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Zebrafish embryo test
Zebrafish embryo test is an emerging model for toxicological studies because represents the complex dynamic, interactive and multi-organ events that occur in adults. This model represents an alternative system which already replaces studies with animal experiments concerning ethical / legal issues. According to new European directive for human and environment protection – REACH (Registration, Evaluation, Authorisation and restriction of CHemicals) we must consider 3R concept - Reduction, Refinement and Replacement of laboratory animals with alternative models in vitro.

We offer a protocol brochure (Slovene language) for all main toxicological assays on zebrafsih embryos and a CD presentation. For more information, please write to metka.filipic@nib.si or tina.elersek@nib.si.

At National Institute of Biology in Ljubljana we have introduced an acute toxicity test with zebrafish embryos. Further more, we have developed expanded tests which combine different biological levels:

Acute toxicity assay

The evaluation of the acute toxicity LC50 of selected compounds is carried out by using standardised recommendation SIST ISO 7346-3:1998, 5667-16 and OECD guideline (OECD Draft). This test uses four morphological endpoints of acute lethality after 24 and 48 hours post fertilisation.

Developmental toxicity assay

Based on deep knowledge of the zebrafish embryology we assess also sublethal and teratogenic effects. These effects indicate potential hazardous long term effects of chemicals.

Organ-Specific toxicity assay

The purpose of these assays is to determine whether toxicity is focused on any specific organ. We can evaluate both, morphological and functional endpoints. In zebrafish, the heartbeat starts around 24 hours after fertilisation. Other organs (brain, eyes, blood vessels…) are formed in the first 48 hours of development, which enables us observation of nevrotoxicity, cardiotoxicity, etc.

Nanotoxicity

Zebrafish model represents fast and efficient system for nano-material toxicity assessment based on morphological and physiological developmental endpoints. Namely, nano-particles are small enough to pass the chorion of zebrafish embryos.

Genotoxicity

Genotoxicity assessment is based on newly optimised comet assay which notice DNA damage on single embryo after 24 hours. This method is extremely sensitive; can assess DNA damages at nano-gram concentration.

Ecotoxicity

Ecotoxicity studies are performed in accordance to European OECD Guidelines. Guidelines OECD 210 and 212 recommend zebrafish as a suitable species for environmental toxicity assessment. A proposal for Fish Embryo Toxicity test (OECD draft) is now under review as a replacement for the Acute Toxicity test on fish (OECD 203). These tests are very suitable for environmental toxicity determination based on fast answers concerning pollution problems, toxic cyanobacterial blooms, etc.

Toxicogenomics

Toxicogenomics is an emerging area allowing further in-depth studies into genetic bases of toxicology. This approach is based on gene expression changes (transcriptomics) reflected by toxic compound in organism. We analyse transcription of specific Danio rerio human homologues for p53, p21 (pak2a in pak2b), gadd45a in mdm2. The analyses of expression are done by RT-PCR.

Custom made assays

Beside already mentioned assays, we offer special assays which are focused on specific organism responses. Assays are adapted in exposure duration and selection of specific observed endpoints.

The main advantages of zebrafish embryo model for toxicological studies are:

  • complexity of all physiological and morphological interactions that occur in adult in vivo,
  • process connection in complete organism (embryo) represents additional advantages in comparison to cell line systems in vitro,
  • in comparison to rodents, faster development and higher genetic homology to human,
  • good correlation to mammal models,
  • small size (~ 1 mm), weight (< 1 mg) and transparency of embryos enables simple microscopic observation,
  • assays are carried out in multiwall plates which enables lower consumption of testing compounds and have great potential for automatic manipulation of time-consuming processes,
  • high popularity of zebrafish system enables increasing data base and information flow.