Global Quantitative Analysis of Protein Phosphorylation Status in Fish Exposed to Microcystin

Abstract

The hepatotoxins, microcystins (MCs) are potent inhibitors of protein phosphatases PP1 and PP2A. These nonribosomal peptides are getting more and more attention because of their acute toxicity and potent tumor-promoting activity. These toxins are produced by freshwater cyanobacteria. Herein, we report a toxicological study conducted on aquatic animal models such as the medaka fish. To date, the detailed mechanisms underlying the toxicity of microcystins are unknown. MC-leucine-arginine (MC-LR) is the most toxic and the most commonly encountered variant of MCs in aquatic environment. It has been used for toxicological investigations on the liver of intoxicated medaka. We performed differential proteome analyses of MC-LR-treated and untreated medaka fish to investigate the mechanisms of establishment of early responses to the toxin. The identification of proteins involved in these early responses might constitute candidates of biomarkers of MC-LR exposure. Cytosolic proteins from livers of exposed or nonexposed medaka were resolved by 2D electrophoresis and detected using stains specific for phosphoproteins and for whole protein content. Overall, 15 spots were found to vary significantly on the proteomic 2D maps or on the phosphoproteomic 2D maps. Of these 15 proteins, only two could not be identified by mass spectrometry. Among the other proteins that were identified, phenylalanine hydroxylase and keratin 18 (type I) showed variations in phoshoryl content in agreement with inhibition of PP2A activity after exposure of the fish to MC-LR. The other identified proteins exhibited variations in their expression level. The identified proteins appear to be involved in cytoskeleton assembly, cell signalling, oxidative stress, and apoptosis. The functional implications of responses to MC-LR exposure of these proteins are discussed. The methodology described in this report should be widely used to a number of tissues and organisms, thus helping in the search for biomarkers of MC-LR contamination.

Metabolic changes in Medaka fish induced by cyanobacterial exposures in mesocosms: an integrative approach combining proteomic and metabolomic analyses

“…The significant dysregulation of the expression of glutathione-S-transferases (GSTs), cytochrome P450 (CYP450) and peroxiredoxin (PRX), which are involved in detoxification processes and redox homeostasis, are observed for fish exposed to both strains with even more contrasted effects in females. Dysregulation of these stress response associated proteins has already been observed in fish exposed to MC or MC-producing cyanobacterial blooms 31 – 34 and our results show here that similar stress responses may be induced by non-MC-producing cyanobacteria. Furthermore, Dysregulations of these proteins at the transcriptome level have also been pointed out in various fish species exposed to MCs.…”

Combined effects of the cyanobacterial toxin microcystin-LR and environmental factors on life-history traits of indigenous cladoceran Moina macrocopa

“…Although the highest exposure (500 µg MC‐LR/L) significantly affected the fecundity of M. macrocopa , the number of offspring increased, unlike the general pattern of reproductive toxicity caused by chronic exposure of MCs to M. macrocopa . The toxicity of MCs to daphnids is mediated by inhibition of protein phosphatase leading to deregulation in cells , binding to adenosine triphosphate synthetase affecting the energy‐sustaining ability of the cells , inactivating chymotrypsin enzymes suppressing digestion activities , inhibiting filter feeding activity, impairing food uptake , and provoking oxidative stress, known as the more general effect of cyanotoxins . Dao et al hypothesized that MCs could decrease energy availability for maintenance, growth, and reproduction because under exposure to MCs, additional energy is spent for physiological reactions, such as biotransformation and antioxidant enzyme activities, toxin excretion, or mechanisms of repairing damages.…”

Environmental OMICS: Current Status and Future Directions

“…In addition to the expression profiling studies, identification of modifications at the gene, protein, and metabolite levels is another important application of OMICS to environmental toxicology and human health research [17,18,19]. It is known that protein carbonylation and phosphorylation are among the major signal transduction pathways in cell biology.…”