Chenglong Ji

Proteomic and metabolomic analysis reveal gender-specific responses of mussel Mytilus galloprovincialis to 2,2',4,4'-tetrabromodiphenyl ether (BDE 47).

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame-retardants (BFRs) that are widely used in industrial products and have posed potential risk on the coastal environment of the Laizhou Bay in China. They are of great concern due to their toxicities, such as hepatotoxicity, carcinogenecity, neurotoxicity, immunotoxicity and endocrine disrupting effects in animals. In this work, we focused on the gender-specific responses of BDE 47 in mussel Mytilus galloprovincialis using a combined proteomic and metabolomic approach. Metabolic responses indicated that BDE 47 mainly caused disturbance in energy metabolism in male mussel gills. For female mussel samples, disruption in both osmotic regulation and energy metabolism was found in terms of differential metabolic profiles. Proteomic responses revealed that BDE 47 induced cell apoptosis and reduced reactive oxygen species (ROS) production in both male and female mussels, disturbance in protein homeostasis in male mussels as well as disturbance in female mussel proteolysis based on the differential proteomic biomarkers. Overall, these results confirmed the gender-specific responses in mussels to BDE 47 exposures. This work demonstrated that an integrated metabolomic and proteomic approach could provide an important insight into the toxicological effects of environmental pollutant to organisms.

Responses of Mytilus galloprovincialis to bacterial challenges by metabolomics and proteomics

Pathogens can cause diseases and lead to massive mortalities of aquaculture animals and substantial economic loss. In this work, we studied the responses induced by Micrococcus luteus and Vibrio anguillarum in gill of mussel Mytilus galloprovincialis at protein and metabolite levels. Metabolic biomarkers (e.g., amino acids, betaine, ATP) suggested that both M. luteus and V. anguillarum induced disturbances in energy metabolism and osmotic regulation. The unique and some more remarkably altered metabolic biomarkers (threonine, alanine, aspartate, taurine, succinate) demonstrated that V. anguillarum could cause more severe disturbances in osmotic regulation and energy metabolism. Proteomic biomarkers (e.g., goose-type lysozyme 2, matrilin, ependymin-related protein, peptidyl-prolyl cis-trans isomerases) indicated that M. luteus caused immune stress, and disturbances in signaling pathways and protein synthesis. However, V. anguillarum mainly induced oxidative stress and disturbance in energy metabolism in mussel gills indicated by altered procollagen-proline dioxygenase, protein disulfide isomerase, nucleoside diphosphate kinases, electron transfer flavoprotein and glutathione S-transferase. This work confirmed that an integration of proteomics and metabolomics could provide an insightful view into the effects of pathogens to the marine mussel M. galloprovincialis.

Proteomic and metabolomic responses of clam Ruditapes philippinarum to arsenic exposure under different salinities

Arsenic (As) contamination is a severe problem in the intertidal zones of the Bohai Sea (China) with wide salinity variation. In the present study, we combined proteomics and metabolomics to characterize the differential responses of arsenic in clam Ruditapes philippinarum under different salinities (31.1, 23.3 and 15.6 psu). Both proteomic and metabolomic responses indicated that varying salinities could significantly affect the toxicological responses of clams to As. Metabolic biomarkers revealed that the environmentally relevant arsenic (20 μg L(-1)) exposure induced disturbance in energy metabolism and/or osmotic regulation under different salinities, whereas protein biomarkers indicated oxidative stress, cellular injury and apoptosis and disturbance in energy metabolism. In addition, the up-regulated proteins including ATP synthase, succinyl-CoA synthetase and nucleoside diphosphate kinase were validated by related metabolites, succinate and ATP, which confirmed the disturbance in energy metabolism in clam gills at low salinity (15.6 psu). These findings provide important insights into toxicological effects of environmental contaminant at molecular levels using combined proteomics and metabolomics.

Proteomic and metabolomic responses in hepatopancreas of Mytilus galloprovincialis challenged by Micrococcus luteus and Vibrio anguillarum

