Beauvericin (BEA) and enniatin B (ENNB)-induced impairment of mitochondria and lysosomes - potential sources of intracellular reactive iron triggering ferroptosis in Atlantic salmon primary hepatocytes

Beauvericin (BEA) and enniatin B (ENNB) are emerging mycotoxins frequently detected in plant-based fish feed. With ionophoric properties, they have shown cytotoxic potential in mammalian models. Sensitivity in fish is still largely unknown. Primary hepatocytes isolated from Atlantic salmon (Salmo salar) were used as a model and exposed to BEA and ENNB (0.05–10 μM) for 48 h. Microscopy, evaluation of cell viability, total ATP, total H2O2, total iron content, total Gpx enzyme activity, and RNA sequencing were used to characterize the toxicodynamics of BEA and ENNB. Both mycotoxins became cytotoxic at ≥ 5 μM, causing condensation of the hepatocytes followed by formation of blister-like protrusions on the cell's membrane. RNA sequencing analysis at sub-cytotoxic levels indicated BEA and ENNB exposed hepatocytes to experience increased energy expenditure, elevated oxidative stress, and iron homeostasis disturbances sensitizing the hepatocytes to ferroptosis. The present study provides valuable knowledge disclosing the toxic action of these mycotoxins in Atlantic salmon primary hepatocytes.acceptedVersio

Long-term effects of environmentally relevant doses of 2,2',4,4',5,5' hexachlorobiphenyl (PCB153) on neurobehavioural development, health and spontaneous behaviour in maternally exposed mice

Background: Polychlorinated biphenyls (PCBs) are widespread in the environment, human food and breast milk. Seafood is known to contain nutrients beneficial for the normal development and function of the brain, but also contaminants such as PCBs which are neurotoxic. Exposure to non-coplanar PCBs during brain development can disrupt spontaneous behaviour in mice and lead to hyperactive behaviour. Humans are chronically exposed to the highest relative levels of organochlorines in early childhood during brain development, though usually at doses which do not give clinical symptoms of toxicity. This study aimed to elucidate the developmental and behavioural effects of 2,2’,4,4’,5,5’ hexachlorobiphenyl (PCB153) in mice, mimicking human exposure during gestation and lactation. Methods: Environmentally relevant doses of PCB153 were added to the experimental diets. Feed concentrations were approximately 0.5, 6.5, and 1500 μg PCB153/kg feed, representing a realistic and a worst case scenario of frequent consumption of contaminated fish. The study also investigated the effects of maternal nutrition, i.e. a standard rodent diet versus a high inclusion of salmon. Mice pups were examined for physical- and reflex development, sensorimotor function and spontaneous behaviour from five days after birth until weaning. A selection of pups were followed until 16 weeks of age and tested for open field behaviour and the acoustic startle response (ASR) with prepulse inhibition (PPI). Blood thyroid hormones and liver enzymes, blood lipids and PCB153 content in fat were examined at 16 weeks. Statistical analyses modelled the three way interactions of diet, PCB exposure and litter size on behaviour, using generalized linear models (GLM) and linear mixed effect models (LME). The litter was used as a random variable. Non-parametric tests were used for pair wise comparisons of biochemical analyses. Results: Litter size consistently influenced pup development and behaviour. Few lasting PCB153 related changes were observed, but results indicated effects on synchronization of physical development. Perinatal PCB153 exposure appeared to reduce habituation and cause aggression in males, though not statistically significant. Conclusions: Litter size and maternal diet influenced physical development and function more than PCB153 in perinatally exposed mouse pups and supports the developmental importance of maternal care and the social environment

Sampling protocol for the determination of nutrients and contaminants in fish and other seafood – The EAF-Nansen Programme

