Profiling DNA methylation patterns of zebrafish liver associated with parental high dietary arachidonic acid.

Diet has been shown to influence epigenetic key players, such as DNA methylation, which can regulate the gene expression potential in both parents and offspring. Diets enriched in omega-6 and deficient in omega-3 PUFAs (low dietary omega-3/omega-6 PUFA ratio), have been associated with the promotion of pathogenesis of diseases in humans and other mammals. In this study, we investigated the impact of increased dietary intake of arachidonic acid (ARA), a physiologically important omega-6 PUFA, on 2 generations of zebrafish. Parental fish were fed either a low or a high ARA diet, while the progeny of both groups were fed the low ARA diet. We screened for DNA methylation on single base-pair resolution using reduced representation bisulfite sequencing (RRBS). The DNA methylation profiling revealed significant differences between the dietary groups in both parents and offspring. The majority of differentially methylated loci associated with high dietary ARA were found in introns and intergenic regions for both generations. Common loci between the identified differentially methylated loci in F0 and F1 livers were reported. We described overlapping gene annotations of identified methylation changes with differential expression, but based on a small number of overlaps. The present study describes the diet-associated methylation profiles across genomic regions, and it demonstrates that parental high dietary ARA modulates DNA methylation patterns in zebrafish liver.

Parental high dietary arachidonic acid levels modulated the hepatic transcriptome of adult zebrafish (Danio rerio) progeny.

Disproportionate high intake of n-6 polyunsaturated fatty acids (PUFAs) in the diet is considered as a major human health concern. The present study examines changes in the hepatic gene expression pattern of adult male zebrafish progeny associated with high levels of the n-6 PUFA arachidonic acid (ARA) in the parental diet. The parental generation (F0) was fed a diet which was either low (control) or high in ARA (high ARA). Progenies of both groups (F1) were given the control diet. No differences in body weight were found between the diet groups within adult stages of either F0 or F1 generation. Few differentially expressed genes were observed between the two dietary groups in the F0 in contrast to the F1 generation. Several links were found between the previous metabolic analysis of the parental fish and the gene expression analysis in their adult progeny. Main gene expression differences in the progeny were observed related to lipid and retinoid metabolism by PPARα/RXRα playing a central role in mediating changes to lipid and long-chain fatty acid metabolism. The enrichment of genes involved in ß-oxidation observed in the progeny, corresponded to the increase in peroxisomal ß-oxidative degradation of long-chain fatty acids in the parental fish metabolomics data. Similar links between the F0 and F1 generation were identified for the methionine cycle and transsulfuration pathway in the high ARA group. In addition, estrogen signalling was found to be affected by parental high dietary ARA levels, where gene expression was opposite directed in F1 compared to F0. This study shows that the dietary n-3/n-6 PUFA ratio can alter gene expression patterns in the adult progeny. Whether the effect is mediated by permanent epigenetic mechanisms regulating gene expression in developing gametes needs to be further investigated.

High dietary arachidonic acid levels induce changes in complex lipids and immune-related eicosanoids and increase levels of oxidised metabolites in zebrafish (Danio rerio).

This study explores the effect of high dietary arachidonic acid (ARA) levels (high ARA) compared with low dietary ARA levels (control) on the general metabolism using zebrafish as the model organism. The fatty acid composition of today's 'modern diet' tends towards higher n-6 PUFA levels in relation to n-3 PUFA. Low dietary n-3:n-6 PUFA ratio is a health concern, as n-6 PUFA give rise to eicosanoids and PG, which are traditionally considered pro-inflammatory, especially when derived from ARA. Juvenile zebrafish fed a high-ARA diet for 17 d had a lower whole-body n-3:n-6 PUFA ratio compared with zebrafish fed a low-ARA (control) diet (0·6 in the control group v. 0·2 in the high-ARA group). Metabolic profiling revealed altered levels of eicosanoids, PUFA, dicarboxylic acids and complex lipids such as glycerophospholipids and lysophospholipids as the most significant differences compared with the control group. ARA-derived hydroxylated eicosanoids, such as hydroxy-eicosatetraenoic acids, were elevated in response to high-ARA feed. In addition, increased levels of oxidised lipids and amino acids indicated an oxidised environment due to n-6 PUFA excess in the fish. To conclude, our results indicate that an ARA-enriched diet induces changes in complex lipids and immune-related eicosanoids and increases levels of oxidised lipids and amino acids, suggesting oxidative stress and lipid peroxidation.

Dietary vitamin A supplementation ameliorates the effects of poly-aromatic hydrocarbons in Atlantic salmon (Salmo salar).

