Marc Edery

Proteomic and phosphoproteomic analysis of cellular responses in medaka fish (Oryzias latipes) following oral gavage with microcystin-LR ☆

Chronic and subchronic toxicity resulting from exposure to microcystins (MCs) receives increasing attention due to the risk of bioaccumulation of these toxins by aquatic animals, including fish. The mechanisms of action of MCs that target the liver, involve modifications of protein phosphorylation resulting from phosphatases 1 and 2A inhibition. Therefore, studying phosphoprotein modifications by using a specific phosphoprotein stain Pro-Q Diamond in fish liver contaminated with MC-leucine-arginine (MC-LR), the most toxic MC, should help dissecting disturbed signaling and metabolic networks. We have recently used this technology to identify several proteins that are modulated either in expression or phosphorylation in the liver of medaka following short-term exposure to MC-LR by balneation. In the present study, we have decided to use an alternative way of introducing the toxin into fish; that is by gavage (force-feeding). This was first achieved using tritiated MC-LR and allowed us to quantify the quantity of toxin incorporated into fish and to demonstrate that the toxin is mainly accumulated in liver. Afterwards a proteomics study limited to liver cytosolic proteins of contaminated animals showed that several proteins were up or down regulated either in quantity or in phosphorylation or both. Some of them had been previously detected as modified in balneation experiments but new molecules were identified as involved in signal transduction pathways activated by the toxin. In addition, in the conditions used (5 microg toxin/g body weight) anatomopathological studies supported a process of apoptonecrosis established after 24h, which was suggested to proceed by the evolution of some of the proteins after 2h contamination.

Pathological modifications following sub-chronic exposure of medaka fish (Oryzias latipes) to microcystin-LR.

Microcystins (MCs) are toxic monocyclic heptapeptides produced by many cyanobacteria. MCs, especially MC-LR, cause toxic effects in animals and are a recognized potent cause of environmental stress and health hazard in aquatic ecosystems when heavy blooms of cyanobacteria appear. Consequently, one of the major problems is the chronic exposure of fish to cyanotoxins in their natural environment. The present experiment involving chronic exposure confirmed initial findings on acute exposure to MC contamination: exacerbated physiological stress and tissue damage in several tissues of exposed medaka fish. The gonads were affected specifically. In female gonads the modifications included reduction of the vitellus storage, lysis of the gonadosomatic tissue and disruption of the relationships between the follicular cells and the oocytes. In the males, spermatogenesis appeared to be disrupted. This is the first report showing that a cyanotoxin can affect reproductive function, and so can impact on fish reproduction and thus fish stocks.

Proteomic study of the effects of microcystin-LR on organelle and membrane proteins in medaka fish liver

The microcystin-leucine-arginine toxin (MC-LR) is produced by cyanobacteria that sometimes bloom in water reservoirs. It targets the liver, thus posing potential health risks to human and animals. Microcystin inhibits the protein phosphatases PP1 and PP2A, leading to diverse cellular deregulation processes. A proteomic approach was applied to the medaka fish (Oryzias latipes) to obtain an overview of the effects of MC-LR on the liver. As membrane and organelle proteins are major structural and functional components of several cell signalling pathways, we decided to investigate here the membrane and organelle-enriched fractions from the livers of control and MC-LR treated medaka fish. Seventeen proteins were identified by proteomic analysis as being modulated in response to MC-LR treatment. This is the first time for eight of them to be reported as being involved in MC-LR effects: prohibitin, fumarylacetoacetase, protein disulfide isomerase A4 and A6, glucose regulated protein 78kDa, 40S ribosomal protein SA, cytochrome b5, and ATP synthase mitochondrial d subunit. These proteins are involved in protein maturation or in the response to oxidative stress highlighting the role of organelles in protein processing and the complex cooperation associated with oxidative stress.

Localization of microcystin-LR in medaka fish tissues after cyanotoxin gavage.

Microcystins (MCs) are toxic monocyclic heptapeptides produced by many cyanobacteria. Over 70 MCs have been successfully isolated and identified, of which MC-LR is the most commonly occurring toxin. Microcystins, especially MC-LR, cause toxic effects in mammals, birds and fish and are a recognized potent cause of environmental stress and pose a potential health hazard in aquatic ecosystems when heavy blooms of cyanobacteria appear. They also constitute a public health threat to people via drinking water and food chains. The concentrations of MC-LR can be very low, even in fish displaying severely disrupted tissues, which makes it essential to devise selective and sensitive histochemical methods for identifying and localizing MC-LR in target organs, such as liver and intestine. The aim of the study reported here was to analyze the presence of MC-LR in contaminated fish tissues using immunohistochemical methods. The present experiment involving subacute exposure confirmed our initial hypothesis that subacute and acute exposure to microcystin contamination can exacerbate physiological stress, induce sustained pathological damage, and affect the immune response in exposed medaka fish.

