Dietary Selenomethionine Reduce Mercury Tissue Levels and Modulate Methylmercury Induced Proteomic and Transcriptomic Alterations in Hippocampi of Adolescent BALB/c Mice.

Methylmercury (MeHg) is a well-known environmental contaminant, particularly harmful to the developing brain. The main human dietary exposure to MeHg occurs through seafood consumption. However, seafood also contains several nutrients, including selenium, which has been shown to interact with MeHg and potentially ameliorate its toxicity. The aim of this study was to investigate the combined effects of selenium (as selenomethionine; SeMet) and MeHg on mercury accumulation in tissues and the effects concomitant dietary exposure of these compounds exert on the hippocampal proteome and transcriptome in mice. Adolescent male BALB/c mice were exposed to SeMet and two different doses of MeHg through their diet for 11 weeks. Organs, including the brain, were sampled for mercury analyses. Hippocampi were collected and analyzed using proteomics and transcriptomics followed by multi-omics bioinformatics data analysis. The dietary presence of SeMet reduced the amount of mercury in several organs, including the brain. Proteomic and RNA-seq analyses showed that both protein and RNA expression patterns were inversely regulated in mice receiving SeMet together with MeHg compared to MeHg alone. Several pathways, proteins and RNA transcripts involved in conditions such as immune responses and inflammation, oxidative stress, cell plasticity and Alzheimer's disease were affected inversely by SeMet and MeHg, indicating that SeMet can ameliorate several toxic effects of MeHg in mice.

RNA sequencing and proteomic profiling reveal different alterations by dietary methylmercury in the hippocampal transcriptome and proteome in BALB/c mice.

Methylmercury (MeHg) is a highly neurotoxic form of mercury (Hg) present in seafood. Here, we recorded and compared proteomic and transcriptomic changes in hippocampus of male BALB/c mice exposed to two doses of MeHg. Mice were fed diets spiked with 0.28 mg MeHg kg-1, 5 mg MeHg kg-1, or an unspiked control diet for 77 days. Total mercury content was significantly (P < 0.05) increased in brain tissue of both MeHg-exposed groups (18 ± 2 mg Hg kg-1 and 0.56 ± 0.06 mg Hg kg-1). Hippocampal protein and ribonucleic acid (RNA) expression levels were significantly altered both in tissues from mice receiving a low dose MeHg (20 proteins/294 RNA transcripts) and a high dose MeHg (61 proteins/876 RNA transcripts). The majority but not all the differentially expressed features in hippocampus were dose dependent. The combined use of transcriptomic and proteomic profiling data provided insight on the influence of MeHg on neurotoxicity, energy metabolism, and oxidative stress through several regulated features and pathways, including RXR function and superoxide radical degradation.

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.

Analysis of immunoglobulin and T cell receptor gene expression in ballan wrasse (Labrus bergylta) revealed an extraordinarily high IgM expression in the gut.

The serum IgM concentration of ballan wrasse is relatively high, estimated to approximately 13 mg/ml in adult wild fish of 800 g. The present study revealed an unusual high abundance of IgM mRNA in the gut of ballan wrasse. Initially, transcripts encoding IgM, IgT, IgD, TCRα, TCRδ and CD3ε were quantified by RT-qPCR in several tissues of wild caught fish (approx. 800 g), indicating an elevated immune activity in hindgut and an extraordinarily high expression of IgM. Subsequently, a new RT-qPCR analysis was performed on the entire intestine, cut into four different segments, of reared fish (32-100 g). The analysis indicated immune activity along the entire intestine, but not as strong as in the hindgut. Furthermore, similar to the larger fish, the relative abundance of IgM transcripts was higher in the hindgut than in kidney and spleen, although the absolute level of IgM was in general higher in the larger fish. The secreted form of IgM was completely dominant in comparison to the membrane bound form of IgM and the other analysed genes. IgM was purified from gut mucus and external mucosal surfaces by magnetic beads coated with protein A. Mucus IgM reacted with rabbit antisera raised against serum IgM and contained subunits of the same size. Regarding the elevated immune activity in the intestine it is tempting to speculate on a possible compensatory strategy in this lineage of stomach-less fish, and that natural antibodies have an important role in the first line defence.

