Molecular adaptation to high pressure in cytochrome P450 1A and aryl hydrocarbon receptor systems of the deep-sea fish Coryphaenoides armatus.

Limited knowledge of the molecular evolution of deep-sea fish proteomes so far suggests that a few widespread residue substitutions in cytosolic proteins binding hydrophilic ligands contribute to resistance to the effects of high hydrostatic pressure (HP). Structure-function studies with additional protein systems, including membrane bound proteins, are essential to provide a more general picture of adaptation in these extremophiles. We explored molecular features of HP adaptation in proteins binding hydrophobic ligands, either in lipid bilayers (cytochrome P450 1A - CYP1A) or in the cytosol (the aryl hydrocarbon receptor - AHR), and their partners P450 oxidoreductase (POR) and AHR nuclear translocator (ARNT), respectively. Cloning studies identified the full-length coding sequence of AHR, CYP1A and POR, and a partial sequence of ARNT from Coryphaenoides armatus, an abyssal gadiform fish thriving down to 5000m depth. Inferred protein sequences were aligned with many non-deep-sea homologs to identify unique amino acid substitutions of possible relevance in HP adaptation. Positionally unique substitutions of various physicochemical properties were found in all four proteins, usually at sites of strong-to-absolute residue conservation. Some were in domains deemed important for protein-protein interaction or ligand binding. In addition, some involved removal or addition of beta-branched residues; local modifications of beta-branched residue patterns could be important to HP adaptation. In silico predictions further suggested that some unique substitutions might substantially modulate the flexibility of the polypeptide segment in which they are found. Repetitive motifs unique to the abyssal fish AHR were predicted to be rich in glycosylation sites, suggesting that post-translational changes could be involved in adaptation as well. Recombinant CYP1A and AHR showed functional properties (spectral characteristics, catalytic activity and ligand binding) that demonstrate proper folding at 1atm, indicating that they could be used as deep-sea fish protein models to further evaluate protein function under pressure. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone".

Effects of polychlorobiphenyls, polybromodiphenylethers, organochlorine pesticides and their metabolites on vitamin A status in lactating grey seals.

Polychlorobiphenyls (PCBs), polybromodiphenylethers (PBDEs) and organochlorine pesticides (OCPs), such as dichlorodiphenyltrichloroethane (DDT) and hexachlorobenzene (HCB), are considered as endocrine disruptors in laboratory and wild animals. This study investigated whether these compounds and their hydroxylated metabolites (HO-PCBs and HO-PBDEs) may affect the homoeostasis of vitamin A, a dietary hormone, in the blubber and serum of twenty lactating grey seals sampled at early and late lactation on the Isle of May, Scotland. The effect of naturally produced compounds such as the methoxylated (MeO)-PBDEs was also examined. Vitamin A levels in inner blubber (37±9 µg/g wet weight (ww) and 92±32 µg/g ww at early and late lactation, respectively) and serum (408±143 and 390±98 ng/ml at early and late lactation, respectively) appeared to be positively related to ΣPCBs, ΣPBDEs and several individual PCB and PBDE congeners in inner blubber and serum. These findings may suggest enhanced mobilisation of hepatic retinoid stores and redistribution in the blubber, a storage site for vitamin A in marine mammals. We have also reported that serum concentrations of ΣHO-PCBs and 4-OH-CB107 tended to increase with circulating vitamin A levels. Although the direction of the relationships may sometimes differ from those reported in the literature, our results are in agreement with previous findings highlighting a disruption of vitamin A homoeostasis in the blubber and bloodstream following exposure to environmental pollutants. The fact that vitamin A and PCBs appeared to share common mechanisms of mobilisation and transfer during lactation in grey seals (Debier et al., 2004; Vanden Berghe et al., 2010) may also play a role in the different relationships observed between vitamin A and lipophilic pollutants.

Dietary methylmercury and fatty acids affect the lipid metabolism of adipose tissue and liver in rainbow trout.

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.

Precision-cut liver slices to investigate responsiveness of deep-sea fish to contaminants at high pressure.

