Dietary protein affects tissue accumulation of mercury and induces hepatic Phase I and Phase II enzyme expression after co-exposure with methylmercury in mice.

Methylmercury (MeHg) is a ubiquitous environmental contaminant, well known for its neurotoxic effects. MeHg can interact with several nutrients in the diet and affect nutrient metabolism, however the interaction between MeHg and dietary proteins has not been thoroughly investigated. Male BALB/c mice were fed diets based on either casein, cod or chicken as protein sources, which were or were not spiked with MeHg (3.5 mg Hg kg-1). Following 13 weeks of dietary exposure to MeHg, the animals accumulated mercury in varying degrees depending on the diet, where the levels of mercury were highest in the mice fed casein and MeHg, lower in mice fed cod and MeHg, and lowest in mice fed chicken and MeHg in all tissues assessed. Assessment of gut microbiota revealed differences in microbiota composition based on the different protein sources, however, the introduction of MeHg eliminated this difference. Proteomic profiling of liver tissue uncovered the influence of the dietary protein sources on a range of enzymes related to Phase I and Phase II detoxification mechanisms, suggesting an impact of the diet on MeHg metabolism and excretion. Also, enzymes linked to pathways including methionine and glycine betaine cycling, which in turn impact the production of glutathione, an important MeHg conjugation molecule, were up-regulated in mice fed chicken as dietary protein. Our findings indicate that dietary proteins can affect expression of hepatic enzyme that potentially influence MeHg metabolism and excretion, highlighting the relevance of considering the dietary composition in risk assessment of MeHg through dietary exposure.

Congener-specific accumulation of persistent organic pollutants in marine fish from the Northeast Atlantic Ocean.

Bioaccumulation of persistent organic pollutants (POPs) in marine fish may pose a health risk to human consumers. Using data from ∼8400 individuals of 15 fish species collected in the North-East Atlantic Ocean (NEAO), we assessed concentrations of individual POP congeners, including dioxins, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). POPs analyses were performed with accredited methods using high-resolution gas chromatography/high-resolution mass spectrometry, gas chromatography/tandem mass spectrometry (GC-MS/MS) and GC/MS. The results showed that POPs congener composition profiles were more influenced by fish species than by geography. However, due to long range transport from emissions at lower latitudes, lighter congeners made a larger contribution to the total POPs concentrations in the northernmost areas compared to southern regions. A model was developed to elucidate the relative effects of several factors on POPs concentrations and showed that variation among and within fish species was associated with fat content, fish size, trophic position, and latitude. For the first time, POPs concentrations were shown to increase nonlinearly with fat content, reaching an asymptotic plateau when fat content was > 10%. This study explored detailed POP congener profiles and the factors associated with POPs accumulation in commercially relevant fish harvested from the NEAO.

Sampling of Atlantic salmon using the Norwegian Quality cut (NQC) vs. Whole fillet; differences in contaminant and nutrient contents.

Risk- benefit assessments of seafood require high-quality food composition data. In accordance with EU regulations, Atlantic salmon (Salmo salar) has conventionally been sampled using the Norwegian Quality Cut (NQC), a sub-cut of the middle section of the fish, in Norwegian surveillance programs. By comparing the contents of nutrients and contaminants in 34 samples of farmed Atlantic salmon, we aimed to evaluate the representativeness of the NQC compared with the whole fillet. Of the 129 analytes evaluated, eight single analytes, in addition to 25 different fatty acids, showed significant differences between the cuts. Significant differences were evident for total fat, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and sum PCB-6, but not for the sum of dioxins and dioxin-like PCBs. We further suggest that the NQC may still be used in large-scale sampling of Atlantic salmon, and that the whole fillet would be preferable when analysing the content of nutrients.

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.

Temporal variations in the nutrient content of Norwegian farmed Atlantic salmon (Salmo salar), 2005-2020.

