Interplay between dietary lipids and cadmium exposure in aquatic biota : a multi-model study

The aim of this work was to investigate if and how (i) dietary lipids and Cd affect fatty acid homeostasis and metabolism, (ii) the individual/tissue/cell fatty acid profile modulates the Cd sensitivity, in rainbow trout (Oncorhynchus mykiss) and the large water flea (Daphnia magna), using both in vitro and in vivo approaches. We successfully modulated the fatty acid profile of the experimental models. Overall, both the fatty acid supply and the Cd exposure influenced fatty acid homeostasis and metabolism. The fatty acid profile influenced individual/tissue and cell sensitivity to Cd. In vitro enrichments in specific polyunsaturated fatty acids (PUFA) (e.g. alpha-linolenic acid (ALA, 18:3n-3) and eicosapentaenoic acid (EPA, 20:5n-3)) conferred protection against Cd while enrichment in others (e.g. linoleic acid (LA, 18:2n-6)) had no impact. PUFA-derived cytoprotection was not linked to a reduced cell Cd burden but would rather occur through a reduction of the oxidative stress induced by Cd and a differential induction of the eicosanoid cascade. In vivo, rainbow trout fed the ALA- and EPA-enriched diets seemed to be respectively the least and the most affected by Cd. Fish fed the ALA-enriched diet had a higher detoxifying ability against Cd and a lower hepatic Cd burden. In contrast, fish fed the EPA-enriched diet had impaired growth performances and a decreased total hepatic antioxidant capacity under Cd exposure. In vivo, liposome feeding increased adult daphnid tolerance to Cd. The presence of EPA in liposomes did not increase adult tolerance to Cd. Offspring's tolerance to Cd was influenced by the brood number and the maternal diet. It was positively correlated with the PUFA level in body neutral lipids, especially ALA. Overall, this work provides new information about interactions between the nature of dietary lipids and the biological responses to Cd in aquatic biota.(AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 202

Body lipid composition modulates acute cadmium toxicity in Daphnia magna adults and juveniles.

Long chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA, 20:5n-3) affect zooplankton fitness and ability to cope with environmental stressors. However, the impact of LC-PUFAs on zooplankton sensitivity to chemical stressors is unknown. Here, we aimed to document the interaction between EPA and cadmium (Cd), as model chemical stressor, in Daphnia magna. A life-history experiment was performed in which daphnid neonates were raised into adulthood on three diets of different lipid composition: (i) algae mix; (ii) algae mix supplemented with control liposomes; (iii) algae mix supplemented with liposomes containing EPA. Juveniles (3rd, 4th and 5th brood) released by daphnids during this life-history experiment were sampled, challenged with Cd during 48 h and their immobility was assessed. At the end of this life-history experiment, another immobilisation test was performed with adults from each treatment. Daphnids absorbed, incorporated and transferred ingested EPA to their offspring. Liposome feeding increased adult tolerance to Cd. The presence of EPA in liposomes did not increase adult tolerance to Cd. Offspring's tolerance to Cd was influenced by the brood number and the maternal diet. It was positively correlated with the PUFA level in body neutral lipids, especially alpha-linolenic acid (ALA, 18:3n-3) and negatively correlated with the saturated fatty acid level in body neutral lipids, especially stearic acid (18:0). Overall, these results emphasize the importance of dietary lipids and maternal transfer of body lipids in D. magna sensitivity to Cd and highlight the need to take into account these parameters in ecotoxicological studies and risk assessment

The fatty acid profile of rainbow trout liver cells modulates their tolerance to methylmercury and cadmium