While deep-sea fish accumulate high levels of persistent organic pollutants (POPs), the toxicity associated with this contamination remains unknown. Indeed, the recurrent collection of moribund individuals precludes experimental studies to investigate POP effects in this fauna. We show that precision-cut liver slices (PCLS), an in vitro tool commonly used in human and rodent toxicology, can overcome such limitation. This technology was applied to individuals of the deep-sea grenadier Coryphaenoides rupestris directly upon retrieval from 530-m depth in Trondheimsfjord (Norway). PCLS remained viable and functional for 15 h when maintained in an appropriate culture media at 4 °C. This allowed experimental exposure of liver slices to the model POP 3-methylcholanthrene (3-MC; 25 µM) at levels of hydrostatic pressure mimicking shallow (0.1 megapascal or MPa) and deep-sea (5-15 MPa; representative of 500-1500 m depth) environments. As in shallow water fish, 3-MC induced the transcription of the detoxification enzyme cytochrome P4501A (CYP1A; a biomarker of exposure to POPs). This induction was diminished at elevated pressure, suggesting a limited responsiveness of C. rupestris toward POPs in its native environment. This very first in vitro toxicological investigation on a deep-sea fish opens the route for understanding pollutants effects in this highly exposed fauna.

Differential changes of fat-soluble vitamins and pollutants during lactation in northern elephant seal mother-pup pairs.

We investigated the changes of vitamins A and E as well as PCBs and DDTs during lactation in northern elephant seal (Mirounga angustirostris) mother-pup pairs. On average, milk vitamin A concentrations were 6 times higher during late lactation than during early lactation, a pattern that differs dramatically from terrestrial mammals. Vitamin A concentrations also significantly increased in the inner blubber throughout lactation, whereas they remained constant in the outer blubber. Similar dynamics were observed for PCBs and DDTs in maternal blubber and milk. Blubber appears to be an important storage site for vitamin A and organochlorines in seals and a direct transfer of those molecules to the mammary gland may occur. The dynamics of vitamin A, PCBs and DDTs differed from those of vitamin E. There was a significant drop in milk vitamin E concentrations between early and late lactation, which is the usual pattern observed in terrestrial mammals. The dynamics of vitamin E in the blubber layers also differed from those of vitamin A, suggesting different mechanisms of mobilization and transfer into the milk.

Kinetic studies of peroxiredoxin 6 from Arenicola marina: rapid oxidation by hydrogen peroxide and peroxynitrite but lack of reduction by hydrogen sulfide.

Arenicola marina lives in marine environments where hydrogen peroxide concentrations reach micromolar levels. The annelid also forms reactive species through metabolic pathways. Its antioxidant systems include a cytosolic peroxiredoxin, peroxiredoxin 6 (AmPrx6 or AmPRDX6) that shows high homology to the mammalian 1-Cys peroxiredoxin. Previous work confirmed the peroxidase activity of AmPrx6 in the presence of dithiotreitol. Herein, we performed an in vitro kinetic characterization of the recombinant enzyme. AmPrx6 reduced hydrogen peroxide and peroxynitrite with rate constants of 1.1×10(7) and 2×10(6)M(-1)s(-1), respectively, at pH 7.4 and 25°C. Reduction of tert-butyl hydroperoxide was slower. The pK(a) of the peroxidatic thiol of AmPrx6 was determined as 5.1±0.2, indicating that it exists as thiolate, the reactive species, at physiological pH. The reductive part of the catalytic cycle was also explored. Hydrogen sulfide, present in millimolar concentrations in marine sediments where the annelid lives and that is able to reduce the mammalian 1-Cys peroxiredoxin, did not support AmPrx6 peroxidase activity. The enzyme was not reduced by other potential physiological reductants tested. Our data indicate that in this annelid, Prx6 could contribute to peroxide detoxification in the presence of a so far unidentified reducing counterpart.

QuoVidi: An open-source web application for the organization of large-scale biological treasure hunts.

Learning biology, and in particular systematics, requires learning a substantial amount of specific vocabulary, both for botanical and zoological studies. While crucial, the precise identification of structures serving as evolutionary traits and systematic criteria is not per se a highly motivating task for students. Teaching this in a traditional teaching setting is quite challenging especially with a large crowd of students to be kept engaged. This is even more difficult if, as during the COVID-19 crisis, students are not allowed to access laboratories for hands-on observation on fresh specimens and sometimes restricted to short-range movements outside their home. Here, we present QuoVidi, a new open-source web platform for the organization of large-scale treasure hunts. The platform works as follows: students, organized in teams, receive a list of quests that contain morphologic, ecologic, or systematic terms. They have to first understand the meaning of the quests, then go and find them in the environment. Once they find the organism corresponding to a quest, they upload a geotagged picture of their finding and submit this on the platform. The correctness of each submission is evaluated by the staff. During the COVID-19 lockdown, previously validated pictures were also submitted for evaluation to students that were locked in low-biodiversity areas. From a research perspective, the system enables the creation of large image databases by the students, similar to citizen science projects. Beside the enhanced motivation of students to learn the vocabulary and perform observations on self-found specimens, this system allows instructors to remotely follow and assess the work performed by large numbers of students. The interface is freely available, open-source and customizable. Unlike existing naturalist platforms, allows the educators to fully customize the quests of interest. This enables the creation of multiple teaching scenarios, without being bound to a fixed scope. QuoVidi can be used in other disciplines with adapted quests and we expect it to be of interest in many classroom settings.