The changes in the feed of farmed Atlantic salmon (Salmo salar) towards a more plant-based diet affect the nutritional value of the fillets. By compiling the contents of a range of nutrients in 1108 samples of Norwegian farmed Atlantic salmon collected between 2005 and 2020, we found that the median contents of eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) have decreased by > 60%. However, farmed Atlantic salmon remains a considerable source of EPA and DHA, with one and two portions being sufficient to meet the weekly adequate intake of EPA and DHA for adults (175 g) and two-year-olds (80 g), respectively. Farmed Atlantic salmon also remains a considerable source of protein, selenium, vitamin B12, and vitamin D3. Together, we demonstrate that farmed Atlantic salmon can contribute substantially to the nutrient intake of the consumers. These data are important for the Norwegian food composition table and future risk-benefit assessments on fatty fish consumption.

Co-occurrence of contaminants in marine fish from the North East Atlantic Ocean: Implications for human risk assessment.

Marine fish from the North East Atlantic Ocean (NEAO) are nutrient rich and considered a valuable economic resource. However, marine fish are also a major dietary source of several contaminants, including persistent organic pollutants (POPs) and heavy metals. Using one of the world's largest seafood datasets (n > 25,000 individuals), comprising 12 commercially important fish species collected during 2006-2019 in the NEAO, we assessed the co-occurrence of elements and POPs, and evaluated potential risks to human consumers. Several positive correlations between concentrations of mercury (Hg), dioxins, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) were observed. Concentrations of Hg, dioxins, PCBs and PBDEs increased from North to South and associations between marine sediment contamination, sea temperature, and fish Hg and POPs concentrations were identified using multi-linear regression (MLR) models. In general, Hg concentrations in fillet and liver of fish were positively associated with increases in both sediment contamination and sea temperature. POPs concentrations in both fillet and liver were positively associated with increases in sediment contamination, and only POPs concentrations in the liver of benthopelagic and demersal species were found to be positively correlated with sea temperature. Using a probabilistic approach to estimate human contaminant exposure from seafood, we showed that intake of pelagic species posed the highest risk of dioxins and dioxin-like PCBs (DL-PCBs) exposure, while intake of benthopelagic and demersal species posed the highest risk of Hg exposure. This study can serve as a model to further understand the distribution, co-occurrence, and trends of contaminants in seafood harvested from the NEAO and their potential risks to human consumers.

Intakes of Fish and Long-chain n-3 Polyunsaturated Fatty Acid Supplements During Pregnancy and Subsequent Risk of Type 2 Diabetes in a Large Prospective Cohort Study of Norwegian Women.

OBJECTIVE: To investigate associations between intakes of total fish, lean fish, fatty fish, and long-chain n-3 polyunsaturated fatty acid (LCn-3PUFA) supplements and risk of type 2 diabetes in women after pregnancy. Furthermore, we sought to compare the estimated intakes of methylmercury (MeHg) and sum of dioxins and dioxin-like polychlorinated biphenyls (dl-PCBs) with tolerable weekly intakes (TWI). RESEARCH DESIGN AND METHODS: Women free of diabetes at baseline (n = 60,831) who participated in the population-based Norwegian Mother, Father and Child Cohort Study (MoBa) were prospectively evaluated for incident type 2 diabetes, identified on the basis of medication usage >90 days after delivery, ascertained through the Norwegian Prescription Database. Dietary intake data were obtained with a validated 255-item food-frequency questionnaire (FFQ), which assessed habitual diet during the first 4-5 months of pregnancy. Intakes of MeHg and sum of dioxins and dl-PCBs were derived with use of a contaminant database and the FFQ. RESULTS: Median age was 31 years (interquartile range 27, 34) at time of delivery, and follow-up time was 7.5 years (6.5, 8.5). Type 2 diabetes occurred in 683 (1.1%) participants. Multivariable Cox regression analyses identified lower risk of type 2 diabetes with increasing energy-adjusted lean fish intake, 25 g/1,000 kcal (25 g/1,000 kcal: hazard ratio 0.71, 95% CI 0.53-0.95, P = 0.022). However, in stratified analyses, a lower risk was found only in women with prepregnancy BMI ≥25 kg/m2. There were no associations between intake of total fish, fatty fish, or LCn-3PUFA supplements and type 2 diabetes. MeHg intake was low, but the intake of the sum of dioxins and dl-PCBs (picograms of toxic equivalents/kilograms of body weight/week) exceeded the TWI set by the European Food Safety Authority (EFSA) for the majority of participants. CONCLUSIONS: Intake of lean fish, but not fatty fish or LCn-3PUFA supplements, was associated with lower risk of pharmacologically treated type 2 diabetes in Norwegian women who were overweight or obese. Fatty fish, which contain dioxins and dl-PCBs, did not increase the risk of type 2 diabetes, but the exceedance of the EFSA TWI for dioxins and dl-PCBs is a health concern.