Effects of a toxic cyanobacterial bloom (Planktothrix agardhii) on fish: Insights from histopathological and quantitative proteomic assessments following the oral exposure of medaka fish (Oryzias latipes)

Cyanobacterial toxic blooms often occur in freshwater lakes and constitute a potential health risk to human populations, as well as to fish and other aquatic organisms. Microcystin-LR (the cyanotoxin most commonly detected in the freshwater environment) is a potent hepatotoxin, deregulating the kinase pathway by inhibiting phosphatases 1 and 2A. Although toxicological effects have been clearly linked to the in vitro exposure of fish to purified microcystins, cyanotoxins are produced by the cyanobacteria together with numerous other potentially toxic molecules, and their overall and specific implications for the health of fish have still not been clearly established and remain puzzlingly difficult to assess. The medaka fish (Oryzias latipes) was chosen as an in vitro model for studying the effects of a cyanobacterial bloom on liver protein contents using a gel free quantitative approach, iTRAQ, in addition to pathology examinations on histological preparations. Fish were gavaged with 5 μL cyanobacterial extracts (Planktothrix agardhii) from a natural bloom (La Grande Paroisse, France) containing 2.5 μg equiv. MC-LR. 2h after exposure, the fish were sacrificed and livers were collected for analysis. Histological observations indicate that hepatocytes present glycogen storage loss, and cellular damages, together with immunological localization of MCs. Using a proteomic approach, 304 proteins were identified in the fish livers, 147 of them with a high degree of identification confidence. Fifteen of these proteins were statistically significantly different from those of controls (gavaged with water only). Overall, these protein regulation discrepancies clearly indicate that oxidative stress and lipid regulation had occurred in the livers of the exposed medaka fish. In contrast to previous pure microcystin-LR gavage experiments, marked induction of vitellogenin 1 protein was observed for the first time with a cyanobacterial extract. This finding was confirmed by ELISA quantification of vitellogenin liver content, suggesting that the Planktothrix bloom extract had induced the occurrence of an endocrine-disrupting effect.

iTRAQ-based proteomic study of the effects of microcystin-LR on medaka fish liver.

Microcystins are cyanotoxins that occur in ground water and thus pose a potential health risk. Microcystin‐LR (microcystin‐leucine‐arginine) is a potent hepatotoxin, and is suspected of being a tumour promoter. Poisoning with this toxin causes several dysfunctions in hepatocytes by inhibiting protein phosphatases 1 and 2A, and notably produces oxidative stress, disrupts the cytoskeleton, and deregulates mitogen‐activated protein kinase pathway. Medaka fish (Oryzias latipes) was chosen as a model for studying the effects of this cyanotoxin on liver proteins using a gel‐free approach, iTRAQ. Fish were gavaged with microcystin‐LR. Two hours later, 325 proteins could be identified by Scaffold Q+ and 32 proteins revealed statistically significant variations above a ∣0.2∣ threshold of log2 ratio by comparison with control. These proteins are mostly involved in the translation and maturation of proteins, lipid metabolism and detoxification. Notably, apolipoproteins are deregulated which indicates a possible alteration of chylomicron‐mediated transport.

Toxins and stress in fish: Proteomic analyses and response network

Fish models are increasingly used in toxicological studies in the laboratory as well as in the field. In addition to contributing to the analysis of toxicity mechanisms, one major aim is to select biomarkers from among the metabolic responses to toxic agents observed that could be useful for surveying the aquatic environment. Since proteomics is a developing field in toxicological research, it seems opportune to explore the data obtained using this approach. This article proposes an overview of proteomic studies of fish exposed to environmental stressors comprising a cyanotoxin and the response networks observed. We tend to take a broad view of how proteins communicate and function within the cell, often encompassing large numbers of proteins that operate in pathways. We start by presenting and discussing the data from four experiments in which the medaka fish was treated under the same conditions with the cyanotoxin, microcystin-LR (MC-LR). Liver proteins were analyzed using two techniques: 2D electrophoresis and LCMSMS. In the second and main part of our paper, the proteomic data obtained from fish contaminated with chemicals, including those reported above concerning the medaka fish intoxicated with MC-LR, are considered in the round in order to identify fish responses to chemical stress. A tentative general overview of how groups of proteins work together depending on exposure and/or subcellular location is proposed, with the inclusion of MC-LR data obtained in mice for comparison.