The polyunsaturated fatty acid (PUFA) composition of fish tissues, which generally reflects that of thediet, affects various cellular properties such as membrane structure and fluidity, energy metabolism andsusceptibility to oxidative stress. Since these cellular parameters can play an important role in the cellularresponse to organic and inorganic pollutants, a variation of the PUFA supply might modify the toxicityinduced by such xenobiotics. In this work, we investigated whether the cellular fatty acid profile has animpact on the in vitro cell sensitivity to two environmental pollutants: methylmercury and cadmium.Firstly, the fatty acid composition of the rainbow trout liver cell line RTL-W1 was modified by enrichingthe growth medium with either alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3),docosahexaenoic acid (DHA, 22:6n-3), linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6) or docos-apentaenoic acid (DPA, 22:5n-6). These modified cells and their control (no PUFA enrichment) were thenchallenged for 24 h with increasing concentrations of methylmercury or cadmium. We observed that (i)the phospholipid composition of the RTL-W1 cells was profoundly modulated by changing the PUFA con-tent of the growth medium: major modifications were a high incorporation of the supplemented PUFA inthe cellular phospholipids, the appearance of direct elongation and desaturation metabolites in the cellu-lar phospholipids as well as a change in the gross phospholipid composition (PUFA and monounsaturatedfatty acid (MUFA) levels and n-3/n-6 ratio); (ii) ALA, EPA and DPA enrichment significantly protected theRTL-W1 cells against both methylmercury and cadmium; (iv) DHA enrichment significantly protectedthe cells against cadmium but not methylmercury; (v) AA and LA enrichment had no impact on the celltolerance to both methylmercury and cadmium; (vi) the abundance of 20:3n-6, a metabolite of the n-6 biotransformation pathway, in phospholipids was negatively correlated to the cell tolerance to bothmethylmercury and cadmium. Overall, our results highlighted the importance of the fatty acid supply onthe tolerance of fish liver cells to methylmercury and cadmium

Transcriptional effects of phospholipid fatty acid profile on rainbow trout liver cells exposed to methylmercury

Lipids, and their constitutive fatty acids, are key nutrients for fish health as they provide energy, maintain cell structure, are precursors of signalling molecules and act as nuclear receptor ligands. These specific roles may be of crucial importance in a context of exposure to pollutants. We recently showed that the fatty acid profile of rainbow trout liver cell phospholipids modulates sensitivity to an acute methylmercury challenge. In order to investigate mechanisms of effects, we herein tested whether specific polyunsaturated fatty acids may protect cells from methylmercury through decreasing intracellular mercury accumulation and/or enhancing cellular defences (e.g. via modulation of gene expression patterns). We also investigated the inverse relationship and assessed the impact of methylmercury on cellular fatty acid metabolism. To do so, the fatty acid composition of rainbow trout liver cell phospholipids was first modified by incubating them in a medium enriched in a specific polyunsaturated fatty acid (PUFA) from either the n-3 family (alpha-linolenic acid, ALA; eicosapentaenoic acid, EPA) or the n-6 family (linoleic acid, LA; arachidonic acid, AA). Cells were then exposed to methylmercury (0.15 or 0.50 μM) for 24 h and sampled thereafter for assessing phospholipid fatty acid profile, intracellular total mercury burden, and expression pattern of genes involved in fatty acid metabolism, synthesis of polyunsaturated fatty acid-derived signalling molecules and stress response. We observed that cells incorporated the given polyunsaturated fatty acid and some biotransformation products in their phospholipids. MeHg had few impacts on this cellular phospholipid composition. None of the PUFA enrichments affected the cellular mercury burden, suggesting that the previously observed cytoprotection conferred by ALA and EPA was not linked to a global decrease in cellular accumulation of mercury. Fatty acid enrichments and methylmercury exposure both modulated gene expression patterns. Genes involved in the synthesis of polyunsaturated fatty acid-derived signalling molecules, in stress response and the orphan cytochrome P450 20A1 were identified as possible sites of interaction between fatty acids and methylmercury in rainbow trout liver cells

Fish cell lines : a screening tool for multiple stress scenarios. Interaction between fatty acid profile and heavy metals in rainbow trout liver (RTL-W1)