Seafood plays a central role in global food and nutrition security. However, there is a lack of data on the concentration of nutrients and contaminants in fish and other seafood, especially in low- and middle-income countries. In order to assess the potential risks and benefits associated with seafood intake, reliable and up-to-date food composition data is crucial. The quality of food composition data is affected by several factors, such as sampling protocols and the suitability and quality of the methods applied for sample preparation and analysis. In this paper, we describe the sampling methodology and protocols related to the sampling of fish and other seafood and the corresponding analytical methods used to analyse the nutrient and contaminant content of such species. For nutrients, the determination of protein, fat, ash, energy, fatty acids, cholesterol, and amino acids is described, in addition to analyses for determination of the vitamin and mineral content in fish and other seafood. For contaminants, analyses for the determination of organic pollutants and microplastics are described. The methodology described in this paper is used for sampling data through scientific surveys in low- and middle-income countries with research vessel Dr. Fridtjof Nansen under the EAF-Nansen Programme. The Programme aims to improve knowledge on the nutritional composition of fish and ensure the fish is safe to consume. •In this paper, we describe the sampling protocols used for sampling fish and other seafood during scientific surveys under the EAF-Nansen Programme. •This paper describes the methodology and quality control for analysing nutrients and contaminants in fish and other seafood.publishedVersio

Enniatin B and beauvericin affect intestinal cell function and hematological processes in Atlantic salmon (Salmo salar) after acute exposure

Unintentional use of mold-infested plant-based feed ingredients are sources of mycotoxins in fish feeds. The presence of the emerging mycotoxins ENNB and BEA in Norwegian commercial fish feeds and plant-based feed ingredients has raised concerns regarding the health effects on farmed Atlantic salmon (Salmon salar). Atlantic salmon pre-smolts were exposed to non-lethal doses of BEA and ENNB (ctrl, 50 and 500 μg/kg feed for 12 h), after which total RNA sequencing of the intestine and liver was carried out to evaluate gut health and identify possible hepatological changes after acute dietary exposure. ENNB and BEA did not trigger acute toxicity, however ENNB caused the onset of pathways linked to acute intestinal inflammation and BEA exposures caused the onset of hepatic hematological disruption. The prevalence and concentration of ENNB found in today's commercial feed could affect the fish health if consumed over a longer time-period.publishedVersio

Cerebral gene expression and neurobehavioural development after perinatal exposure to an environmentally relevant polybrominated diphenylether (BDE47)

Nutrients in seafood are known to be beneficial for brain development. Effects of maternal exposure to 2,2′,4,4′ tetrabromo diphenylether (BDE47) was investigated, alongside the potential ameliorating impact of seafood nutrients, through assessment of neurobehaviour and gene expression in brain and liver. Developing mice were exposed during gestation and lactation via dams dosed through casein- or salmon-based feed, spiked with BDE47. Two concentrations were used: a low level (6 μg/kg feed) representing an environmentally realistic concentration and a high level (1,900 μg/kg feed) representing a BDE47 intake much higher than expected from frequents consumption of contaminated seafood. Experimental groups were similar with respect to reproductive success, growth and physical development. Minor, transient changes in neurobehavioural metrics were observed in groups given the highest dose of BDE47. No significant differences in behaviour or development were seen on postnatal day 18 among maternally exposed offspring. Cerebral gene expression investigated by microarray analyses and validated by RT-qPCR showed low fold changes for all genes, despite dose-dependent accumulation of BDE47 in brain tissue. The gene for glutamate ammonia ligase was upregulated compared to control in the casein-based high BDE47diet, suggesting potential impacts on downstream synaptic transmission. The study supported a previously observed regulation of Igfbp2 in brain with BDE47 exposure. Genes for hepatic metabolic enzymes were not influenced by BDE47. Potential neurotoxic effects and neurobehavioural aberrations after perinatal exposure to high levels of BDE47 were not readily observed in mice pups with the present experimental exposure regimes and methods of analysis

Program for fremmedstoffer i fôrmidler av fisk - Rapport for prøver innsamlet i 2021