Methylmercury displays pro-adipogenic properties in rainbow trout preadipocytes.

Methylmercury (MeHg) is a ubiquitous contaminant largely found in aquatic environments, especially in species at high trophic level such as salmonids. The aim of this study was to evaluate the effects of MeHg on adipocyte differentiation and lipid metabolism in rainbow trout. Primary cultured preadipocytes were exposed to increasing concentrations of MeHg during six days with or without a hormonal cocktail. Main results showed a dose-dependent intracellular accumulation of neutral lipids with a preferential uptake of n-3 polyunsaturated fatty acids. Interestingly, this accumulation occurred after a fairly low uptake of MeHg by preadipocytes and was maintained after the cellular exposure to MeHg. In membrane phospholipids, arachidonic acid (20:4 n-6) was released in a dose-dependent manner. At the transcriptional level, the expression of several adipocyte-specific genes (perilipin 2 and apolipoprotein Eb) as well as lipid-related genes (fatty acid synthase and fatty acid binding protein 11a) was up-regulated in preadipocytes exposed to MeHg. These results highlight for the first time the disrupting effect of MeHg in trout adipocyte metabolism, providing new insights regarding the role of environmental pollutants in adipose tissue dysfunction and related pathologies.

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.

Response of Alvinella pompejana to variable oxygen stress: a proteomic approach.

Alvinella pompejana is one of the most emblematic species of the animal communities colonizing the deep-sea hydrothermal vents of the East Pacific Rise. This extreme environment is characterized by high temporal variability of its physical-chemical parameters. Among these, the variation in concentration of available oxygen should lead to a specific physiological adaptive response of the animal. To evaluate the mechanisms of this response at a molecular level, a classical 2-DE-based proteomic approach has been implemented. After collection (Garrett-18S and -17S vent sites) animals were reconditioned in a high-pressure chamber pressurized at 260 bar and then the oxygen concentration was regulated to a constant value corresponding to hypoxia, normoxia or hyperoxia for 7 h. The soluble proteins from gills were then analyzed by 2-DE. The protein content of spots showing specific changes following oxygen concentration variation was determined based on comparison of MS/MS sequence data with a recently established A. pompejana ESTs database. Fifteen proteins, belonging mainly to three families, cytoskeleton protein, enzymes of energetic metabolism and heat shock proteins, have been identified as potentially involved in the response to the change in oxygen concentration. The significance of the relatively small set of proteins modulated by oxygen variations is discussed in the context of a potential universal cellular response to stress.

Insights into metazoan evolution from Alvinella pompejana cDNAs.

BACKGROUND: Alvinella pompejana is a representative of Annelids, a key phylum for evo-devo studies that is still poorly studied at the sequence level. A. pompejana inhabits deep-sea hydrothermal vents and is currently known as one of the most thermotolerant Eukaryotes in marine environments, withstanding the largest known chemical and thermal ranges (from 5 to 105°C). This tube-dwelling worm forms dense colonies on the surface of hydrothermal chimneys and can withstand long periods of hypo/anoxia and long phases of exposure to hydrogen sulphides. A. pompejana specifically inhabits chimney walls of hydrothermal vents on the East Pacific Rise. To survive, Alvinella has developed numerous adaptations at the physiological and molecular levels, such as an increase in the thermostability of proteins and protein complexes. It represents an outstanding model organism for studying adaptation to harsh physicochemical conditions and for isolating stable macromolecules resistant to high temperatures. RESULTS: We have constructed four full length enriched cDNA libraries to investigate the biology and evolution of this intriguing animal. Analysis of more than 75,000 high quality reads led to the identification of 15,858 transcripts and 9,221 putative protein sequences. Our annotation reveals a good coverage of most animal pathways and networks with a prevalence of transcripts involved in oxidative stress resistance, detoxification, anti-bacterial defence, and heat shock protection. Alvinella proteins seem to show a slow evolutionary rate and a higher similarity with proteins from Vertebrates compared to proteins from Arthropods or Nematodes. Their composition shows enrichment in positively charged amino acids that might contribute to their thermostability. The gene content of Alvinella reveals that an important pool of genes previously considered to be specific to Deuterostomes were in fact already present in the last common ancestor of the Bilaterian animals, but have been secondarily lost in model invertebrates. This pool is enriched in glycoproteins that play a key role in intercellular communication, hormonal regulation and immunity. CONCLUSIONS: Our study starts to unravel the gene content and sequence evolution of a deep-sea annelid, revealing key features in eukaryote adaptation to extreme environmental conditions and highlighting the proximity of Annelids and Vertebrates.

