxidised types. Since we knew from previous studies that nanoparticles cause mainly oxidative stress, we investigated induction of reactive oxygen species (ROS) in different test systems.

Graphene platelet suspensions were subjected to transgenic yeasts that can reveal different modes of action for ROS induction. They were further used to expose algae and also zebrafish embryos. The assays made use of dyes that react with ROS after metabolic activation to fluorescent molecules. Fluorescence is hence proportional to the strength of ROS induction and can be determined by means of microscopy or photometric measurement. For zebrafish investigations we used a new protocol from colleagues in Spain on semi-quantitative evaluation of ROS induction.

Our study showed that graphene nanoplatelets do not induce ROS induction in yeasts and zebrafish up to a nominal concentration of 2 mg/L. However, higher concentrations were not yet tested, since this concentration would be likely the one for environmental application according to the sorption studies conducted by the UPB. Algae gave weak ROS induction, but with high variability and hence not significant.

While our results give a first good indication that graphene platelets are suitable for soil and sediment remediation, these were only initial investigations. Further studies will test higher concentrations, as graphene platelets are likely to accumulate. Thus, the relevant environmental concentrations could clearly exceed the applied. Also, more sensitive assays using yeasts and zebrafish will be carried out. But most importantly, we will measure the exposure concentration, since chances exist that the real concentrations of platelets were lower than the one of the initial suspension.

Saturday, February 06, 2016

An evidence on failure in sediment risk assessment

Recently, we put out a paper on an ecotoxicological investigation of sediments from an oxbow lake of the River Rhine. What looks just like another research paper about a study of contaminated sediments holds an important finding about standard sediment assessment: it might fail.

We tested the samples using a comprehensive bioassay battery that goes far beyond the regulatory requirements. Besides the commonly employed test on immobilisation of Daphnia magna, we used assays to reveal estrogenicity, teratogenicity, embryotoxicity and cytotoxicity. Surprisingly, all tests gave clear effects of the sediments, except for the standard test with daphnids.

So, are sediments a risk? Read our discussion and conclusions of this important topic in our paper published in the Journal of Soils and Sediments.