Mattilsynet er oppdragsgiver for dette overvåkningsprogrammet, som har som formål å undersøke innholdet av fremmedstoffer i fôrmidler av fisk, og som gjennomføres ved Havforskningsinstituttet. Overvåkingsprogrammet supplerer «Program for overvåking av fiskefôr» med data på uønskede stoffer i fôrmidler av fisk som er produsert i Norge og tatt ut på produksjonsvirksomhetene. I 2021 ble 16 prøver analysert: 6 prøver av fiskeolje, 2 prøver av fiskemel og 8 prøver av fiskeprotein konsentrat. Fremmedstoffene som ble inkludert i 2021 var dioksiner og furaner (PCDD/F), dioksinlignende PCB (dl-PCB), PCB6, polybromerte diphenyleter (PBDE), Perfluorerte forbindelser (PFAS), klorerte pesticider, og metaller (kvikksølv, bly, kadmium og arsen). Prøvene ble også analysert for ethoxyquin, ethoxyquin dimer, medisinrester og salmonella. Resultatene viste at innholdet av alle organiske miljøgifter analysert i fiskemel, fiskeolje og fiskeprotein-konsentrat var lave. Ingen av fiskemelene, fiskeoljene eller fiskeprotein konsentratene hadde nivå av dioksiner, sum dioksiner og dl-PCB, eller PCB6 over grenseverdien. Resultatene for 2021 viste lave nivåer av metaller, som innbefatter ingen overskridelser av grenseverdiene for arsen, kvikksølv, kadmium eller bly i fiskemel eller fiskeprotein konsentrat. Det ble ikke påvist salmonella, ethoxyquin eller ethoxyquin dimer i noen av prøvene analysert i 2021. Generelt var nivåene av uønskede stoffer i de fôrmidler av fisk som analysert i dette overvåkningsprogrammet i 2021 sammenlignbare med resultatene fra prøvene som ble tatt ut på fôrfabrikkpublishedVersio

Tolerance and dose-response assessment of subchronic dietary ethoxyquin exposure in Atlantic salmon (Salmo salar L.)

Ethoxyquin (EQ; 6-Ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline) has been used as an antioxidant in feed components for pets, livestock and aquaculture. However, possible risks of EQ used in aquafeed for fish health have not yet been characterized. The present study investigated the toxicity and dose-response of subchronic dietary EQ exposure at doses ranging from 41 to 9666 mg EQ/kg feed in Atlantic salmon (Salmo salar L.). Feed at concentrations higher than 1173 mg EQ/kg were rejected by the fish, resulting in reduced feed intake and growth performance. No mortality was observed in fish exposed to any of the doses. A multi-omic screening of metabolome and proteome in salmon liver indicated an effect of dietary EQ on bioenergetics pathways and hepatic redox homeostasis in fish fed concentrations above 119 mg EQ/kg feed. Increased energy expenditure associated with an upregulation of hepatic fatty acid â-oxidation and induction and carbohydrate catabolic pathways resulted in a dose-dependent depletion of intracytoplasmic lipid vacuoles in liver histological sections, decreasing whole body lipid levels and altered purine/pyrimidine metabolism. Increased GSH and TBARS in the liver indicated a state of oxidative stress, which was associated with activation of the NRF2-mediated oxidative stress response and glutathione-mediated detoxification processes. However, no oxidative DNA damage was observed. As manifestation of altered energy metabolism, the depletion of liver intracytoplasmic lipid vacuoles was considered the critical endpoint for benchmark dose assessment, and a BMDL10 of 243 mg EQ/kg feed was derived as a safe upper limit of EQ exposure in Atlantic salmon

Program for fremmedstoffer i fôrmidler av fisk - Rapport for prøver innsamlet i 2022

