Sea Bass Primary Cultures versus RTgill-W1 Cell Line: Influence of Cell Model on the Sensitivity to Nanoparticles

Determination of acute toxicity to vertebrates in aquatic environments is mainly performed following OECD test guideline 203, requiring the use of a large number of fish and with mortality as endpoint. This test is also used to determine toxicity of nanomaterials in aquatic environments. Since a replacement method for animal testing in nanotoxicity studies is desirable, the feasibility of fish primary cultures or cell lines as a model for nanotoxicity screenings is investigated here. Dicentrarchus labrax primary cultures and RTgill-W1 cell line were exposed to several concentrations (0.1 to 200 ug/mL) of different nanoparticles (TiO2, polystyrene and silver), and cytotoxicity, metabolic activity and reactive oxygen species formation were investigated after 24 and 48 h of exposure. Protein corona as amount of protein bound, as well as the influence of surface modification (-COOH, -NH2), exposure media (Leibovitz’s L15 or seawater), weathering and cell type were the experimental variables included to test their influence on the results of the assays. Data from all scenarios was split based on the significance each experimental variable had in the result of the cytotoxicity tests, in an exploratory approach that allows for better understanding of the determining factors affecting toxicity. Data shows that more variables significantly influenced the outcome of toxicity tests when the primary cultures were exposed to the different nanoparticles. Toxicity tests performed in RTgill-W1 were influenced only by exposure time and nanoparticle concentration. The whole data set was integrated in a biological response index to show the overall impact of nanoparticle exposures.This study was supported by a postdoctoral grant to AJ-R (Basque Government; POSDOC program 2017–2019), Basque Country, Spain; and Federal Ministry of Education and Research, BMBF (NanoUmwelt, grant agreement Nº 030150B), Germany

Hepato(Geno)toxicity assessment of nanoparticles in a HepG2 liver spheroid model

(1) In compliance with the 3Rs policy to reduce, refine and replace animal experiments, the development of advanced in vitro models is needed for nanotoxicity assessment. Cells cultivated in 3D resemble organ structures better than 2D cultures. This study aims to compare cytotoxic and genotoxic responses induced by titanium dioxide (TiO), silver (Ag) and zinc oxide (ZnO) nanoparticles (NPs) in 2D monolayer and 3D spheroid cultures of HepG2 human liver cells. (2) NPs were characterized by electron microscopy, dynamic light scattering, laser Doppler anemometry, UV-vis spectroscopy and mass spectrometry. Cytotoxicity was investigated by the alamarBlue assay and confocal microscopy in HepG2 monolayer and spheroid cultures after 24 h of NP exposure. DNA damage (strand breaks and oxidized base lesions) was measured by the comet assay. (3) Ag-NPs were aggregated at 24 h, and a substantial part of the ZnO-NPs was dissolved in culture medium. Ag-NPs induced stronger cytotoxicity in 2D cultures (EC 3.8 µg/cm 2) than in 3D cultures (EC > 30 µg/cm 2), and ZnO-NPs induced cytotoxicity to a similar extent in both models (EC 10.1-16.2 µg/cm 2). Ag- and ZnO-NPs showed a concentration-dependent genotoxic effect, but the effect was not statistically significant. TiO-NPs showed no toxicity (EC > 75 µg/cm 2). (4) This study shows that the HepG2 spheroid model is a promising advanced in vitro model for toxicity assessment of NPs

Risk assessment of grilled and barbecued food

When grilling, more harmful substances can be formed than when frying in a pan. For most people, there is a low risk associated with eating grilled food. Grilling food at a high temperature and/or on a campfire, often, and eating a lot of it may damage one's health. Distance to the heat source, how early food is placed onto the grill, and the type of fuel used can affect the formation of harmful substances. It is known that heat treatment such as grilling and frying can give rise to unwanted toxic compounds in the food, so-called process-induced contaminants. Grilling is a common way of preparing food in Norway, and the grilling season has become longer. The food selection has become ever wider and sales of different types of grills are increasing. Many factors may thus have changed since VKM's previous assessment of health risks from the consumption of grilled food, which was published in 2007. To be able to give current and relevant advice to consumers and others who sell or offer grilled food, the Norwegian Food Safety Authority has asked VKM for updated knowledge about the formation of process-induced contaminants in different food products by different grilling methods, and an assessment of what risk this may pose. Main findings: Harmful substances can be formed at high cooking temperatures. When grilling, the temperature is higher and less controllable than when frying in a pan. There is good evidence that two groups of genotoxic and carcinogenic substances, heterocyclic amines (HAA) and polycyclic aromatic hydrocarbons (PAH), are formed in higher concentrations in grilled food than in fried food. VKM has assessed PAH, for which there are good studies of occurrence in barbecued food. PAHs are formed when fat burns after dripping from the food onto the heat source. PAHs can also be released from coal, briquettes and wood. The smoke with PAH settles on the food. The occurrence of PAH in grilled food varies greatly and depends on how the food is grilled. The concentration of PAH is highest in very well-done meat with a high fat content, such as pork ribs and hamburgers. The highest concentration of PAH was found in sausage grilled on a campfire. This is caused by unburnt fat or soot/smoke from the fire that stick to the sausage. By avoiding fat dripping directly onto the heat source, preventing smoke from coming into contact with the food, and not overcooking the food, the amount of PAH in grilled food can be reduced. When using charcoal, the PAH emission is higher immediately after lighting, and the amount of PAH in the food can be reduced by waiting sufficiently long before start grilling. It is not the number of times you grill that is a critical factor, but how. A "worst-case" scenario shows that the annual consumption of more than 15-25 meals that have been grilled in a way that produces a high PAH incidence may provide too low margin of exposure* for the risk to be low. If, on the other hand, you prefer lean barbecue food that is not overcooked, then you can eat it more than 100 times a year according to our calculations and still have a high enough margin of exposure. * Margin of exposure is the ratio between the lowest dose (reference point) that causes increased cancer in experimental animals and the calculated intake. Exposure margin below 10,000 is considered a public health concern. Norsk: Ved grilling kan det dannes flere helseskadelige stoffer enn ved steking i panne. For de fleste er det lav risiko knyttet til å spise grillmat.Risk assessment of grilled and barbecued foodpublishedVersionacceptedVersionacceptedVersio

