Ligand-activated receptors regulate numerous genes, and mediate effects of a broad set of endogenous and exogenous chemicals in vertebrates. Understanding the roles of these transcription factors in ...zebrafish (Danio rerio) is important to the use of this non-mammalian model in toxicological, pharmacological, and carcinogenesis research. Response to a potential agonist for the pregnane X receptor (Pxr) pregnenolone (PN) was examined in developing zebrafish, to assess involvement of Pxr in regulation of selected genes, including genes in cytochrome P450 subfamilies CYP2 and CYP3. We also examined interaction of Pxr and the aryl hydrocarbon receptor (Ahr) signaling pathways. Pregnenolone caused a dose-dependent increase in mRNA levels of pxr, ahr2, CYP1A, CYP2AA1, CYP2AA12, CYP3A65, and CYP3C1, most of which peaked at 3 µM PN. The well-known Ahr agonist 3,3',4,4',5-pentachlorobiphenyl (PCB126) also upregulated expression of pxr, ahr2, CYP1A, CYP2AA12, CYP3A65, and CYP3C1 in a dose-dependent manner. Inhibition of pxr translation by morpholino antisense oligonucleotides (MO) suppressed PN-induced expression of pxr, ahr2, CYP3A65, and CYP3C1 genes. Levels of CYP2AA1 and CYP2AA12 mRNA were increased in the control-MO group exposed to PN; this was prevented by knocking down Pxr. Similarly, Ahr2-MO treatment blocked PCB126-induced mRNA expression of pxr, CYP1A, CYP2AA12, CYP3A65, and CYP3C1. The present study shows self-regulation of pxr by PN in developing zebrafish. Selected zebrafish CYP1, CYP2 (including several CYP2AAs) and CYP3 genes appear to be under the regulation of both Pxr and Ahr2.
Hydrostatic pressure (HP) increases linearly with depth in aquatic environments, so that many fish species routinely experience moderate-to-high HP levels (i.e., from a few to dozens of MPa). ...Biological effects of this thermodynamic variable are evidenced by a reduced functionality of many biomolecular systems, even in barotolerant and barophilic species. It is likely that environmentally-relevant HP levels (i.e., above atmospheric) could also modulate the responsiveness to and toxic effects of pollutants in fish. Still, only a few laboratories have investigated this possibility. The already-published ecobarotoxicological studies have brought strong support to the notion that HP can indeed modulate pollutant response in shallow-water and deep-sea animals. A careful reassessment of toxicity responses is therefore required. To quantify the exact influence of HP in marine fish toxicology, a research framework is proposed that should ensure the collection of meaningful data for risk assessment, using standard toxicity testing and mechanistic approaches.
•Hydrostatic pressure increases linearly with depth in the water column.•Hydrostatic pressure influences the static and dynamic behavior of biomolecules.•Ecotoxicological responses of marine fishes can be affected by hydrostatic pressure.•Ecobarotoxicological studies with marine fishes are needed for risk assessment.•A framework for ecobarotoxicological studies with marine fishes is proposed.
Zebrafish express five cytochrome P450 1 genes: CYP1A, CYP1B1, CYP1C1, CYP1C2, inducible by aryl hydrocarbon receptor agonists, and CYP1D1, a constitutively expressed CYP1A-like gene. We examined ...substrate selectivity of CYP1s expressed in yeast.
CYP1s were expressed in W(R) yeast, engineered to over-express P450 reductase, via pYES/DEST52 and via pYeDP60. Microsomal fractions from transformed yeast were examined for activity with fluorogenic substrates, benzoapyrene and testosterone. Modeling and docking approaches were used to further evaluate sites of oxidation on benzoapyrene and testosterone.
CYP1s expressed in yeast dealkylated ethoxy-, methoxy-, pentoxy- and benzoxy-resorufin (EROD, MROD, PROD, BROD). CYP1A and CYP1C2 had the highest rates of EROD activity, while PROD and BROD activities were low for all five CYP1s. The relative rates of resorufin dealkylation by CYP1C1, CYP1C2 and CYP1D1 expressed via pYeDP60 were highly similar to relative rates obtained with pYES/DEST52-expressed enzymes. CYP1C1 and CYP1C2 dealkylated substituted coumarins and ethoxy-fluorescein-ethylester, while CYP1D1 did not. The CYP1Cs and CYP1D1 co-expressed with epoxide hydrolase oxidized BaP with different rates and product profiles, and all three produced BaP-7,8,9,10-tetrol. The CYP1Cs but not CYP1D1 metabolized testosterone to 6β-OH-testosterone. However, CYP1D1 formed an unidentified testosterone metabolite better than the CYP1Cs. Testosterone and BaP docked to CYP homology models with poses consistent with differing product profiles.
