Frédéric Silvestre

Oxidative stress, protein carbonylation and heat shock proteins in the black tiger shrimp, Penaeus monodon, following exposure to endosulfan and deltamethrin.

The impact of commonly used pesticides, endosulfan and deltamethrin, on the molecular stress level in black tiger shrimp Penaeus monodon, was assessed using classical oxidative stress biomarkers, protein carbonylation profiles, and levels of heat shock proteins. Results showed that 4 days exposure to 0.1 μg L(-1) deltamethrin significantly (p<0.05) increased lipid peroxidation (LPO) level in gills (64.3 ± 3.2 compared to 34.2 ± 5.3 nmol MDA equiv.g(-1) tissue at day 0). However, no pesticide treatment had significant effect on the activities of antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST). Carbonylated protein profiles were determined on gills following 2,4-dinitrophenylhydrazine derivatization and 2D-PAGE along with Western blotting. Immunoblotting with dinitrophenol-specific antibody revealed 17 protein spots carbonylated in response to 4 days exposure to 0.1 μg L(-1) deltamethrin while 24 protein spots specifically oxidized at day 0 were no longer detected after deltamethrin treatment. On the other hand, endosulfan exposure at 0.1 and 1 μg L(-1) induced up to 2.1-fold increase of HSP90 level in muscle. This approach is providing new insights into the molecular impacts of deltamethrin and endosulfan on an economically important crustacean. While deltamethrin has shown a pro-oxidant effect in gills, endosulfan exposure rather induced proteotoxic effects in muscles. This argues that LPO level, protein carbonylation specificities, and HSP90 levels may be potential discriminating biomarkers to assess the chemical stress level in farm shrimp.

Combined effects of deltamethrin, temperature and salinity on oxidative stress biomarkers and acetylcholinesterase activity in the black tiger shrimp (Penaeus monodon)

This study aimed to investigate the interactions of two abiotic factors (temperature and salinity) and deltamethrin (pyrethroid pesticide) exposure on some oxidative stress biomarkers as well as on acetylcholinesterase activity (AChE) in hepatopancreas, gills and muscle of black tiger shrimp (Penaeus monodon). A combination of three temperatures (24, 29 and 34°C), two salinities (15 and 25 ppt), and the absence or presence of 0.1 μg L(-1) deltamethrin was applied on shrimp during 4 d under laboratory conditions. Lipid peroxidation level (LPO) and glutathione S-transferase activity (GST) were not affected by combined effect of temperature, salinity and deltamethrin in any of the studied tissues. Deltamethrin impaired other tested oxidative stress biomarkers, i.e. total glutathione (tGSH), catalase (CAT), glutathione peroxidase (GPx). tGSH level significantly increased in hepatopancreas due to deltamethrin exposure mainly at 34°C, while pesticide effects on tGSH and CAT activity in gills were influenced by both temperature and salinity. In addition, GPx activity in hepatopancreas decreased after deltamethrin treatment mainly at 24°C. Finally, AChE in muscle was strongly inhibited by deltamethrin at all tested temperatures and salinities. These novel findings demonstrate that interactions between abiotic factors and a commonly used pesticide exposure should be taken into account when analyzing some widespread biomarkers in black tiger shrimp.

Protein Expression Profiling in the African Clawed Frog Xenopus laevis Tadpoles Exposed to the Polychlorinated Biphenyl Mixture Aroclor 1254

