Workshop Report 30

Zebrafish Model applications to address what is relevant for multiple generational studies

Peter Alestrøm
Professor of Biochemistry,
Norwegian University of Life Sciences,
The understanding of how toxicants, alone and in combinations, affect human health is a complex issue and demands the use of both in vitro and in vivo model systems. Among in vivo models, mice have dominated much of the research, with zebrafish (Danio rerio) coming up as the second most used laboratory animal model. Among several advantages of zebrafish as vertebrate model organisms is the option for automatic high-throughput screening of compound effects in 96-384 well plate formats. A Zebrafish Embryo Acute Toxicity (FET) Test has been established (OECD Guideline 236, 2013) for screening standardisation. There is increased understanding of how epigenetics influences gene expression and as such can lead to adverse health outcomes without changes in the genetic code. It appears that much of the epigenetic control mechanisms of DNA methylation, histone modifications and non-coding RNAs are highly conserved among the vertebrates. Results from two ongoing projects, which aim at mapping transgenerational effects from induced epigenetic changes and the cause of inherited changed phenotypes, were presented. The Centre of Excellence for Environmental Radioactivity (CoE CERAD) at the Norwegian University of Life Sciences (http://cerad.nmbu.no) addresses how low dose gamma radiation in combination with secondary environmental stressors can affect ecosystems and human health. In this project the zebrafish model will be used in a multi-generational set-up up to F4. The Institute for Environmental Studies at VU Amsterdam (J. Legler/J. Kamstra) assesses trans-generational effects of the phthalate metabolite mono(2ethylhexyl)phthalate (MEHP) and the DNA methylation inhibitor 5-azacytidine (5AC) from early embryonic exposed zebrafish up to F2. In both projects, global methylation will be measured with LC-MS. Site specific methylation will be assessed with reduced representation bisulphite sequencing (RRBS). Additionally in the CERAD project, transgenerational effect on histone modifications and transcriptome, including the miRNAome will be assessed with NGS methods, in order to provide a thorough mapping of the epigenetic landscape with and without exposure to radiation.
In summary, epigenetic landscapes can be subdivided into three levels: (i) DNAmethylation/hydroxymethylation, (ii) histone PTM and variants (histone code), and (iii) ncRNAs (short and long ncRNAs). Histone and ncRNA levels each have high complexity. Cross-talk between the epigenetic marker levels suggests higher sum complexity and is more difficult to predict outcome. Transgenerational epigenetic inheritance is still not well proven. Therefore, more basic research is needed and zebrafish is a good vertebrate model for epigenetic transgenerational studies.

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