Several studies have reported on the interaction between vitamin A (VA) and aryl hydrocarbon receptor (AhR)-binding toxicants, including poly-aromatic hydrocarbons (PAHs). In aquaculture, the use of plant oils in novel aquafeeds can increase PAH levels while simultaneously lowering natural VA background levels, causing the need to supplement plant oil-based feeds with synthetic VA. To study dietary VA-PAH interactions, Atlantic salmon (initial weight 195±0.15g) were fed four identical plant-based diets that were supplemented with PAHs (100 and 10mgkg(-1) benzo[a]pyrene (BaP) and phenanthrene (Phe), respectively) or VA (retinyl acetate 8721IUkg(-1)) separately or combined for 2.5 months in a 2×2 factorial design, with triplicate net-pens per diet. Dietary PAH significantly reduced hepatic VA storage, and VA-enriched diets restored hepatic VA. There was a significant PAH-VA interaction effect on hepatic BaP, but not Phe, accumulation, with reduced hepatic BaP concentrations in fish fed VA+PAH compared to fish fed PAH alone. Concurrently, PAH and VA significantly interacted in their effects on CYP1A phase I biotransformation as observed from increased ethoxyresorufin-O-deethylase (EROD) activity, increased CYP1A protein concentration, and elevated transcription (cyp1a1 gene expression) in fish fed PAH+VA compared to PAH alone. Dietary VA supplementation alone had no significant effect on CYP1A phase I biotransformation. Metabolomic assessment showed that dietary VA caused a restoration of metabolic intermediates involved in energy metabolism that were affected by dietary PAH. Moreover, a PAH-induced growth inhibition was partially ameliorated by dietary VA supplementation. In conclusion, dietary VA interacted with PAH toxicity on the level of CYP1A-mediated detoxification, hepatic PAH accumulation, energy allocation, and growth.

Vitamin A and arachidonic acid altered the skeletal mineralization in Atlantic cod (Gadus morhua) larvae without any interactions on the transcriptional level.

The main object of this study was to evaluate the impact of different levels of vitamin A (VA) and arachidonic acid (ARA) in relation to eicosapentaenoic acid (EPA) on mineralization and gene expression in Atlantic cod larvae (Gadus morhua). First-feeding larvae were fed enriched rotifers from start-feeding until 29 days post hatch (dph). Larvae in four tanks were fed one of the following diets: control (EPA/ARA ratio: 15.8, 0.9µg VA g(-1)), control+VA (EPA/ARA ratio: 15.8, 7.8µg VA g(-1)), High ARA (EPA/ARA ratio: 0.9, 1.5µg VA g(-1)) or High ARA+VA (EPA/ARA ratio: 0.9, 12.0µg VA g(-1)). Larvae fed High ARA+VA were shorter at 29dph compared to the other groups and had significantly less mineralized bones when comparing larvae of similar size, showing interaction effects between VA and ARA. Although transcriptomic analysis did not reveal any interaction effects, a higher number of genes were differentially expressed in the high ARA fed larvae compared to control+VA fed larvae. Furthermore, bglap1, bglap2 and col10a1 were all down-regulated in larvae fed High ARA-diets and to a greater extent than larvae fed VA supplemented diet, indicating an additive effect on mineralization. In conclusion, this study showed that the dietary increase in ARA and VA altered the skeletal metabolism during larval development, most likely through signaling pathways specific for each nutrient rather than an interaction. The present study also demonstrates that VA could affect the larval response to ARA, even within the accepted non-toxic/non-deficient range.

Arachidonic acid enhances turnover of the dermal skeleton: studies on zebrafish scales.

In fish nutrition, the ratio between omega-3 and omega-6 poly-unsaturated fatty acids influences skeletal development. Supplementation of fish oils with vegetable oils increases the content of omega-6 fatty acids, such as arachidonic acid in the diet. Arachidonic acid is metabolized by cyclooxygenases to prostaglandin E2, an eicosanoid with effects on bone formation and remodeling. To elucidate effects of poly-unsaturated fatty acids on developing and existing skeletal tissues, zebrafish (Danio rerio) were fed (micro-) diets low and high in arachidonic acid content. Elasmoid scales, dermal skeletal plates, are ideal to study skeletal metabolism in zebrafish and were exploited in the present study. The fatty acid profile resulting from a high arachidonic acid diet induced mild but significant increase in matrix resorption in ontogenetic scales of adult zebrafish. Arachidonic acid affected scale regeneration (following removal of ontogenetic scales): mineral deposition was altered and both gene expression and enzymatic matrix metalloproteinase activity changed towards enhanced osteoclastic activity. Arachidonic acid also clearly stimulates matrix metalloproteinase activity in vitro, which implies that resorptive effects of arachidonic acid are mediated by matrix metalloproteinases. The gene expression profile further suggests that arachidonic acid increases maturation rate of the regenerating scale; in other words, enhances turnover. The zebrafish scale is an excellent model to study how and which fatty acids affect skeletal formation.