In vitro Lipolysis and Leptin Production of Elephant Seal Blubber Using Precision-Cut Adipose Tissue Slices.

Adipose tissue plays key roles in energy homeostasis. Understanding its metabolism and regulation is essential to predict the impact of environmental changes on wildlife health, especially in fasting-adapted species. However, in vivo experimental work in wild vertebrates can be challenging. We have developed a novel in vitro approach of precision-cut adipose tissue slices from northern elephant seal (Mirounga angustirostris) as a complementary approach to whole animal models. Blubber biopsies were collected from 14 pups during early and late post-weaning fast (Año Nuevo, CA, United States), precision-cut into 1 mm thick slices and maintained in culture at 37°C for at least 63 h. The slices exhibited an efficient response to ß-adrenergic stimulation, even after 2 days of culture, revealing good in vitro tissue function. The response to lipolytic stimulus did not vary between regions of outer and inner blubber, but was higher at early than at late fast for inner blubber slices. At early fast, lipolysis significantly reduced leptin production. At this stage, inner blubber slices were also more efficient at producing leptin than outer blubber slices, especially in the non-lipolytic condition. This model will aid the study of adipose tissue metabolism and its response to environmental stressors in marine mammals.

In vitro and in vivo studies of 6,8-(diaryl)imidazo[1,2-a]pyrazin-3(7H)-ones as new antioxidants.

A series of 5-aryl and 3,5-diaryl-2-amino-1,4-pyrazines and the derived imidazopyrazinones has been synthesized to study the chemical oxidative degradation of the bicyclic systems in vitro. Imidazopyrazinones mainly degraded following two independent pathways producing their precursors, namely aminopyrazines, and the corresponding amidopyrazines, respectively. Despite the fact that there is no influence of the substituent of the 3-aryl group on the ratio of the products aminopyrazine/amidopyrazine, diarylimidazopyrazinones and diarylaminopyrazines are good antioxidants in vivo. They protected against microvascular damages in ischemia/reperfusion with similar efficiencies.

Cloning and characterization of Arenicola marina peroxiredoxin 6, an annelid two-cysteine peroxiredoxin highly homologous to mammalian one-cysteine peroxiredoxins.

Peroxiredoxins (PRDXs) are a superfamily of thiol-dependent peroxidases found in all phyla. PRDXs are mechanistically divided into three subfamilies, namely typical 2-Cys, atypical 2-Cys, and 1-Cys PRDXs. To reduce peroxides, the N-terminal peroxidatic Cys of PRDXs is first oxidized into sulfenic acid. This intermediate is reduced by forming a disulfide bond either with a resolving Cys of another monomeric entity (typical 2-Cys) or of the same molecule (atypical 2-Cys). In 1-Cys PRDXs, the resolving Cys is missing and the sulfenic acid of the peroxidatic Cys is reduced by a heterologous thiol-containing reductant. In search of a homolog of human 1-Cys PRDX6 in Arenicola marina, an annelid worm living in intertidal sediments, we have cloned and characterized a PRDX exhibiting high sequence homology with its mammalian counterpart. However, A. marina PRDX6 possesses five Cys among which two Cys function as peroxidatic and resolving Cys of typical 2-Cys PRDXs. Thus, A. marina PRDX6 belongs to a transient group exhibiting sequence homologies with mammalian 1-Cys PRDX6 but must be mechanistically classified into typical 2-Cys PRDXs. Moreover, PRDX6 is highly expressed in tissues directly exposed to the external environment, suggesting that this PRDX may be of particular importance for protection against exogenous oxidative attacks.

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.

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 (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.

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-1 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-1) 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).

High hydrostatic pressure influences the in vitro response to xenobiotics in Dicentrarchus labrax liver.