Mattilsynet er oppdragsgiver for dette overvåkningsprogrammet, som har som formål å undersøke innholdet av fremmedstoffer i fôrmidler av fisk, og som gjennomføres ved Havforskningsinstituttet. Overvåkingsprogrammet supplerer «Program for overvåking av fiskefôr» med data på uønskede stoffer i fôrmidler av fisk som er produsert i Norge og tatt ut på produksjonsvirksomhetene. I 2022 ble 20 prøver analysert: 9 prøver av fiskeolje og 11 prøver av fiskemel. Fremmedstoffene som det ble analysert for i 2022 var dioksiner og furaner (PCDD/F), dioksinlignende PCB (dl-PCB), PCB6 (ndl-PCB), polybromerte diphenyleter (PBDE), perfluorerte forbindelser (PFAS) og klorerte pesticider (dieldrin/aldrin, DDT, toksafen, klordan, endosulfan, sum heptaklor, sum HCH og HCB). Fiskemel ble i tillegg analysert for metaller (kvikksølv, bly, kadmium og arsen), syntetiske antioksidanter (ethoxyquin, ethoxyquin dimer, BHA og BHT), prosesserte animalske proteiner (PAP), vitamin E og Salmonella. Resultatene viste at konsentrasjonene av alle organiske miljøgifter i fiskemel og fiskeolje var lave. Ingen av fiskemelene eller fiskeoljene hadde konsentrasjoner av PCDD/PCDF («sum dioksiner»), PCDD/PCDF + dl-PCB («sum dioksiner og dioksinlignende PCB» eller PCB6 over de øvre grenseverdiene. Resultatene for 2022 viste lave nivå av metaller i fiskemel, og ingen overskridelser av grenseverdiene for arsen, kvikksølv, kadmium eller bly. Det ble ikke påvist salmonella, PAP, ethoxyquin eller ethoxyquin dimer i noen av prøvene analysert i 2022. Generelt var nivåene av uønskede stoffer i fôrmidler av fisk analysert i 2022 sammenlignbare med resultatene fra tidligere års undersøkelser.Program for fremmedstoffer i fôrmidler av fisk - Rapport for prøver innsamlet i 2022publishedVersio

Seasonal variations in mercury, cadmium, lead and arsenic species in Norwegian blue mussels (Mytilus edulis L.) – Assessing the influence of biological and environmental factors

Blue mussels (Mytilus edulis L.) can accumulate undesirable substances, including the potentially toxic elements (PTEs) cadmium (Cd), mercury, (Hg), lead (Pb), arsenic (As) and As species. In this study, the levels of PTEs and As species were determined in samples of blue mussels to assess the influence of environmental and biological factors, and evaluate the potential risk associated with blue mussels in terms of food and feed safety.publishedVersio

Subchronic dietary exposure to ethoxyquin dimer induces microvesicular steatosis in male BALB/c mice

The use of the synthetic antioxidant ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline; EQ) in animal feed results in the presence of EQ residues and metabolites, including the EQ dimer (1,8′-bi(6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline); EQDM) in animal food products. To investigate the toxicity and dose-response of dietary exposure to EQDM, male BALB/c mice were exposed to one of six dietary doses of EQDM, ranging from 0.015 to 518 mg/kg body weight/day for 90 days. Doses above 10 mg/kg body weight/day affected whole body lipid metabolism resulting in increased liver weights and decreased adipose tissue mass. Metabolomic screening of livers revealed alterations indicating incomplete fatty acid β-oxidation and hepatic oxidative stress. Histopathological evaluation and biochemical analyses of the liver confirmed the development of microvesicular steatosis and activation of the glutathione system. Hepatic protein profiling and pathway analyses suggested that EQDM-induced responses are mediated through activation of CAR/PXR nuclear receptors and induction of a NRF2-mediated oxidative stress response. Based on the development of microvesicular steatosis as the critical endpoint, a Reference Point for dietary EQDM exposure was established at 1.1 mg/kg body weight/day (BMDL10) from benchmark dose modelling. Applying an uncertainty factor of 200, an Acceptable Daily Intake of 0.006 mg EQDM/kg body weight was proposed.publishedVersio