Cytokine gene expression and prostaglandin production in head kidney leukocytes isolated from Atlantic cod (Gadus morhua) added different levels of arachidonic acid and eicosapentaenoic acid.

Primary head kidney leukocytes from Atlantic cod were isolated to evaluate the use of arachidonic acid and eicosapentaenoic acid by cyclooxygenases and the production of prostaglandins E2 and E3. The expression of cyclooxygenase genes and selected interleukin genes like Interleukin 1ß, Interleukin 6, interleukin 8 and interleukin 10 were monitored. Increasing concentrations of eicosapentaenoic acid and arachidonic acid in equal amounts increased cyclooxygenase2 transcription as well as cell secretion of prostaglandin E2. Even though the ratio of the two fatty acids was 1:1, the ratio between prostaglandin E2 and E3 was 50:1. The addition of arachidonic acid alone increased prostaglandin E2 secretion but did not induce cyclooxygenase2 transcription. However, when the concentration of eicosapentaenoic acid was increased, maintaining arachidonic acid constant, both prostaglandin E3 and prostaglandin E2 production was induced and the prostaglandin E2 production was higher than in cell cultures only added arachidonic acid. An up-regulation of cyclooxygenase2 transcription was also observed. The addition of the two fatty acids also affected the immune response by alteration of leukocytic cytokines gene expression. According to our results the Cyclooxygenase in cod seem to prefer arachidonic acid as substrate. Therefore, we suggest that the shift from marine oils (rich in n-3 fatty acids) to plant oils (higher in n-6 fatty acids) in the diet of commercially reared Atlantic cod could have negative effects on the whole organism through the increase in the production of prostaglandins belonging to those derived from n-6 fatty acids.

Retinoic acid induces two osteocalcin isoforms and inhibits markers of osteoclast activity in Atlantic cod (Gadus morhua) ex vivo cultured craniofacial tissues.

Nutritional status including vitamin A could explain some of the developmental deformities observed in cultivated teleosts, including Atlantic cod (Gadus morhua). In the present study we aimed to investigate the transcriptional effect of retinoic acid (RA) on bone related genes using Atlantic cod craniofacial explants tissue cultures. Two different osteoblast specific osteocalcin/bone gla protein isoforms were discovered in cod. Transcription of both isoforms was up-regulated following RA treatment of 65 dph cod lower jaw explants. In contrast, transcripts coding for genes related to bone resorption and osteoclast activity, matrix metalloproteinase 9 and cathepsin K were down-regulated following RA treatment. This could be linked to the decreased transcriptional ratio between receptor activator of nuclear factor kappa-B ligand rankl and osteoprotegerin observed in the same tissue samples. RA treatment of juvenile explants had no effect on runt-related transcription factor 2 and osterix mRNA levels. However, osterix was significantly down-regulated in 25 dph cod head explants following RA treatment. In situ hybridizations revealed differential spatial distribution of the two isoforms and the predominant expression of cathepsin K in bone surrounding tissues. The present study indicates that RA causes a shift in the balance between osteoclast activity and osteoblast activity in favor of the latter.

Transcriptional evidence for low contribution of oil droplets to acute toxicity from dispersed oil in first feeding Atlantic cod (Gadus morhua) larvae.

We evaluated the potential contribution of oil droplets to the toxicity of dispersed oil to first feeding fish larvae. Atlantic cod larvae were exposed to five concentrations of either artificially weathered (200°C residue) dispersed oil (D1-D5) containing oil droplets [medium size 11-13 µm based on volume] and water-soluble fraction [WSF] or the filtered dispersions containing only the corresponding equilibrium WSFs only (W1-W5). The larvae were exposed for 4 days and harvested for transcriptional analysis at 13 days post hatching. The most significant differently expressed genes were observed in cod larvae exposed to the highest concentration of the dispersed oil (containing 10.41 ± 0.46 µg ∑PAH/L), with CYP1A showing the strongest response. Functional analysis further showed that the top scored network as analyzed with Ingenuity Pathway Analysis was "Drug Metabolism, Endocrine System Development and Function, Lipid Metabolism". Oil exposure also increased the expression of genes involved in bone resorption and decreased the expression of genes related to bone formation. In conclusion, oil exposure affects drug metabolism, endocrine regulation, cell differentiation and proliferation, apoptosis, fatty acid biosynthesis and tissue development in Atlantic cod larvae. The altered gene transcription was dominated by the WSF and the corresponding oil droplet fraction only had a moderate contribution to the observed changes.