UNLABELLED The outbreak of pathogens can induce diseases and lead to massive mortalities of aquaculture animals including fish, mollusk and shrimp. In this work, the responses induced by Micrococcus luteus and Vibrio anguillarum were investigated in hepatopancreas of mussel Mytilus galloprovincialis using proteomics and metabolomics. Metabolic biomarkers demonstrated that M. luteus and V. anguillarum injections could induce osmotic stress and disturbance in energy metabolism. And the uniquely and more markedly altered metabolic biomarkers (glutamine, succinate, aspartate, glucose, ATP, homarine and tyrosine) indicated that V. anguillarum could cause more severe disturbances in osmotic regulation and energy metabolism. The differentially altered proteins meant that M. luteus and V. anguillarum induced different effects in mussels. However, the common proteomic biomarkers, arginine kinase and small heat shock protein, demonstrated that these two bacteria induced similar effects including oxidative stress and disturbance in energy metabolism in M. galloprovincialis. In addition, some metabolic biomarkers, ATP and glutamine, were confirmed by related proteins including arginine kinase, ATP synthase, nucleoside diphosphate kinase and glutamine synthetase in bacteria-challenged mussels. This study demonstrated that proteomics and metabolomics could provide an insightful view into the effects of environmental pathogens to the marine mussel M. galloprovincialis. BIOLOGICAL SIGNIFICANCE The outbreak of pathogens can lead to diseases and massive mortalities of aquaculture animals including fish, mollusk and shrimp. The mussel M. galloprovincialis distributes widely along the Bohai coast and is popularly consumed as delicious seafood by local residents. This bivalve has become one of the important species in marine aquaculture industry in China. Therefore a study on pathogen-induced effects is necessary. In the present study, an integrated metabolomic and proteomic approach was used to elucidate the differential effects induced by the representative Gram-positive (M. luteus) and Gram-negative (V. anguillarum) bacteria in M. galloprovincialis.

A metabolomic investigation on arsenic-induced toxicological effects in the clam Ruditapes philippinarum under different salinities

Arsenic is an important contaminant in the Bohai marine ecosystem due to the anthropogenic activities. In this work, we investigated the toxicological effects of arsenic in Ruditapes philippinarum under different seawater salinities using NMR-based metabolomics. Under normal salinity (31.1 ppt), arsenic decreased the levels of amino acids (glutamate, β-alanine, etc.), and increased the levels of betaine and fumarate. The metabolic biomarkers including decreased threonine, histidine, ATP and fumarate were found in the muscles of arsenic-treated clams under medium salinity (23.3 ppt). However, only elevated ATP and depleted succinate were detected in the arsenic-exposed clam samples under low salinity (15.6 ppt). These differential metabolic biomarkers indicated that arsenic could induce osmotic stress and disturbance in energy metabolism in clam under normal and medium salinities. However, arsenic caused only disturbance in energy metabolism in clam under low salinity. Overall, our results demonstrated that seawater salinity could influence the toxicological effects of arsenic.

Proteomic and metabolomic responses of Pacific oyster Crassostrea gigas to elevated pCO2 exposure.

UNLABELLED The gradually increased atmospheric CO2 partial pressure (pCO2) has thrown the carbonate chemistry off balance and resulted in decreased seawater pH in marine ecosystem, termed ocean acidification (OA). Anthropogenic OA is postulated to affect the physiology of many marine calcifying organisms. However, the susceptibility and metabolic pathways of change in most calcifying animals are still far from being well understood. In this work, the effects of exposure to elevated pCO2 were characterized in gills and hepatopancreas of Crassostrea gigas using integrated proteomic and metabolomic approaches. Metabolic responses indicated that high CO2 exposure mainly caused disturbances in energy metabolism and osmotic regulation marked by differentially altered ATP, glucose, glycogen, amino acids and organic osmolytes in oysters, and the depletions of ATP in gills and the accumulations of ATP, glucose and glycogen in hepatopancreas accounted for the difference in energy distribution between these two tissues. Proteomic responses suggested that OA could not only affect energy and primary metabolisms, stress responses and calcium homeostasis in both tissues, but also influence the nucleotide metabolism in gills and cytoskeleton structure in hepatopancreas. This study demonstrated that the combination of proteomics and metabolomics could provide an insightful view into the effects of OA on oyster C. gigas. BIOLOGICAL SIGNIFICANCE The gradually increased atmospheric CO2 partial pressure (pCO2) has thrown the carbonate chemistry off balance and resulted in decreased seawater pH in marine ecosystem, termed ocean acidification (OA). Anthropogenic OA is postulated to affect the physiology of many marine calcifying organisms. However, the susceptibility and metabolic pathways of change in most calcifying animals are still far from being understood. To our knowledge, few studies have focused on the responses induced by pCO2 at both protein and metabolite levels. The pacific oyster C. gigas, widely distributed throughout most of the worlds oceans, is a model organism for marine environmental science. In the present study, an integrated metabolomic and proteomic approach was used to elucidate the effects of ocean acidification on Pacific oyster C. gigas, hopefully shedding light on the physiological responses of marine mollusk to the OA stress.