Hydrostatic pressure (HP) increases by about 1 atmosphere (0.1MPa) for each ten-meter depth increase in the water column. This thermodynamical parameter could well influence the response to and effects of xenobiotics in the deep-sea biota, but this possibility remains largely overlooked. To grasp the extent of HP adaptation in deep-sea fish, comparative studies with living cells of surface species exposed to chemicals at high HP are required. We initially conducted experiments with precision-cut liver slices of a deep-sea fish (Coryphaenoides rupestris), co-exposed for 15h to the aryl hydrocarbon receptor (AhR) agonist 3-methylcholanthrene at HP levels representative of the surface (0.1MPa) and deep-sea (5-15MPa; i.e., 500-1500m depth) environments. The transcript levels of a suite of stress-responsive genes, such as the AhR battery CYP1A, were subsequently measured (Lemaire et al., 2012; Environ. Sci. Technol. 46, 10310-10316). Strikingly, the AhR agonist-mediated increase of CYP1A mRNA content was pressure-dependently reduced in C. rupestris. Here, the same co-exposure scenario was applied for 6 or 15h to liver slices of a surface fish, Dicentrarchus labrax, a coastal species presumably not adapted to high HP. Precision-cut liver slices of D. labrax were also used in 1h co-exposure studies with the pro-oxidant tert-butylhydroperoxide (tBHP) as to investigate the pressure-dependence of the oxidative stress response (i.e., reactive oxygen production, glutathione and lipid peroxidation status). Liver cells remained viable in all experiments (adenosine triphosphate content). High HP precluded the AhR agonist-mediated increase of CYP1A mRNA expression in D. labrax, as well as that of glutathione peroxidase, and significantly reduced that of heat shock protein 70. High HP (1h) also tended per se to increase the level of oxidative stress in liver cells of the surface fish. Trends to an increased resistance to tBHP were also noted. Whether the latter observation truly reflects a protective response to oxidative stress will be addressed in future co-exposure studies with both surface and deep-sea fish liver cells, using additional pro-oxidant chemicals. Altogether, data on CYP1A inducibility with D. labrax and C. rupestris support the view that high HP represses AhR signaling in marine fishes, and that only species adapted to thrive in the deep-sea have evolved the molecular adaptations necessary to counteract to some extent this inhibition.

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 the diet, affects various cellular properties such as membrane structure and fluidity, energy metabolism and susceptibility to oxidative stress. Since these cellular parameters can play an important role in the cellular response to organic and inorganic pollutants, a variation of the PUFA supply might modify the toxicity induced by such xenobiotics. In this work, we investigated whether the cellular fatty acid profile has an impact 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 enriching the 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 docosapentaenoic acid (DPA, 22:5n-6). These modified cells and their control (no PUFA enrichment) were then challenged for 24h 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 content of the growth medium: major modifications were a high incorporation of the supplemented PUFA in the cellular phospholipids, the appearance of direct elongation and desaturation metabolites in the cellular phospholipids as well as a change in the gross phospholipid composition (PUFA and monounsaturated fatty acid (MUFA) levels and n-3/n-6 ratio); (ii) ALA, EPA and DPA enrichment significantly protected the RTL-W1 cells against both methylmercury and cadmium; (iv) DHA enrichment significantly protected the cells against cadmium but not methylmercury; (v) AA and LA enrichment had no impact on the cell tolerance 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 both methylmercury and cadmium. Overall, our results highlighted the importance of the fatty acid supply on the tolerance of fish liver cells to methylmercury and cadmium.

Impacts of antibiotics on in vitro UVA-susceptibility of human skin fibroblasts.

Many studies of UVA-induced cell damage use skin cells obtained during plastic surgery. As the skin is contaminated by micro-organisms, antibiotics need to be added to primary skin cell culture media. This study analysed the impact of the most widely used agents, penicillin, streptomycin, and amphotericin B deoxycholate (amB), on UVA-irradiated human skin fibroblasts. The results show that the presence of amB in cell culture media increases the susceptibility of fibroblasts to UVA and the intracellular level of reactive oxygen species, even when cells are irradiated in amB-free saline. This photosensitising effect of amB can be prevented if the antifungal agent is removed from the culture medium at least 24 hours before irradiation. Moreover, the use of streptomycin during cell culture partly protects cells against the UVA-induced mortality linked to amB. Acellular tests on lipid micelles suggest that this protective effect could result from an inhibition of lipid peroxidation by the antibacterial agent. In conclusion, antibiotics should be used with care in cell culture media if the cells are to be used in physiological studies of fine mechanisms in UVA-susceptibility of skin cells. In other cases, cells should be maintained in antibiotic-free media for 24 hours before irradiation.