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.

Fatty fish, hair mercury and cognitive function in Norwegian preschool children: Results from the randomized controlled trial FINS-KIDS.

BACKGROUND: The toxic effects of prenatal methylmercury (MeHg) include neurological abnormalities and developmental delay of which infants and children are particular susceptible. Studies on the effects of low and moderate exposure show conflicting results. Seafood is the main dietary source of MeHg, but also contributes with nutrients regarded as beneficial for development. OBJECTIVES: To measure the change in total hair mercury concentration (THHg) after an intervention of lunch meals with fatty fish or meat in Norwegian preschool children, and to examine the associations between THHg and cognitive function. METHODS: Children (n = 232) 4-6 years old were randomized to lunch meals with fatty fish (n = 114) or meat (n = 118) three times a week for 16 weeks. THHg was determined using a Direct Mercury Analyzer, and cognitive function was assessed by the Wechsler Preschool and Primary Intelligence Scale-III (WPPSI-III) pre- and post-intervention. Linear mixed effect models were used to analyze changes in THHg and WPPSI-III scores. RESULTS: The mean (SD) THHg pre-intervention was 0.373 (0.204) mg kg-1. Children in the fish group had an increase in THHg (change 0.162, 95% CI 0.111, 0.213 mg kg-1), whereas children in the meat group had decreased THHg (-0.053, 95% CI -0.103, -0.002 mg kg-1). There were no notable associations between THHg and the WPPSI-III raw scores at baseline or after 16 weeks of the fish/meat intervention. CONCLUSIONS: Lunch meals including fatty fish led to a significant increase in THHg, but the values remain below the point of departures used for risk assessment by the EFSA, WHO and US-EPA. We observed no associations between THHg and cognitive function.

Factors influencing risk assessments of brominated flame-retardants; evidence based on seafood from the North East Atlantic Ocean.

Brominated flame-retardants (BFRs) such as polybrominated diphenyl ethers (PBDE) and hexabromocyclododecane (HBCD) are considered hazardous to human health. Due to their persistence, they are still present in the environment and in biota and seafood is major contributor of BFRs to human exposure. Here, we used data from >9700 samples of wild and farmed fish, fish feed and fish feed ingredients collected from the North Atlantic between 2006 and 2016 aiming to investigate factors influencing the risk assessments of BFRs. Due to most representative number of analyses, PBDEs were the main focus of investigation. Mean ∑PBDE in fillet samples ranged from below quantification in Atlantic cod fillet to 2.0 µg kg-1 in Atlantic halibut. The main congener contributing to the ∑PBDE in all species was BDE 47. Factors affecting the level of BFR in seafood were multifaceted, and the levels were within species mainly determined by fish age, geographical origin and time of sampling. BDE 47, 99, 153 and HBCD were selected for margin of exposure (MOE) evaluation. When other sources of BFR than seafood were excluded, our risk assessment showed low risk at the current dietary intake of seafood. However, the dietary intake of BDE 99 may be of concern for toddlers when all sources are considered. The choice of fish species, dietary studies, choice of statistics, as well as exposure from other sources than seafood, were all factors that influenced the final MOE of BFRs. We propose the use of regression on order statistics as a tool for risk assessment, to illustrate means and spreads in large surveillance datasets to avoid the issue of measurements below the limit of quantification. A harmonized, updated evaluation of the risk associated with exposure to BFRs from diet, air and dust is warranted, where the fish species most commonly consumed also is taken into consideration.

Lower levels of Persistent Organic Pollutants, metals and the marine omega 3-fatty acid DHA in farmed compared to wild Atlantic salmon (Salmo salar).

