Zebrafish brain proteomics reveals central proteins involved in neurodegeneration.

Understanding the complex biology of the brain requires analyzing its structural and functional complexity at the protein level. The large-scale analysis of the brain proteome, coupled with characterization of central brain proteins, provides insight into fundamental brain processes and processes linked to neurodegenerative diseases. Here we provide a map of the zebrafish brain proteome by using two-dimensional gel electrophoresis (2DE), followed by the identification of 95 brain proteins using mass spectrometry (LC-ESI MS/MS). Our data show extensive phosphorylation of brain proteins but less prominent glycosylation. Furthermore, ~51% of the identified proteins are predicted to have one or more ubiquitination sites whereas ~90% are predicted to have one or more SUMOylation sites. Our findings provide a valuable proteome map of the zebrafish brain and associated posttranslational modifications demonstrating that zebrafish proteomic approaches can aid in our understanding of proteins central to important neuronal processes and those associated with neurodegenerative disorders.

Cod (Gadus morhua) muscle proteome cataloging using 1D-PAGE protein separation, nano-liquid chromatography peptide fractionation, and linear trap quadrupole (LTQ) mass spectrometry.

Because Atlantic cod (Gadus morhua) has high economic value and its protein-rich muscle tissue is a food source, an increased understanding of the effects and consequences of environmental, nutritional, biological, and industrial factors on meat quality is necessary. To gain insight into cod muscle tissue protein composition, a large-scale proteomics approach has been used. One-dimensional polyacrylamide gel electrophoresis, nanoflow liquid chromatography peptide separation, and linear trap quadrupole mass spectrometry were used to identify 4804 peptides, which retrieved 9113 cod expressed sequence tags (ESTs), which in turn were mapped to 446 unique proteins. The same data set identified 3924 proteins from the zebrafish protein database, which highlights the complementary value of the two approaches. The generated data sets will act as a foundation for studies related to physiological status assessment of cod under different environmental conditions, screening for diseases, and biomarker identification for assessment of fish quality during industrial processing and preservation.

Multixenobiotic resistance, acetyl-choline esterase activity and total oxyradical scavenging capacity of the Arctic spider crab, Hyasaraneus, following exposure to bisphenol A, tetra bromo diphenyl ether and diallyl phthalate.

The Arctic has become a sink for persistent organic pollutants (POPs) originating from lower latitudes, and relatively high levels have been found in different biota. Recent studies have identified detrimental effects on wildlife including endocrine disruption, impairment of enzyme activity, and reduced immune function. The Arctic spider crab, Hyas araneus, shown interesting potential for its use as sentinel organism in polar ecosystems. This study investigated the effect of 2,2',4,4'-tetra bromo diphenyl ether (BPDE), bisphenol A (BPA), and diallyl phthalte (DPA) on H. araneus in a three weeks exposure study. Expression of multixenibiotic resistance (MXR) proteins has been studied using the C219 monoclonal antibody which allows identifying an immunoreactive protein of 40 kDa in the digestive gland while no such protein could be observed in the gills. Expression of this protein was increased by exposure to DPA (+75%; p<0.05, n=10). All compounds significantly affected muscle acetylcholine esterase (AChE) activity (p<0.05, n=10) with 50 microg/L DPA having the strongest effect by lowering the value to 37% of control. The total oxyradical scavenging capacity measured in the digestive gland toward peroxyl, hydroxyl and peroxynitrite was also significantly reduced indicating a decreased resistance to oxidative stress generated by DPA (p<0.05, n=5). These results thus suggest the potential detrimental effects of DPA even at concentration as low as 50 microg/L on H. araneus.

Mass spectrometric profiling - a diagnostic tool in fish?

The development of rapid and sensitive diagnostic tools to assess the effect of stressors on organisms is a principal objective of environmental proteomics. This study is focused on evaluating the potential of using surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS) to assess stress in Atlantic salmon (Salmo salar). Plasma and mucus samples were taken from fish that had previously been maintained in a range of high density conditions, together with control fish maintained under low density conditions. Samples were collected during the post-density stress period for protein profile analysis. The mass spectra were analysed to evaluate reproducibility and to search for condition specific changes in protein expression. Multivariate analysis of the peak relative intensity data indicated a segregation of the data into three entities in accordance with the density level fish had been subjected to during the density stress period. This segregation was seen in both plasma and mucus data.

