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SIPHINIFES-SIP ved HI

Inheritance of epigenetic patterns under the influence of diets and contaminants

Alternative title: null

Awarded: NOK 16.0 mill.

This project was established to gain deeper knowledge on how nutrients and contaminants alter the epigenetic DNA methylation pattern in fish and humans. WP1: In WP1 we investigated how arachidonic acid (ARA) in parental feed affect offspring (F1) generation through epigenetic changes. Using zebrafish, we found that the diets changed the metabolism of parents, and affected gene expression and DNA methylation in both generations. Results from metabolic profiling revealed a general shift in lipid profiles in F0. High dietary ARA levels did not affect the body weight of zebrafish. DNA methylation and gene expression profiles in F1 were associated with the parental diet, whereas the effect on gene expression was less strong in parents than in progeny. Several links were found between the metabolic profiles in parents and both DNA methylation and gene expression of the progeny, which suggested an impact of parental diet on the progeny during early embryonic development. The PhD involved in this project defended her thesis in March 2018 at which she got her doctoral degree. The overall results from these studies suggest that increased levels of arachidonic acid in the parental diet impact metabolic pathways involved in lipid handling and retinoid signalling in adult offspring. WP2: We have focused on the 1C nutrients folate, vitamin B12, vitamin B6, choline and methionine effects on epigenetic regulation. Using zebrafish we studied mature F1 from nutritional deficient parents and observed a pale red liver color. Histological examination revealed a higher inclusion of lipid in hepatocytes. RNA sequencing revealed changes in steroid biosynthesis and mitochondrial proteins due to parental feed. DNA methylation turned out to be very sensitive to parental 1C nutrient level, especially in promoter regions and less in CpG island shores. Differential methylation was identified at 2869 CpG sites, among others the ppp2r2ba revealed strong hypomethylation of 20 CpG sites in all replicate samples. This gene is implicated in the negative control of cell growth and division and defects have been linked to degeneration of central nervous system. It is possible that the differences in DNA methylation were established early in development and these may have regulatory effects in other tissues. Using Atlantic salmon we included three different levels of 1-C nutrients, and the genes in the steroid biosynthetic pathway responded in a nutrient gradient specific gene expression pattern. DNA have been purified and RRBS performed from the same livers as mentioned above. We are now in the process of analysing the DNA methylation pattern, adapting the zebrafish pipeline for RRBS scripts for the salmon genome WP3: The goal of WP3 was to identify differences in the methylome in saliva samples collected from children, correlated with Hg status and seafood consumption in mothers, and to study interactions between MeHg and polyunsaturated fatty acids in zebrafish. Unfortunately, the first part of the project has been plagued by technical issues stemming from the integrity of the provided saliva samples. A study using zebrafish demonstrated transgenerational effects of MeHg on DNA methylation in F1 and F2 embryos. WP4: The goal of WP4 was to determine possible changes of epigenetic patterns caused by a set of emerging contaminants relevant for seafood safety, using zebrafish and Atlantic salmon exposure experiments. The results clearly show that the gene expression patterns and the epigenome of fish can be modulated by environmental contaminants. The endocrine disruptor bisphenol A (BPA) affects behavior, and differentially methylated (DM) sites especially in protocadherins, related to axon targeting, synaptic development and neuronal survival in zebrafish larvae. Metabolic pathways most significantly affected by BPA exposure were phosphatidylinositol signaling system, followed by VEGF and MAPK signaling pathways. In addition, we tested other contaminants like 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE), chlorpyrifos (CPF) and tris(2,3-dibromopropyl) phosphate (TDBPP), and identified numerous differentially methylated sites. For example, hedgehog signaling pathway and oocyte meiosis were significantly affected by CPF, while the adrenergic signaling in cardiomyocytes pathway was significantly affected by TDBPP. Follow-up studies with Atlantic salmon liver cells have demonstrated distinct effects of endocrine-disrupting chemicals on DNA methylation mechanisms. Together these results show that epigenetic regulation in fish is highly sensitive to both nutrient levels and toxicants.

This project was established to gain deeper knowledge on how nutrients and contaminants alter the epigenetic DNA methylation pattern in fish. Together these results show that epigenetic regulation in fish is highly sensitive to both nutrient levels and toxicants, and have led the way towards new research areas on developmental programming in terms of either abiotic or biotic environmental changes. The results have gained new insight at the molecular level of epigenetic modification, both in terms of nutritional programming and metabolic programming (toxicants). We have shown that these environmental changes, when applied during early stages of development, permanently adjust the physiology in terms of gene expression and liver histology at later life stages. As such, the results have shed new light towards focus on broodstock nutrition and early environmental stressors for fish.

Epigenetics is the changes in phenotype due to processes arising independent of DNA sequence. This project will establish new knowledge on how nutrients and contaminants, alone or together, alter the epigenetic patterns in Atlantic cod, A. salmon and in h umans. Diets for aquacultured species are now based on plant ingredients. With plant proteins, it is likely that the one-carbon cycle is affected and possibly also the DNA methylation. Omega-6 levels increases with the use of plant oils and tilt the balan ce between omega-3 and -6 fatty acids. These are essential but have different effects on health and development. Therefore, zebrafish will be fed diets with either low levels of B-vitamins and methionine or with high levels of omega-6 fatty acids and be a model for the epigenetic changes driven by these nutrients. Based on this, analysis of samples from life cycle experiments (A. cod and A. salmon), running in other projects. A good nutritional status ameliorates several effects of contaminants, but the m echanisms behind is largely unknown. Epigenetics is likely to be one. A feeding trial with A. salmon juveniles will be done with diets spiked with methyl donors and co-factors in the one-carbon cycle (i.e. B5, B6 and B12). These will then be exposed to ne w and emerging toxicants (in feeds and feedstuffs for aquaculture, like pesticides) through their diet to elucidate how an optimal diet can produce a fish with higher resilience. The choice of contaminants will be based on a screen using zebrafish embryos /larvae exposed to a set of emerging toxicants (in accordance with considerations from EFSA). Seafood consumers get both methylmercury (MeHg) and omega-3 fatty acids (n-3PUFAs). n-3PUFAs have been shown to ameliorate the effects of contaminants. Data ob tained by analysing epigenetic patterns in human samples, correlated to the intake of seafood and the levels of MeHg, will be the basis for experimental studies using zebrafish focusing on neural development.

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SIPHINIFES-SIP ved HI