Differential metabolic responses of clam Ruditapes philippinarum to Vibrio anguillarum and Vibrio splendidus challenges

Clam Ruditapes philippinarum is one of the important marine aquaculture species in North China. However, pathogens can often cause diseases and lead to massive mortalities and economic losses of clam. In this work, we compared the metabolic responses induced by Vibrio anguillarum and Vibrio splendidus challenges towards hepatopancreas of clam using NMR-based metabolomics. Metabolic responses suggested that both V. anguillarum and V. splendidus induced disturbances in energy metabolism and osmotic regulation, oxidative and immune stresses with different mechanisms, as indicated by correspondingly differential metabolic biomarkers (e.g., amino acids, ATP, glucose, glycogen, taurine, betaine, choline and hypotaurine) and altered mRNA expression levels of related genes including ATP synthase, ATPase, glutathione peroxidase, heat shock protein 90, defensin and lysozyme. However, V. anguillarum caused more severe oxidative and immune stresses in clam hepatopancreas than V. splendidus. Our results indicated that metabolomics could be used to elucidate the biological effects of pathogens to the marine clam R. philippinarum.

Evaluation of protein extraction protocols for 2DE in marine ecotoxicoproteomics

In ecotoxicoproteomics, an accurate and reproducible extraction of proteins is a critical step for 2DE analysis and further protein identification using MS. The criteria for the assessment of protein extraction quality include protein yield, protein spots resolved in a 2DE gel, matched protein spots in replicate gels, reproducibility, and compatibility with MS. In this work, we evaluated three protein extraction systems, straightforward lysis buffer, trichloroacetic acid–acetone, and TRIzol reagent with some modifications, for the protein extraction from three animal species including mussel Mytilus galloprovincialis, flounder Paralichthys olivaceus, and polychaete Nereis diversicolor used in marine ecotoxicology. Our results indicated that these methods could extract significantly different protein profiles. The method using TRIzol reagent resulted in the most matched protein spots resolved in four replicate 2DE gels and highest reproducibilities for the gill of M. galloprovincialis and liver of P. olivaceus. However, a modified trichloroacetic acid–acetone solvent system was best for the whole soft tissue of N. diversicolor. This work provides the fundamental information of the extraction quality of protein extraction protocols from different marine animals, which may facilitate the selection of a suitable protein extraction protocol for ecotoxicoproteomics.

An Integrated Proteomic and Metabolomic Study on the Chronic Effects of Mercury in Suaeda salsa under an Environmentally Relevant Salinity

As an environmental contaminant, mercury is of great concern due to its high risk to environmental and human health. The halophyte Suaeda salsa is the dominant plant in the intertidal zones of the Yellow River Delta (YRD) where has been contaminated by mercury in some places. This study aimed at evaluating the chronic effects of mercury (Hg2+, 20 µg L−1) and the influence of an environmentally relevant salinity (NaCl, 500 mM) on mercury-induced effects in S. salsa. A total of 43 protein spots with significant changes were identified in response to Hg2+, salinity and combined Hg2+ and salinity. These proteins can be categorized into diverse functional classes, related to metabolic processes, photosynthesis, stress response, protein fate, energy metabolism, signaling pathways and immunosuppression. Metabolic responses demonstrated that Hg2+ could disturb protein and energy metabolisms in S. salsa co-exposed with or without salinity. In addition, both antagonistic and synergistic effects between Hg2+ and salinity were confirmed by differential levels of proteins (magnesium-chelatase and ribulose-l,5-bisphosphate carboxylase/oxygenase) and metabolites (valine, malonate, asparagine, glycine, fructose and glucose) in S. salsa. These findings suggest that a combination of proteomics and metabolomics can provide insightful information of environmental contaminant-induced effects in plants at molecular levels.

A metabolomic study on the responses of daphnia magna exposed to silver nitrate and coated silver nanoparticles.

We examined the short-term toxicity of AgNPs and AgNO3 to Daphnia magna at sublethal levels using (1)H NMR-based metabolomics. Two sizes of polyvinylpyrrolidone-coated AgNPs (10 and 40nm) were synthesized and characterized and their Ag(+) release was studied using centrifugal ultrafiltration and inductively coupled plasma mass spectrometry. Multivariate statistical analysis of the (1)H NMR spectra showed significant changes in the D. magna metabolic profiles following 48h exposure to both AgNP particle sizes and Ag(+) exposure. Most of the metabolic biomarkers for AgNP exposure, including 3-hydroxybutyrate, arginine, lysine and phosphocholine, were identical to those of the Ag(+)-exposed groups, suggesting that the dominant effects of both AgNPs were due to released Ag(+). The observed metabolic changes implied that the released Ag(+) induced disturbance in energy metabolism and oxidative stress, a proposed mechanism of AgNP toxicity. Elevated levels of lactate in all AgNP-treated but not in Ag(+)-treated groups provided evidence for Ag-NP enhanced anaerobic metabolism. These findings show that (1)H NMR-based metabolomics provides a sensitive measure of D. magna response to AgNPs and that further targeted assays are needed to elucidate mechanisms of action of nanoparticle-induced toxicity.