Comparison of protein expression in plasma from nonylphenol and bisphenol A-exposed Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus) by use of SELDI-TOF.

The overall objective of this study was to compare the expression of plasma proteins in juvenile cod and turbot after a 3 week exposure to two different chemicals known to be estrogenic: 4-nonylphenol (NP, 29 microg/L) and bisphenol A (BPA, 59 microg/L). ProteinChip) array technology in combination with surfaced enhanced laser desorption ionisation-time of flight (SELDI-TOF) was used to investigate general responses in plasma proteins. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) was used to analyse two specific biomarkers of estrogenic exposure, vitellogenin (Vtg) and zona radiata protein (Zrp) in plasma. Both methods revealed clear species specific responses. In cod, 67% of significantly altered proteins showed the same response (up or down regulated) in NP and BPA exposed animals (males and females combined). The rest were either specific to NP (10%), BPA (19%) or they showed opposite responses to the two chemicals (4%). In contrast, only 20% of significantly altered proteins were common for NP and BPA exposed turbot: 60% were altered only in NP and 17% only in BPA. Furthermore, in BPA exposed cod, 77% of the responses were common for male and females, whereas turbot showed only 21% similarity for the two genders. However, NP exposed male and female turbot showed 88% similarity in responses. As gender was not determined in NP exposed cod, gender specific responses could not be determined. ELISA results supported that cod responded clearly to both chemicals as a large increase was observed in Vtg and Zrp levels. Turbot responded strongly to NP, but seemed only slightly affected by BPA. Overall, the results indicated that cod are more sensitive or respond with less specificity to estrogenic chemicals than turbot. The relatively large degree of common responses in NP and BPA exposed cod may indicate that in cod BPA have similar mode of action as NP. Generally, the results show the potential of SELDI-TOF as a tool for comparing multiple responses, and for identifying exposure as well as gender specific responses.

Induction of micronuclei and other nuclear abnormalities in mussels exposed to bisphenol A, diallyl phthalate and tetrabromodiphenyl ether-47.

Analysis of micronuclei, nuclear buds, bi-polynucleated and fragmented-apoptotic cells was performed in gills of blue mussels exposed for 3 weeks to sublethal concentrations of bisphenol A, diallyl phthalate (for the both nominal concentration 50 ppb) and to tetrabromodiphenyl ether-47 (nominal concentration 5 ppb). Fourteen specimens from each treatment and control group were used for the analysis. Our results demonstrated a significant increase in micronuclei frequency after the treatment with bisphenol A (P=0.0243), diallyl phthalate (P=0.0005) and tetrabromodiphenyl ether-47 (P<0.0001; Mann-Whitney U-test). Induction of bi-nucleated (P=0.0028), fragmented-apoptotic (P=0.0004) cells and nuclear buds (P=0.0101) was found in mussels exposed to tetrabromodiphenyl ether-47 while treatment with diallyl phthalate increased the level of fragmented-apoptotic cells (P=0.0283). Bisphenol A was the only agent that resulted only in induction of micronuclei but not any other kind of nuclear injuries.

The BEEP Stavanger Workshop: Mesocosm exposures.

Within the BEEP project (Biological Effects of Environmental Pollution in Marine Ecosystems) the Work Package 1 was addressed to the development of new and more sensitive biomarkers of exposure in several sentinel organisms. Within this framework, common mesocosm exposures of organic pollutants relevant for marine ecosystems were conducted in the facilities of Akvamiljø a/s (Stavanger, Norway). In the first experiment, Atlantic cod (Gadus morhua), turbot (Scophthalmus maximus) and shore crab (Carcinus maenas) were exposed to nonylphenol, North Sea crude oil and a combination of crude oil and alkylated phenols. Mussels (Mytilus edulis) were exposed to North Sea crude oil and a combination of crude oil, alkylated phenols and PAHs. In the second experiment, Atlantic cod, turbot, mussel and spider crab (Hyas araneus) were exposed to the plasticizers bisphenol A and diallyl phatalate and the brominated flame retardant BDE-47. The main purpose of the present study was to provide the 30 participating Institutes with samples which had been exposed to defined contaminant concentrations in a controlled laboratory exposure for 3 weeks. This paper describes the mesocosm experimental design, the transplantation and treatment of the organisms, and the contaminant exposures.