In aquatic ecosystems, environmental conditions (e.g. temperature, food quality, water quantity...) are seldom optimal and change overtime. Though these variations may affect organisms' capacity to cope with chemical contamination, they are rarely taken into account in classical ecotoxicological tests and most of the regulation on chemicals is based on single substance test performed under optimal conditions. Nevertheless, different environmental conditions (e.g. food quality) can modify the characteristics of an organism (e.g. nutritional status) and thus can influence both the entrance of chemicals within this organism and its ability to cope with it. In particular, in fish, fatty acid composition can modulate membrane fluidity and thus influence contaminant uptake. Due to their different antioxidant properties, fatty acids are also likely to influence cells' sensibility to oxidative stress induced by contaminants. Fish fatty acid profile is influenced strongly by nutrition and temperature, thus, fish from the same species may present strong differences in their fatty acid profile and may respond differently to contamination. Few studies have been exploring this hypothesis therefore an in vitro approach was set up to better explore the role of a wide range of fatty acids in fish response to contaminants such as metals. Fish hepatocytes from the same cell line (RTL-W1) but with up to 7 different fatty acid profiles were then obtained by enriching the growth medium with 6 different fatty acids of interest. Though these cells were from the same cell line; the modification of their fatty acid profile changed certain of their properties (e.g. membrane fluidity) and modified their response to increasing concentrations of mercury (HgCl2 or CH3HgCl) or cadmium (CdCl2). For instance, cells enriched with the omega-3 fatty acid: alpha-linolenic acid showed a stronger resistance to organic mercury (EC50CH3HgCl = 6.6 µM) than non-enriched cells (EC50CH3HgCl = 2.9 µM) but a similar resistance to cadmium and inorganic mercury. The use of a fish cell line allowed screening the influence of various types of fatty acid profiles on heavy metals' sensitivity of fish. Based on these result, the multiple stress scenarios more likely to present a risk for the environment have been selected for further mechanistic (in vitro) and ecotoxicologic (in vivo) investigation

Exploring the interactions between polyunsaturated fatty acids and cadmium in rainbow trout liver cells: a genetic and proteomic study.

Polyunsaturated fatty acids (PUFAs) have key biological roles in fish cells. We recently showed that the phospholipid composition of rainbow trout liver cells (RTL-W1 cell line) modulates their tolerance to an acute cadmium (Cd) challenge. Here, we investigated (i) the extent to which PUFAs and Cd impact fatty acid homeostasis and metabolism in these cells and (ii) possible mechanisms by which specific PUFAs may confer cytoprotection against Cd. First, RTL-W1 cells were cultivated for one week in growth media spiked with 50 μmol L of either alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), linoleic acid (LA, 18:2n-6) or arachidonic acid (AA, 20:4n-6) in order to modulate their fatty acid profile. Then, the cells were challenged with Cd (0, 50 or 100 μmol L) for 24 h prior to assaying viability, fatty acid profile, intracellular Cd content, proteomic landscape and expression levels of genes involved in fatty acid metabolism, synthesis of PUFA-derived signalling molecules and stress response. We observed that the fatty acid supply and, to a lesser extent, the exposure to Cd influenced cellular fatty acid homeostasis and metabolism. The cellular fatty acid composition of fish liver cells modulated their tolerance to an acute Cd challenge. Enrichments in ALA, EPA, and, to a lesser extent, AA conferred cytoprotection while enrichment in LA had no impact on cell viability. The present study ruled out the possibility that cytoprotection reflects a decreased Cd burden. Our results rather suggest that the PUFA-derived cytoprotection against Cd occurs through a reduction of the oxidative stress induced by Cd and a differential induction of the eicosanoid cascade, with a possible role of peroxiredoxin and glutaredoxin (antioxidant enzymes) as well as cytosolic phospholipase A2 (enzyme initiating the eicosanoid cascade)

Environmentally-realistic concentration of cadmium combined with polyunsaturated fatty acids enriched diets modulated non-specific immunity in rainbow trout.