Contaminants and fatty acid levels in farmed- versus wild Atlantic salmon have been a hot topic of debate in terms of food safety. The present study determined dioxins (polychlorinated dibenzo-p-dioxin and dibenzofuran), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), organochlorine pesticides (OCPs), metals and fatty acids in wild and farmed Atlantic salmon. Contaminant levels of dioxins, PCBs, OCPs (DDT, dieldrin, lindane, chlordane, Mirex, and toxaphene), and mercury were higher in wild salmon than in farmed salmon, as were the concentrations of the essential elements selenium, copper, zinc and iron, and the marine omega-3 fatty acid docosahexaenoic acid (DHA). PBDE, endosulfan, pentachlorobenzene, hexachlorobenzene, cadmium and lead levels were low and comparable in both wild and farmed fish, and there was no significant difference in the marine omega-3 fatty acid eicosapentaenoic acid (EPA) concentration. The total fat content was significantly higher in farmed than wild salmon due to a higher content of both saturated and monounsaturated fatty acids, as well as a higher content of omega-6 fatty acids. The omega-3 to omega-6 fatty acid ratio was considerably lower in farmed than wild salmon due to the high level of omega-6 fatty acids. Contaminant concentrations in Atlantic salmon were well below maximum levels applicable in the European Union. Atlantic salmon, both farmed and wild, is a good source of EPA and DHA with a 200g portion per week contributing 3.2g or 2.8g respectively, being almost twice the intake considered adequate for adults by the European Food Safety Authority (i.e. 250mg/day or 1.75g/week).

Contaminant levels in Norwegian farmed Atlantic salmon (Salmo salar) in the 13-year period from 1999 to 2011.

BACKGROUND: Environmental pollutants such as dioxins and PCBs, heavy metals, and organochlorine pesticides are a global threat to food safety. In particular, the aquatic biota can bioaccumulate many of these contaminants potentially making seafood of concern for chronic exposure to humans. OBJECTIVES: The main objective was to evaluate trends of contaminant levels in Norwegian farmed Atlantic salmon in light of the derived tolerable intakes. METHODS: Through an EU-instigated surveillance programme, the Norwegian Food Safety Authority (NFSA) has between 1999 and 2011 collected more than 2300 samples of Norwegian farmed Atlantic salmon (Salmo salar) for contaminant analyses. The fillets of these fish were homogenised and analysed for dioxins, PCBs, heavy metals and organochlorine pesticides. RESULTS: The levels of the contaminants mercury, arsenic, dioxins, dioxin-like PCBs and DDT in Norwegian farmed salmon fillet have decreased during our period of analyses. The levels of cadmium, lead and several organochlorine pesticides were too close to the limit of quantification to calculate time trends. For PCB6 and quantifiable amounts of pesticides, except DDT, stable levels were observed. CONCLUSION: The contaminant levels in Norwegian farmed salmon have generally decreased between 1999 and 2011. Excluding other dietary sources, the levels of dioxins and dioxin-like PCBs in 2011 allowed consumption of up to 1.3kg salmon per week to reach the tolerable weekly intake. The group of contaminants which was the limiting factor for safe consumption of Norwegian farmed salmon, based on currently established TWI values, is the sum of dioxins and dioxin-like PCBs.

Atlantic halibut (Hippoglossus hippoglossus L.) T-cell and cytokine response after vaccination and challenge with nodavirus.

Viral encephalopathy and retinopathy (VER), caused by nodavirus, is one of the major infectious diseases affecting the marine fish farming industry, yet no effective vaccine is available. In this study, we examined the halibut immune response following administration of an experimental vaccine comprising a recombinant nodavirus capsid protein in combination with an oil adjuvant (OA). Four groups of halibut were injected with either: PBS alone, PBS plus OA, 10µg recCP plus OA, or 50µg recCP plus OA. 15 weeks later, half the fish in each group were challenged with nodavirus and the immune response investigated by analysis of: serum levels of recCP-specific halibut immunoglobulins (Igs), and mRNA transcript levels of several T-cell markers (CD3ɛ, Lck, CD4, CD4-2, CD8α and CD8ß) and cytokines (IL-1ß, IL-6, IL-12ßc and IFNγ). Additionally, the presence of nodaviral RNA2 transcripts in the brains of infected halibut was analysed. After vaccination, the level of IL-6 was consistently elevated in the spleens of fish given injections containing the OA. The combination of recCP and OA increased the expression of IL-1ß and IFNγ, as well as the level of recCP-specific Igs in blood plasma. Following challenge with nodavirus, IL-1ß and IFNγ were elevated in halibut spleens after 24h in all groups that had received OA with or without recCP antigen. In brain, a general increase in the expression levels of all T-cell markers and IFNγ was observed following challenge with nodavirus. The viral load at 8 weeks post-challenge was lower in the fish that received 50µg recCP, with 5 out of 8 individuals being negative for nodavirus. Additionally, a better correlation between these markers (apart from the CD8 markers), and the viral RNA2 was also observed in this group, suggesting that the activation of CD4+T-cells might be important in reducing the viral load. In conclusion, this study identifies recCP as a promising candidate antigen for the future development of a vaccine against nodavirus.