Exposure to environmental pollutants such as polychlorinated biphenyls (PCBs) is now taken into account to partly explain the worldwide decline of amphibians. PCBs induce deleterious effects on developing amphibians including deformities and delays in metamorphosis. However, the molecular mechanisms by which they express their toxicity during the development of tadpoles are still largely unknown. A proteomics analysis was performed on developing Xenopus laevis tadpoles exposed from 2 to 5 days postfertilization to either 0.1 or 1 ppm Aroclor 1254, a PCB mixture. Two-dimensional DIGE with a minimal labeling method coupled to nanoflow liquid chromatography-tandem mass spectrometry was used to detect and identify proteins differentially expressed under PCBs conditions. Results showed that 59 spots from the 0.1 ppm Aroclor 1254 condition and 57 spots from the 1 ppm Aroclor 1254 condition displayed a significant increase or decrease of abundance compared with the control. In total, 28 proteins were identified. The results suggest that PCBs induce mechanisms against oxidative stress (peroxiredoxins 1 and 2), adaptative changes in the energetic metabolism (enolase 1, glycerol-3-phosphate dehydrogenase, and creatine kinase muscle and brain types), and the implication of the unfolded protein response system (glucose-regulated protein, 58 kDa). They also affect, at least at the highest concentration tested, the synthesis of proteins involved in normal cytogenesis (α-tropomyosin, myosin heavy chain, and α-actin). For the first time, proteins such as aldehyde dehydrogenase 7A1, CArG binding factor-A, prolyl 4-hydroxylase β, and nuclear matrix protein 200 were also shown to be up-regulated by PCBs in developing amphibians. These data argue that protein expression reorganization should be taken into account while estimating the toxicological hazard of wild amphibian populations exposed to PCBs.

A proteomic analysis of green and white sturgeon larvae exposed to heat stress and selenium.

Temperature and selenium are two environmental parameters that potentially affect reproduction and stock recruitment of sturgeon in the San Francisco Bay/Delta Estuary. To identify proteins whose expression is modified by these environmental stressors, we performed a proteomic analysis on larval green and white sturgeons exposed to 18 or 26 degrees C and micro-injected with Seleno-L-Methionine to reach 8microgg(-)(1) selenium body burden, with L-Methionine as a control. Selenium and high temperature induced mortalities and abnormal morphologies in both species, with a higher mortality in green sturgeon. Larval proteins were separated by two-dimensional gel electrophoresis and differential abundances were detected following spot quantitation and hierarchical cluster analysis. In green sturgeon, 34 of 551 protein spots detected on gels showed a variation in abundance whereas in white sturgeon only 9 of 580 protein spots were differentially expressed (P<0.01). Gel replicates were first grouped according to heat treatment. Fifteen of these spots were identified using MALDI TOF/TOF mass spectrometry. Proteins involved in protein folding, protein synthesis, protein degradation, ATP supply and structural proteins changed in abundance in response to heat and/or selenium. 40S ribosomal protein SA, FK506-binding protein 10, 65kDa regulatory subunit A of protein phosphatase 2, protein disulfide isomerase, stress-induced-phosphoprotein 1, suppression of tumorigenicity 13 and collagen type II alpha 1, were differentially expressed in high temperature treatment only. Serine/arginine repetitive matrix protein 1, creatine kinase, serine peptidase inhibitor Kazal type 5 and HSP90 were sensitive to combined temperature and selenium exposure. Valosin-containing protein, a protein involved in aggresome formation and in protein quality control decreased more than 50% in response to selenium treatment. Potential use of such proteins as biomarkers of environmental stressors in larval sturgeons could indicate early warning signals preceding population decline.

Ecotoxicoproteomics in gills of the sentinel fish species, Cottus gobio, exposed to perfluorooctane sulfonate (PFOS)

The environmental persistence, bioaccumulative tendency and potential toxicity of perfluorooctane sulfonate (PFOS) have generated great concern. This study aimed at evaluating the toxicity of short-term PFOS exposure in gills of the European bullhead Cottus gobio, a candidate sentinel species, by monitoring the response of some enzymes (citrate synthase CS, cytochrome c oxidase CCO, and lactate dehydrogenase LDH), and by undertaking a proteomic analysis using 2D-DIGE. First, a 96-h exposure to 1mg PFOS/L significantly altered the activity of mitochondrial CS and CCO. Second, 2D-DIGE gels were used to compare gills from the control fish group with tissues from fish exposed for 96h to either 0.1 or 1mg PFOS/L. From the 27 protein spots displaying significant changes in abundance following PFOS exposure, a total of 20 different proteins were identified using nano LC-MS/MS and the Peptide and Protein Prophet of Scaffold software. The differentially expressed proteins that were identified are involved in the general stress response, ubiquitin-proteasome system, energy metabolism, and actin cytoskeleton, which provide clues on the cellular pathways and components mainly affected by PFOS. Moreover, our results showed that most proteins were differentially expressed at the low but not at the high PFOS concentration. This work provides insights into the biochemical and molecular events in PFOS-induced toxicity in gill tissue, and suggests that further studies on the identified proteins could provide crucial information to better understand the mechanisms of PFOS toxicity in fish.