Yeast-expressed zebrafish CYP1s will be useful in determining further functionality with endogenous and xenobiotic compounds.
Determining the roles of zebrafish CYP1s in physiology and toxicology depends on knowing the substrate selectivity of these enzymes.
•Zebrafish CYP1 enzymes have been compared in two yeast expression systems.•Fluorogenic substrates, benzoapyrene and testosterone were assayed.•The two yeast systems gave similar relative activities with resorufins.•Docking benzoapyrene and testosterone to CYP1 models supports sites of oxidation.•An unknown testosterone hydroxylated metabolite is produced by CYP1D1.
Cytochrome P450 (CYP) enzymes for which there is no functional information are considered “orphan” CYPs. Previous studies showed that CYP20A1, an orphan, is expressed in human hippocampus and ...substantia nigra, and in zebrafish (Danio rerio) CYP20A1 maternal transcript occurs in eggs, suggesting involvement in brain and in early development. Moreover, hyperactivity is reported in humans with chromosome 2 microdeletions including CYP20A1. We examined CYP20A1 in zebrafish, including impacts of chemical exposure on expression. Zebrafish CYP20A1 cDNA was cloned, sequenced, and aligned with cloned human CYP20A1 and predicted vertebrate orthologs. CYP20A1s share a highly conserved N-terminal region and unusual sequences in the I-helix and the heme-binding CYP signature motifs. CYP20A1 mRNA expression was observed in adult zebrafish organs including the liver, heart, gonads, spleen and brain, as well as the eye and optic nerve. Putative binding sites in proximal promoter regions of CYP20A1s, and response of zebrafish CYP20A1 to selected nuclear and xenobiotic receptor agonists, point to up-regulation by agents involved in steroid hormone response, cholesterol and lipid metabolism. There also was a dose-dependent reduction of CYP20A1 expression in embryos exposed to environmentally relevant levels of methylmercury. Morpholino knockdown of CYP20A1 in developing zebrafish resulted in behavioral effects, including hyperactivity and a slowing of the optomotor response in larvae. The results suggest that altered expression of CYP20A1 might be part of a mechanism linking methylmercury exposure to neurobehavioral deficits. The expanded information on CYP20A1 brings us closer to “deorphanization”, that is, identifying CYP20A1 functions and its roles in health and disease.
•The “orphan” CYP20A1 was cloned from zebrafish and its sequence analyzed.•Knockdown of CYP20A1 reduced an optomotor response and elicited bursts of activity.•Effects of knockdown resemble some features of a microdeletion of CYP20A1 in human.•Expression of CYP20A1 was downregulated by the neurotoxicant methylmercury.•CYP20A1 may be involved in neurobehavioral processes and effects of some chemicals.
Fish isolated cell systems have long been used to predict
in vivo toxicity of man-made chemicals. In present study, we tested the suitability of Precision-Cut Liver Slices (PCLS) as an alternative to ...these models that allows the evaluation of a global tissue response to toxicants, to investigate oxidative stress response to cytochrome P450 1A (CYP1A) induction in fish liver. PCLS of
Salmo salar were exposed for 21
h to increasing doses of 3-methylcholanthrene (3-MC) and Polychlorobiphenyl 126 (PCB 126). 3-MC (25
μM) strongly induced CYP1A transcription. In dose–response analysis (25–100
μM), EROD activity was strongly increased at intermediate 3-MC concentrations. We found the counter-intuitive decline of EROD at the highest 3-MC doses to result from reversible competition with ethoxyresorufin. No increases of H
2O
2 production, antioxidant enzymes activities or oxidative damage to lipids were found with 3-MC treatments. PCLS subjected to PCB 126 (2–200
nM) showed increased contamination levels and a parallel increased CYP1A mRNA synthesis and EROD activity. H
2O
2 production tended to increase but no oxidative damage to lipids was found. As antioxidant enzymes activities declined at the highest PCB 126 dose, it is suggested that longer incubation periods could be required to generate oxidative stress in PCLS.