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.

T Cell Receptor Alpha Chain Genes in the Teleost Ballan Wrasse (Labrus bergylta) Are Subjected to Somatic Hypermutation.

Previously, somatic hypermutation (SHM) was considered to be exclusively associated with affinity maturation of antibodies, although it also occurred in T cells under certain conditions. More recently, it has been shown that SHM generates diversity in the variable domain of T cell receptor (TCR) in camel and shark. Here, we report somatic mutations in TCR alpha chain genes of the teleost fish, Ballan wrasse (Labrus bergylta), and show that this mechanism adds extra diversity to the polymorphic constant (C) region as well. The organization of the TCR alpha/delta locus in Ballan wrasse was obtained from a scaffold covering a single copy C alpha gene, 65 putative J alpha segments, a single copy C delta gene, 1 J delta segment, and 2 D delta segments. Analysis of 37 fish revealed 6 allotypes of the C alpha gene, each with 1-3 replacement substitutions. Somatic mutations were analyzed by molecular cloning of TCR alpha chain cDNA. Initially, 79 unique clones comprising four families of variable (V) alpha genes were characterized. Subsequently, a more restricted PCR was performed to focus on a specific V gene. Comparison of 48 clones indicated that the frequency of somatic mutations in the VJ region was 4.5/1,000 base pairs (bps), and most prevalent in complementary determining region 2 (CDR2). In total, 45 different J segments were identified among the 127 cDNA clones, counting for most of the CDR3 diversity. The number of mutations in the C alpha chain gene was 1.76 mutations/1,000 bps and A nucleotides were most frequently targeted, in contrast to the VJ region, where G nucleotides appeared to be mutational hotspots. The replacement/synonymous ratios in the VJ and C regions were 2.5 and 1.85, respectively. Only 7% of the mutations were found to be linked to the activation-induced cytidine deaminase hotspot motif (RGYW/WRCY).

Characterization of IgM in Norwegian cleaner fish (lumpfish and wrasses).

The use of cleaner fish in Norwegian aquaculture has to a large extent been based on wild catches, but breeding of lumpfish and ballan wrasse is currently increasing. Due to disease problems and required vaccine development, tools to study immune responses and a better understanding of the immune system in these species is demanded. The present study comprises lumpfish (Cyclopterus lumpus) and five species of wrasses: Ballan wrasse (Labrus bergylta), rock cook (Centrolabrus exoletus), cuckoo wrasse (Labrus mixtus), corkwing wrasse (Symphodus melops), and goldsinny wrasse (Ctenolabrus rupestris). We present a comparison of the IgM sequences, phylogenetic relationship to other teleosts and characteristic features of IgM in the species studied. The lumpfish IgM heavy chain sequence was assembled from high throughput cDNA sequencing whereas the wrasse sequences were determined by molecular cloning. The secreted form of the IgM heavy chain from all species consisted of four constant Ig domains. IgM was purified from lumpfish and ballan wrasse sera by gel filtration followed by anion exchange chromatography, and polyclonal sera were produced against these proteins. Antisera against ballan wrasse IgM showed cross-reactivity to all analyzed species of wrasses, some cross-reactivity to lumpfish, very low reaction to salmon, and no reaction to cod. Anti- IgM sera against lumpfish cross-reacted to the light chain of all species studied. Wrasses and lumpfish IgM showed high binding affinities for protein A. IgM concentration in adult ballan wrasse (700-800 g) was measured by single radial immunodiffusion assay and found to be 13.4 mg/ml which is about 36% of the total protein concentration. The IgM concentration in lumpfish (600-3600 g) was estimated to 1-2.6 mg/ml, which corresponds to approximately 3% of the total protein concentration.