The neutral red lysosomal retention assay and Comet assay on haemolymph cells from mussels (Mytilus edulis) and fish (Symphodus melops) exposed to styrene.

Despite the extensive transport of chemicals at sea, there is current lack of knowledge of the fate and effects of many of them on the marine biota. The current regulation that follows the GESAMP-MARPOL classification is mainly based on ecotoxicity assessment from fresh water based studies. Repetitive spills in marine coastal environment from tanker ship loaded with several thousand tonnes of chemicals raised concern about whether the existing freshwater data location can be used to predict the behaviour and the environmental effects of contaminants in marine surroundings. There is a general lack of information of the fate of chemicals at sea. A deviating pattern in marine environment from that in freshwater may have significant consequences for the counteracting actions taken to fight the spill, on staff working on the site of spill as well as on marine life present in the vicinity of the accident. In the present article, an environmental effect study of styrene was conducted as part of the ECOPEL program. We report some biological effects of styrene in laboratory-exposed marine organisms. Styrene was continuously supplied at a nominal concentration of 2mg L(-1) over 7 days to both mussels (Mytilus edulis) and fish (Symphodus mellops). At the end of this period, DNA damage was assessed by the Comet assay performed on blood (fish) and haemolymph (mussel) cells. In mussels, the lysosomal membrane stability was additionally assessed by the neutral red retention time assay (NRRT). Significant biological responses were observed over the studied period in both organisms with these two tests. Hence, the results favour the use of a biomarker-based approach to assess the health conditions in case of spill.

Analysis of micronuclei in blue mussels and fish from the Baltic and North Seas.

Micronuclei (MN) were analyzed in erythrocytes of flounder (Platichthys flesus) and wrasse (Symphodus melops) and in gill cells of blue mussels (Mytilus edulis). The organisms were collected from three study stations in the Baltic Sea and from seven stations in the North Sea (Karmsund area, Norway) 4 times. The statistically significant differences obtained were related to the season, sex of the fish, and sampling locality. Higher MN frequencies were found in fish and mussels collected from the most polluted study stations in the North Sea. The same tendency could be described in the Baltic Sea; however, it was masked by the recent oil spill from the Butinge oil terminal. Our results showing higher MN frequencies in presumably what were the most polluted study locations suggest that MN tests in fish and mussels may be used for the detection of genotoxic effects in a marine environment. The endpoint is well characterized and can be easily recognized, and the technique is convenient to use in field samplings following standard procedures and protocols.

Vitellogenin induction in Cyprinus carpio treated with 17beta-estradiol and 4-nonylphenol.

The aim of the present study was to demonstrate induction of vitellogenin in the common carp (Cyprinus carpio) as a biomarker for monitoring freshwater ecosystems. Sexually undifferentiated specimens of common carp were treated experimentally with 17beta-estradiol and increasing doses of 4-nonylphenol and levels of plasma Vtg were measured in order to: 1) validate an ELISA assay for plasma Vtg in the common carp using the polyclonal rabbit anti-salmon Vtg antibody AA1 (Biosense, Norway); 2) check the sensitivity of carp juveniles in producing Vtg in response to estrogen stimulation. The group treated with 17beta-estradiol showed high induction (156%) with respect to controls, also groups treated with 4-nonylphenol showed induction of Vtg. The group treated with 100 mg kg(-1) b.wt showed an induction of 61%. A statistically significant correlation was found between dose and response. This preliminary study demonstrate a response to the ELISA assay for Vtg in the common carp using rabbit anti-salmon antibody AA1. With further evidence the tested biomarker might be proposed for large scale monitoring of estrogenic effects caused by pollution in urban and industrial effluents.