Proteomic Response to Sublethal Cadmium Exposure in a Sentinel Fish Species, Cottus gobio

The present study aimed at evaluating the toxicity of short-term cadmium (Cd) exposure in the European bullhead Cottus gobio, a candidate sentinel species. Several enzymatic activity assays (citrate synthase, cytochrome c oxidase, and lactate dehydrogenase) were carried out in liver and gills of fish exposed to 0.01, 0.05, 0.25, and 1 mg Cd/L for 4 days. Exposure to high Cd concentrations significantly altered the activity of these enzymes either in liver and/or in gills. Second, 2D-DIGE technique was used to identify proteins differentially expressed in tissues of fish exposed to either 0.01 or 1 mg Cd/L. Fifty-four hepatic protein spots and 37 branchial protein spots displayed significant changes in abundance in response to Cd exposure. A total of 26 and 12 different proteins were identified using nano LC-MS/MS in liver and gills, respectively. The identified differentially expressed proteins can be categorized into diverse functional classes, related to metabolic process, general stress response, protein fate, and cell structure for instance. This work provides new insights into the biochemical and molecular events in Cd-induced toxicity in fish and suggests that further studies on the identified proteins could provide crucial information to better understand the mechanisms of Cd toxicity in fish.

Eco-, geno- and human toxicology of bio-active nanoparticles for biomedical applications

Gene delivery has become an increasingly important strategy for treating a variety of human diseases, including infections, genetic disorders and tumours. To avoid the difficulties of using viral carriers, more and more non-viral gene delivery nanoparticles are developed. Among these new approaches polyethylene imine (PEI) is currently considered as one of the most effective polymer based method solution and considered as the gold standard. The toxicity of nanoparticles is a major concern when used for medical application. In this study we chose two nanoparticles for an in depth toxicological and ecotoxicological evaluation, one well characterized, PEI, and another novel polymer, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA). In the present study we have assessed the toxicity of these cation nanoparticles as such and of the polyplexes - nanoparticles covered with DNA. As these nanoparticles are also frequently used in high volumes in various industries and as such may enter in the environment, we also made an initial assessment of ecotoxicological effects assessment. The following nanoparticles related aspects have been studied during the project: development and characterization, ecotoxicity, general toxicity and specific toxicity. To this end a battery of different tests was used. The conclusion of these tests is that toxicity is varying between different nanoparticles and between different DNA covering ratios. In general, in the different systems tested, the PEI polymer is more toxic than the PDMAEMA polymer. The same difference is seen for the polyplexes and the higher the charge ratio, the more toxic are the polyplexes. Our study also clearly shows the need for a broad spectrum of toxicity assays for a comprehensive risk assessment. Our study has performed such a comprehensive analysis of two biomedical nanoparticles.

Acetylcholinesterase activity as a biomarker of exposure to antibiotics and pesticides in the black tiger shrimp (Penaeus monodon).

This study aimed to assess the potentiality to use cholinesterase activity (ChE) in black tiger shrimp (Penaeus monodon) as a biomarker of exposure to 2 antibiotics (enrofloxacin, furazolidone) and 2 pesticides (endosulfan, deltamethrin), commonly used in Vietnamese farms. ChE from muscle and gills was first characterised using three different substrates and specific inhibitors. Results showed that both tissues possess only one ChE which displays the typical properties of an acetylcholinesterase (AChE). In a second part, shrimp (average weight of 8.8-10 g) were fed with medicated-feed containing 4g enrofloxacin (quinolone) or furazolidone (nitrofuran)/kg for 7 days, or exposed to 3 actual concentrations of endosulfan (0, 0.009, 0.09, 0.9 microg/L) or deltamethrin (0, 0.0007, 0.007, 0.07 microg/L) for 4 days. After treatment, animals were decontaminated during 7 days. We observed that AChE activity in muscle was not significantly affected in shrimp fed with enrofloxacin or furazolidone, while it significantly decreased (up to 28%) in gills of shrimp fed with furazolidone. Following endosulfan and deltamethrin exposure, no significant changes in AChE activity were observed in gills. However, a significant decrease occurred in muscle after 4 days exposure (inhibition of 30% and 49% at 0.9 microg/L endosulfan and 0.07 microg/L deltamethrin, respectively). While muscle AChE activity should be assessed to point out endosulfan or deltamethrin exposure, gill AChE activity impairment could indicate an exposure to furazolidone. The present study underlines the benefits to use AChE as a biomarker of chemotherapeutics as part of an integrated aquaculture management to reach industry sustainability.