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.

Pathogen recognition and mechanisms in Atlantic cod (Gadus morhua) head kidney cells: bacteria (LPS) and virus (poly I:C) signals through different pathways and affect distinct genes.

Understanding pathogen recognition and mechanisms in Atlantic cod are of significant importance for both basic research on wild populations and health management in aquaculture. A microarray approach was utilized to search for effects of viral (polyinosinic acid:polycytidylic acid), bacterial (lipopolysaccharide) and polyclonal activator (phytohaemoagglutinin) stress in Atlantic cod head kidney cells. LPS cell activation increased mRNA expression of interleukin 8; interleukin-1ß; cyclooxygenase 2; leukocyte derived chemotaxin 2; carboxyl-esterase 2 and environmental biomarker cytochrome P450 1A. Mitogen activated protein kinase p38 and cathepsin F were down regulated by LPS. The antiviral responses induced by double stranded RNA clearly increased transcription of Toll like receptor 3 and interferon stimulating gene 15. The phytohaemoagglutinin response seemed to be more non-specific. Special for the phytohaemoagglutinin induction was the increase in major histocompatibility complex class I. CC chemokine type 2 mRNA expression was increased by phytohaemoagglutinin, lipopolysaccharide and polyinosinic acid:polycytidylic acid, while mitogen activated protein kinase p38 and leukocyte derived chemotaxin 2 were down regulated by phytohaemoagglutinin. Oxidative stress related genes like catalase and glutaredoxin and the anti-apoptotic gene Bcl-2 showed no transcriptional changes compared to control in any of the treatments. Eicosanoids like prostaglandin 2, leukotriene B4 and B5 were constitutively produced by cod head kidney cells in vitro. The most remarkable feature of eicosanoid secretion is the higher production of leukotrienes against prostaglandins, indicating that the lipooxygenase pathway is preferred over the cyclooxygenase pathway. Although there were no significant differences in eicosanoid secretion between the groups, polyinosinic acid:polycytidylic acid showed a clear tendency to increase the levels of leukotriene B4 and B5. This study reveals distinct signatures of bacteria and virus transcriptional responses in cod head kidney cells. In addition, the novel finding that cytochrome P450 1A was upregulated during the antibacterial response indicates a connection between immunity and aryl hydrocarbon receptor activation in Atlantic cod.

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.

Molecular cloning of IgT from Atlantic salmon, and analysis of the relative expression of τ, µ, and δ in different tissues.

In the present study, IgT genes of Atlantic salmon were cloned and characterised. Analysis of our sequence data as well as ESTs reported to the databases revealed three distinct IgT heavy chain sub-variants in salmon, as opposed to two of IgM and IgD. The IgT sub-variants in salmon are 76-80% identical to each other, and 75-82% identical to the reported rainbow trout sequences, whereas the similarity to the orthologous molecules in zebrafish, grass carp, mandarin fish, and grouper is 25-41%. The heavy chains of both secreted and membrane anchored forms of salmon IgT include four constant Ig domains, τ1-τ4. This parallels the IgM heavy chains in elasmobranch fish and higher vertebrates, but differs from IgM in teleost fish where the membrane anchored form include only three constant Ig domains, µ1-µ3. The similarity between τ1 and µ1 in salmon is relatively high (52%) when compared to the remaining part of the molecules (τ2-τ4 and µ2-µ4 are 13-24% similar). To compare τ, µ and δ expressions in different tissues (head kidney, thymus, spleen, gill, skin, hind gut, brain and muscle) of Atlantic salmon, RT-qPCR assays were designed and evaluated. The analyses revealed that IgM transcripts are most abundant (up to 200 times more than IgD) followed by IgT (up to 20 times more than IgD) in most tissues. Highest expression of IgM, IgT, and IgD was in head kidney and spleen.