Detection of free and covalently bound microcystins in different tissues (liver, intestines, gills, and muscles) of rainbow trout (Oncorhynchus mykiss) by liquid chromatography-tandem mass spectrometry: method characterization.

So far only a few publications have explored the development of extraction methods of cyanotoxin extracted from complex matrices. With regard to cyanobacterial microcystins (MCs), the data on the contamination of the flesh of aquatic organisms is hard to compare and very limited due to the lack of validated methods. In recent years, evidence that both free and bound fractions of toxin are found in these tissues has highlighted the need to develop effective methods of quantification. Several techniques do exist, but only the Lemieux oxidation has so far been used to investigate complex tissue matrices. In this study, protocols based on the Lemieux approach were adapted for the quantitative chemical analysis of free MC-LR and MMPB derived from bound toxin in the tissues of juvenile trout gavaged with MC-LR. Afterwards, the NF V03 110 guideline was used to characterize the protocols elaborated and evaluate their effectiveness.

Toxicity of harmful cyanobacterial blooms to bream and roach

Aquatic ecosystems are facing increasing environmental pressures, leading to an increasing frequency of cyanobacterial Harmful Algal Blooms (cHABs) that have emerged as a worldwide concern due to their growing frequency and their potential toxicity to the fauna that threatens the functioning of ecosystems. Cyanobacterial blooms raise concerns due to the fact that several strains produce potent bioactive or toxic secondary metabolites, such as the microcystins (MCs), which are hepatotoxic to vertebrates. These strains of cyanobacteria may be potentially toxic to fish via gastrointestinal ingestion and also by direct absorption of the toxin MC from the water. The purpose of our study was to investigate toxic effects observed in fish taken from several lakes in the Ile-de-France region, where MCs-producing blooms occur. This study comprises histological studies and the measurement of MC concentrations in various organs. The histological findings are similar to those obtained following laboratory exposure of medaka fish to MCs: hepatic lesions predominate and include cell lysis and cell detachment. MC concentrations in the organs revealed that accumulation was particularly high in the digestive tract and the liver, which are known to be classical targets of MCs. In contrast concentrations were very low in the muscles. Differences in the accumulation of MC variants produced by blooms indicate that in order to more precisely evaluate the toxic potential of a specific bloom it is necessary not only to consider the concentration of toxins, but also the variants produced.

Oral toxicity of extracts of the microcystin-containing cyanobacterium Planktothrix agardhii to the medaka fish (Oryzias latipes).

As previously demonstrated the medaka fish appears to offer a good model for studies of microcystins (MCs) effects. Since cyanobacterial toxins are released with other molecules in the aquatic environment when the producers are dying, in this study, we performed additional experiments in order to compare the described effects obtained with the pure toxin microcystin-LR (MC-LR), among the most toxic MCs, to those induced by complex extracts of an MCs-producer Planktothrix agardhii, strain PMC 75.02 and a natural bloom containing the MCs-producer P. agardhii. The toxicity of these extracts containing several variants of MC was determined in adult medaka treated by gavage. Extracts of an MCs-free strain of P. agardhii (PMC 87.02) were assayed for comparison. Extracts effects were analysed on two tissues, liver and intestine by means of photon and transmission electron microscopy. MC was localized in these tissues by immunocytochemistry. No effect was detectable with extracts of the MCs-free P. agardhii strain. The two MCs-P. agardhii extracts (strain and natural bloom) were able to induce harmful effects in the liver and intestine of the medaka fish in acute intoxication by gavage. In these target organs as shown by toxin immunolocalization, reactions leading to cell disjunction and lysis were observed apparently associated with an immune reaction implying MC containing macrophages. These effects are similar to those previously described with photonic microscopy in medaka treated with pure MC-LR with additional results obtained under the electron microscope. Since no significant effect was detected with the MCs-free (PMC 87.02) extract, we then conclude that MCs, even in complex association with other cyanobacterial components, should be responsible for the toxic effects observed in treated fish.