Proteomics to assess the role of phenotypic plasticity in aquatic organisms exposed to pollution and global warming.

Nowadays, the unprecedented rates of anthropogenic changes in ecosystems suggest that organisms have to migrate to new distributional ranges or to adapt commensurately quickly to new conditions to avoid becoming extinct. Pollution and global warming are two of the most important threats aquatic organisms will have to face in the near future. If genetic changes in a population in response to natural selection are extensively studied, the role of acclimation through phenotypic plasticity (the property of a given genotype to produce different phenotypes in response to particular environmental conditions) in a species to deal with new environmental conditions remains largely unknown. Proteomics is the extensive study of the protein complement of a genome. It is dynamic and depends on the specific tissue, developmental stage, and environmental conditions. As the final product of gene expression, it is subjected to several regulatory steps from gene transcription to the functional protein. Consequently, there is a discrepancy between the abundance of mRNA and the abundance of the corresponding protein. Moreover, proteomics is closer to physiology and gives a more functional knowledge of the regulation of gene expression than does transcriptomics. The study of protein-expression profiles, however, gives a better portrayal of the cellular phenotype and is considered as a key link between the genotype and the organismal phenotype. Under new environmental conditions, we can observe a shift of the protein-expression pattern defining a new cellular phenotype that can possibly improve the fitness of the organism. It is now necessary to define a proteomic norm of reaction for organisms acclimating to environmental stressors. Its link to fitness will give new insights into how organisms can evolve in a changing environment. The proteomic literature bearing on chronic exposure to pollutants and on acclimation to heat stress in aquatic organisms, as well as potential application of proteomics in evolutionary issues, are outlined. While the transcriptome responses are commonly investigated, proteomics approaches now need to be intensified, with the new perspective of integrating the cellular phenotype with the organismal phenotype and with the mechanisms of the regulation of gene expression, such as epigenetics.

Malachite green toxicity assessed on Asian catfish primary cultures of peripheral blood mononuclear cells by a proteomic analysis.

The potential genotoxic and carcinogenic properties reported for malachite green (MG) and the frequent detection of MG residues in fish and fish products, despite the ban of MG, have recently generated great concern. Additional toxicological data are required for a better understanding of the mechanism of action and a more comprehensive risk assessment for the exposure of fish to this fungicide. To date, the use of fish peripheral blood mononuclear cells (PBMCs) has not been exploited as a tool in the assessment of the toxicity of chemicals. However, PBMCs are exposed to toxicants and can be easily collected by blood sampling. The present study aims at better understanding the effects of MG by a proteomic analysis of primary cultured PBMC from the Asian catfish, Pangasianodon hypophthalmus, exposed to MG. The two lowest concentrations of 1 and 10 ppb were selected based on the MTS (water soluble tetrazolium salts) cytotoxicity test. Using a proteomic analysis (2D-DIGE), we showed that 109 proteins displayed significant changes in abundance in PBMC exposed during 48 h to MG. Most of these proteins were successfully identified by nano LC-MS/MS and validated through the Peptide and Protein Prophet of Scaffold™ software, but only 19 different proteins were considered corresponding to a single identification per spot. Our data suggest that low concentrations of MG could affect the mitochondrial metabolic functions, impair some signal transduction cascades and normal cell division, stimulate DNA repair and disorganize the cytoskeleton. Altogether, these results confirm that the mitochondrion is a target of MG toxicity. Further studies on the identified proteins are needed to better understand the mechanisms of MG toxicity in fish produced for human consumption.