Risk assessment of dioxins, furans, and dioxin-like PCBs in food in Norway - Scientific Opinion of the Panel on Contaminants of the Norwegian Scientific Committee for Food and Environment

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Cyanobakterier og cyanotoksiner i norske drikkevannskilder - (VKM)

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Contaminants in fish from Etnefjord, Norway

1,2,5,6,9,10-hexabromocyclododecane (HBCDD) is one of the most frequently used brominated flame retardants (BFR) and is considered as an emerging environmental pollutant. Recently, high levels of HBCDD were reported in five different fish species from Etnefjord, which is a branch of the Hardangerfjord at the southwest coast of Norway. This report raised some serious concerns and the Norwegian Climate and Pollution Agency (Klif) therefore initiated a survey of HBCDD in cod and flounder from Etnefjorden in order to confirm the findings. In addition to HBCDD, the levels of other environmental pollutants were analyzed in the fish, namely polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), hexachlorocyclohexane (HCH), dichlorodiphenyltrichloroethane (DDT), mercury (Mg), lead (Pb), cadmium (Cd) and arsenic (As). The fish muscle filet and livers were prepared and analyzed using recognized and previously published methods. The levels of HBCDD in the fish were low, representing expected background concentrations. HBCDD was only detected in the cod livers with concentrations ranging 0.3-5.7 ng/g wet weight, corresponding to 1.3-8.2 ng/g on a lipid weight basis. Previously, it was reported a level of 30 000 ng/g lipid weight in cod filet, which is, as far as we know, the highest HBCDD concentration ever reported in cod. Our study could, therefore, not confirm the recent findings that showed high HBCDD concentrations in fish from the Etnefjord area. Also, the levels of the other pollutants were low. For example, the concentrations of DDT, PCB, HCH and Hg can, according to Klifs system for classification of environmental state, be regarded as background levels (class 1, slightly/negligible polluted). In conclusion, there are no recons to believe that cod and flounder in Etnefjorden is exposed to considerable amount of environmental pollutants, neither of HBCDD nor other contaminants. Only further analysis of sediments from the inner site of the fjord can, however, positively reveal a possible HBCDD source. The Norwegian Climate and Pollution Agency. TA-2821/2011

A survey of dioxin-like contaminants in fish from recreational fishing.

The dioxin and dioxin-like compounds are regarded as one of the most toxic group of environmental contaminants. Food for the commercial market is regularly monitored for their dioxin levels and the concentration allowed in food is strictly regulated. Less is known about locally caught fish from recreational fishing, which is often brought home for consumption. This can be fish caught from nearby lakes or streams or fish with marine origin close to industrial areas or harbours that are not regularly monitored for their dioxin levels. In this study, we established collaboration with schools in 13 countries. We received 203 samples of 29 different fish species of which Atlantic cod was the most abundant followed by brown trout and pollock. In general, the majority of samples from the participating countries had low concentrations (between 0.1 and 0.2 pg/g chemical-activated luciferase gene expression toxic equivalency wet weight (CALUX TEQ w.w.)) of dioxins and dioxin-like PCBs. Only 18 samples had concentrations above 1 pg/g CALUX TEQ w.w., and only 2 dab samples had concentration above maximum levels set by the European Commission. The Atlantic cod samples showed a significant reduction in the concentrations of dioxins with increasing latitude indicating less contamination of dioxin and dioxin-like compounds in the north of Norway. The results indicate that a moderate consumption of self-caught fish at presumed non-contaminated sites does not represent a major risk for exposure to dioxins or dioxin-like compounds at concentrations associated with adverse health effects. Recreational fishermen should, however, obtain knowledge about local fish consumption advice

Uptake and effects of 2, 4, 6 - trinitrotoluene (TNT) in juvenile Atlantic salmon (Salmo salar)

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Different Sensitivity of Advanced Bronchial and Alveolar Mono- and Coculture Models for Hazard Assessment of Nanomaterials

For the next-generation risk assessment (NGRA) of chemicals and nanomaterials, new approach methodologies (NAMs) are needed for hazard assessment in compliance with the 3R’s to reduce, replace and refine animal experiments. This study aimed to establish and characterize an advanced respiratory model consisting of human epithelial bronchial BEAS-2B cells cultivated at the air–liquid interface (ALI), both as monocultures and in cocultures with human endothelial EA.hy926 cells. The performance of the bronchial models was compared to a commonly used alveolar model consisting of A549 in monoculture and in coculture with EA.hy926 cells. The cells were exposed at the ALI to nanosilver (NM-300K) in the VITROCELL® Cloud. After 24 h, cellular viability (alamarBlue assay), inflammatory response (enzyme-linked immunosorbent assay), DNA damage (enzyme-modified comet assay), and chromosomal damage (cytokinesis-block micronucleus assay) were measured. Cytotoxicity and genotoxicity induced by NM-300K were dependent on both the cell types and model, where BEAS-2B in monocultures had the highest sensitivity in terms of cell viability and DNA strand breaks. This study indicates that the four ALI lung models have different sensitivities to NM-300K exposure and brings important knowledge for the further development of advanced 3D respiratory in vitro models for the most reliable human hazard assessment based on NAMs