Nutrition is crucial to grow healthy fish particularly in a context of pollution, overcrowding and pathogen risks. Nowadays, the search for food components able to improve fish health is increasingly developing. Here, the influence of four dietary polyunsaturated fatty acids (PUFAs) that are alpha-linolenic acid (ALA, 18:3n-3), linoleic acid (LA, 18:2n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) on the sensitivity of rainbow trout (Oncorhynchus mykiss) juveniles to environmentally realistic cadmium (Cd, 0.3 μg/L) concentration was investigated. Fish diets were designed to ensure the specific abundance of one of these individual PUFAs, and were given for a 4-week pre-conditioning period followed by a 6-week Cd exposure period. Focus was put on growth performance and immune responses following a short (24 h) and a long-term (6 weeks) Cd exposure. For each experimental condition, some fish were submitted to a bacterial challenge (24 h) with Aeromonas salmonicida achromogenes at the end of Cd conditioning period. DHA-enriched diet improved growth performances as compared to LA-enriched diet, but also increased ROS production (after short-term exposure to Cd) that could lead to a higher inflammation status, and some immunity-related genes (at short and long-term exposure). We notably highlighted the fact that even a low, environmentally-realistic concentration, Cd can strongly impact the immune system of rainbow trout, and that specific dietary PUFA enrichment strategies can improve growth performance (DHA-enriched diet), provide protection against oxidative stress (ALA- and EPA-enriched diet) and stimulate non-specific immunity

Interplay between dietary lipids and cadmium exposure in rainbow trout liver: Influence on fatty acid metabolism, metal accumulation and stress response

The present study aimed at investigating interactive effects between dietary lipids and both short- and long-term exposures to a low, environmentally realistic, cadmium (Cd) concentration. Juvenile rainbow trout were fed four isolipidic diets (31.7 g/kg) enriched in either linoleic acid (LA, 18:2n-6), alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3) or docosahexaenoic acid (DHA, 22:6n-3). From the 4th week of this 10-week experiment, the lipid level of the diet was increased (120.0 g/kg) and half of the fish fed each diet were aqueously exposed to Cd (0.3 μg/L) while the other half were not exposed to Cd (control). Fish were sampled and their liver was harvested for fatty acid profile, hepatic Cd and calcium concentrations, total glutathione level and gene expression assessment, either (i) after 4 weeks of feeding and 24 h of Cd contamination (day 29) (short-term Cd exposure) or (ii) after 10 weeks of feeding and 6 weeks of Cd contamination (day 70) (long-term Cd exposure). We found that both dietary lipids and Cd exposure influenced fatty acid homeostasis and metabolism. The hepatic fatty acid profile mostly reflected that of the diet (e.g. n-3/n-6 ratio) with some differences, including selective retention of specific long chain polyunsaturated fatty acids (LC-PUFAs) like DHA and active biotransformation of dietary LA and ALA into LC-PUFAs. Cd effects on hepatic fatty acid profiles were influenced by the duration of the exposure and the nutritional status of the fish. The effects of diet and Cd exposure on the fatty acid profiles were only sparsely explained by variation of the expression pattern of genes involved in fatty acid metabolism. The biological responses to Cd were also influenced by dietary lipids. Fish fed the ALA-enriched diet seemed to be the least affected by the Cd exposure, as they showed a higher detoxifying ability against Cd with an early upregulation of protective metallothionein a (MTa) and apoptosis regulator BCL2-Like1 (BCLx) genes, an increased long-term phospholipid synthesis and turnover and fatty acid bioconversion efficiency, as well as a lower long-term accumulation of Cd in their liver. In contrast, fish fed the EPA-enriched diet seemed to be the most sensitive to a long-term Cd exposure, with an impaired growth performance and a decreased antioxidant capacity (lower glutathione level). Our results highlight that low, environmentally realistic aqueous concentrations of Cd can affect biological response in fish and that these effects are influenced by the dietary fatty acid composition