Dietary methylmercury alters the proteome in Atlantic salmon (Salmo salar) kidney.

BACKGROUND: Methylmercury (MeHg) is an environmental contaminant most known for its severe neurotoxic effects. Although accumulation of MeHg tends to be several folds higher in kidney compared to other tissues, studies on nephrotoxic effects are almost non-existing. In this study we aim to investigate the toxicity of dietary MeHg in kidney of Atlantic salmon (Salmo salar). MATERIAL AND METHODS: Atlantic salmon were exposed to dietary MeHg for a period of 8 weeks, before the fish were euthanized and kidney was sampled for proteomic and real time RT-PCR analysis, as well as for mercury determination. Protein separation was done with 2-D PAGE, and differentially regulated spots were picked for analysis using liquid chromatography MS/MS analysis. Moreover, whole blood and liver tissue were sampled for mercury determination and real time RT-PCR (liver). RESULTS: MeHg exposed fish accumulated significantly more mercury (Hg) than control fish. The proteomic analysis revealed differential abundance of 26 spots in the kidney, and 14 of these protein spots were successfully identified. The proteins identified indicated effects of MeHg on; metabolism, inflammation, oxidative stress, protein-folding, and cell-structural components. Gene expression analysis of selected markers revealed few differentially regulated transcripts in kidney and liver in the exposed fish compared to the control fish. However, the affected transcripts indicated a disruption in the expression of two metabolic markers due to MeHg exposure in liver. CONCLUSION: This study suggests that dietary MeHg has similar effects in kidney as previously shown for other tissues in fish. The effects observed were in markers for oxidative stress, inflammation and energy metabolism. The identification of proteomic markers in this study provides a basis for a better understanding of MeHg-induced nephrotoxicity in fish.

Marine n-3 fatty acids alter the proteomic response to methylmercury in Atlantic salmon kidney (ASK) cells.

Fish based diets have been linked to the amelioration of methylmercury (MeHg) induced symptoms in several epidemiological studies, particularly due to their contents of marine n-3 fatty acids. It has been suggested that n-3 fatty acids may mask the detrimental effects of MeHg due to their beneficial effect on the same biological functions which are negatively affected by MeHg. However, in vitro studies have implied that there may be direct interactions between the marine n-3 FAs and MeHg, which ameliorates MeHg toxicity through interactions at a biological level. To understand how marine n-3 FAs and MeHg interact in fish as a biological system, we wanted to investigate molecular interaction in a fish cell system. Atlantic salmon kidney (ASK) cells were pre-incubated with the marine n-3 FAs docosahexaenoic acid (22:6n-3, DHA) and eicosapentaenoic acid (20:5n-3, EPA) before exposing them to MeHg. Modulating effects of the marine FAs on MeHg toxicity were subsequently assessed using the exploratory technique of proteomics, in a factorial design. Thirty-four differentially regulated proteins were identified. From these; twenty-seven were shown to be differentially regulated by MeHg, twelve were regulated by the fatty acids, and another eight showed interaction effects between MeHg and the FAs. Several of the proteins were concomitantly affected by MeHg- and FA-main effects, as well as interaction effects. Functional annotations and pathway analysis of the proteins revealed that marine n-3 FAs and MeHg concurrently affected the abundance of protein markers relating to such molecular mechanisms as: cell signaling, calcium homeostasis, structural integrity, apoptosis, and energy metabolism. In conclusion, both marine n-3 FAs and MeHg can differentially affect the abundances of the same proteins, indicating modulating effects of EPA and DHA on MeHg metabolism, and possibly on its toxicity.