In the context of climate change, determining the physiological mechanisms of drought-induced mortality in woody plants and identifying thresholds of drought survivorship will improve forecasts of ...forest and agroecosystem die-off. Here, we tested whether continuous measurements of branch diameter variation can be used to identify thresholds of hydraulic failure and physiological recoverability in lavender (
and
×
) plants exposed to severe drought. Two parameters of branch diameter variation were tested: the percentage loss of diameter and the percentage loss of rehydration capacity. In two greenhouse experiments with different growth conditions, we monitored variation in branch diameter in the two lavender species exposed to a series of drought/rewatering cycles that varied in drought-stress intensity. Water potential, stomatal conductance, loss of xylem hydraulic conductance, and electrolyte leakage were also measured. We observed that plants were not able to recover when percentage loss of diameter reached maximum values of 21.3% ± 0.6% during drought, regardless of species and growth conditions. A percentage loss of rehydration capacity of 100% was defined as the point of no recovery, and was observed with high levels of cellular damage as estimated by electrolyte leakage measured at 75.4% ± 9.3% and occurred beyond 88% loss of xylem hydraulic conductance. Our study demonstrates that lavender plants are not able to recover from severe drought when they have used up their elastic water storage. Additionally, drought-induced mortality in these species was not linked to xylem hydraulic failure but rather to high levels of cell damage.
•Whole-body lipid content was decreased by MeHg and increased by LA.•Adipocytes were larger after MeHg exposure and feeding with plant-derived oils.•Fatty acid composition reflected retention and ...bioconversion of dietary fatty acids.•MeHg decreased n-6 PUFA amounts in hepatic membranes of fish rich in n-6 PUFA.
Methylmercury (MeHg) is a pervasive environmental contaminant in aquatic ecosystems that can reach elevated concentrations in fish of high trophic levels, such as salmonids. The present study aims at investigating the individual and combined impacts of dietary MeHg and fatty acids on lipid metabolism in juvenile rainbow trout (Oncorhynchus mykiss) with a focus on two key organs, adipose tissue and liver. MeHg and fatty acids are both known to act on energy homeostasis although little is known about their interplay on lipid metabolism in fish. Fish were fed diets enriched in linoleic acid (LA, 18:2 n-6), α-linolenic acid (ALA, 18:3 n-3), eicosapentaenoic acid (EPA, 20:5 n-3) or docosahexaenoic acid (DHA, 22:6 n-3) for ten weeks, with the addition of MeHg to the diets during the last six weeks (0, 2.4 or 5.5 mg MeHg/kg dry matter). LA and ALA are polyunsaturated fatty acids (PUFA) typical of plant-derived oils whereas EPA and DHA are n-3 long chain PUFA largely found in fish oil, all used in feed formulation in aquaculture. The results showed that the LA-enriched diet induced a higher whole-body lipid content compared to the three other diets. On the contrary, the addition of MeHg led to a significant reduction of the whole-body lipid content, regardless of the diet. Interestingly, the adipocytes were larger both in presence of LA, compared to EPA and DHA, or MeHg, indicating a lipogenic effect of these two compounds. No effect was, however, observed on lipid accumulation per gram of adipose tissue. The fatty acid composition of adipose tissue and liver was significantly modified by the dietary lipids, reflecting both the fatty acid composition of the diets and the high bioconversion capacity of the rainbow trout. Exposure to MeHg selectively led to a release of n-6 PUFA from the hepatic membranes of fish fed the LA-enriched diet, showing a disruption of the pathways using n-6 PUFA. This study highlights the significant impact of MeHg exposure and dietary fatty acids on lipid metabolism in fish. Further investigation is needed to elucidate the underlying mechanisms and to explore the potential involvement of other organs.
•Environmentally-realistic concentration of Cd can impact the immune system of rainbow trout.•DHA-enriched diet improved growth performance and induced higher oxidative stress when fish were exposed ...to Cd.•EPA triggered different inflammatory responses by favoring humoral components and induced anti-inflammatory cytokines.•ALA-enriched diet appeared to be as efficient as other diets in protecting fish against the adverse Cd effects.•Fish fed LA diet were not more affected by Cd than those fed other PUFAs, a finding different fron the in vitro study.
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.
•Fatty acid composition of fish liver cell phospholipids can be manipulated.•Methylmercury (MeHg) has few impacts on fatty acid composition of cell phospholipids.•n-3 and n-6 polyunsaturated fatty ...acids (PUFAs) have no impact on MeHg accumulation.•Fatty acid metabolism and stress response genes are influenced by PUFAs and MeHg.
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 (PUFAs) 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 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 PUFA-derived signalling molecules and stress response. We observed that cells incorporated the given PUFA and some biotransformation products in their phospholipids. Methylmercury 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 PUFA-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.
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