Differential expression profile of membrane proteins in Aplysia pleural–pedal ganglia under the stress of methyl parathion

This study was aimed to analyze the alteration of membrane protein profiles in Aplysia juliana Quoy & Gaimard (A. juliana) pleural–pedal ganglia under MP exposure. Both the results of GC–MS analysis and the activity assay of acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT) reveal that MP toxicological effects on Aplysia left and right pleural–pedal ganglia are different under 7 and 14 days of exposure. Therefore, Aplysia were subjected for exposure at two concentrations (1 and 2 mg/l) of MP for 7 and 14 days for membrane proteomic study. As a result, 19 and 14 protein spots were differentially expressed in A. juliana left pleural–pedal ganglia under 7 and 14 days treatment, and 20 and 14 protein spots found with differential expressions in their right ganglia under the same treatment, respectively. Several proteins with expression variations were detected from both the left and right pleural–pedal ganglia; however, most proteins have distinctive expressions, indicating different mechanisms might be involved in initiating MP toxicology in left and right ganglia. Among the total differential protein spots obtained, 29 proteins were classed as membrane proteins. These proteins are mainly involved in the metabolism process, cell redox homeostasis, signal transduction, immunology, intracellular transport and catalysis, indicating MP toxicity in mollusks seems to be complex and diverse. Some differentially expressed proteins were further confirmed by Western blotting and quantitative real-time PCR. These results might provide renovated insights to reveal the mechanism of MP-induced neurotoxicity, and the novel candidate biomarkers might have potential application for environmental evaluation of MP pollution level.     

Proteomics and 1H NMR-based metabolomics analysis of pathogenic Vibrio vulnificus aquacultures isolated from sewage drains

Vibrio bacteria live in both marine and freshwater habitats and are associated with aquatic animals. Vibrio vulnificus is a pathogenic bacterium that infects people and livestock. It is usually found in offshore waters or within fish and shellfish. This study presents a comparative proteomic analysis of the outer membrane protein (OMP) changes in V. vulnificus proteins after stimulation with sewage from sewage drains. Using two-dimensional electrophoresis followed by MALDI-TOF MS/MS, 32 protein spots with significant differences in abundance were identified and characterized. These identified proteins were found to be involved in various functional categories, including catalysis, transport, membrane proteins progresses, receptor activity, energy metabolism, cytokine activity, and protein metabolism. The mRNA expression levels of 12 differential proteins were further assessed by qRT-PCR. Seven genes including carboxypeptidase, hemoglobin receptor, succinate dehydrogenase iron-sulfur subunit, ATP synthase subunit alpha, thioredoxin, succinyl-CoA synthetase subunit, and alanine dehydrogenase were downregulated upon stimulation, whereas the protein expression levels HupA receptor, type I secretion outer membrane protein, glutamine synthetase, superoxide dismutase, OmpU, and VuuA were upregulated. ¹H NMR spectra showed 18 dysregulated metabolites from V. vulnificus after the sewage stimulation and the pathogenicity was enhanced after that.

     

Genotoxic potential of selected cytostatic drugs in human and zebrafish cells

Due to their increasing use, the residues of anti-neoplastic drugs have become emerging pollutants in aquatic environments. Most of them directly or indirectly interfere with the cell’s genome, which classifies them into a group of particularly dangerous compounds. The aim of the present study was to conduct a comparative in vitro toxicological characterisation of three commonly used cytostatics with different mechanisms of action (5-fluorouracil [5-FU], cisplatin [CDDP] and etoposide [ET]) towards zebrafish liver (ZFL) cell line, human hepatoma (HepG2) cells and human peripheral blood lymphocytes (HPBLs). Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and acridine orange/ethidium bromide staining. All three drugs induced time- and dose-dependent decreases in cell viability. The sensitivity of ZFL and HepG2 cells towards the cytotoxicity of 5-FU was comparable (half maximal inhibitory concentration (IC₅₀) 5.3 to 10.4 μg/mL). ZFL cells were more sensitive towards ET- (IC₅₀ 0.4 μg/mL) and HepG2 towards CDDP- (IC₅₀ 1.4 μg/mL) induced cytotoxicity. Genotoxicity was determined by comet assay and cytokinesis block micronucleus (CBMN) assay. ZFL cells were the most sensitive, and HPBLs were the least sensitive. In ZFL cells, induction of DNA strand breaks was a more sensitive genotoxicity endpoint than micronuclei (MNi) induction; the lowest effective concentration (LOEC) for DNA strand break induction was 0.001 μg/mL for ET, 0.01 μg/mL for 5-FU and 0.1 μg/mL for CDDP. In HepG2 cells, MNi induction was a more sensitive genotoxicity endpoint. The LOEC values were 0.01 μg/mL for ET, 0.1 μg/mL for 5-FU and 1 μg/mL for CDDP. The higher sensitivity of ZFL cells to cytostatic drugs raises the question of the impact of such compounds in aquatic ecosystem. Since little is known on the effect of such drugs on aquatic organisms, our results demonstrate that ZFL cells provide a relevant and sensitive tool to screen genotoxic potential of environmental pollutant in the frame of hazard assessment.     

Gonad differential proteins revealed with proteomics in oyster (Saccostrea cucullata) using alga as food contaminated with cadmium

As mercury and lead, cadmium (Cd) is one of the highly toxic metals in both the ocean and land environments, but its toxicological mechanism in organisms including human is still unclear because of the complex toxicological pathways in vivo. Here, the alga Chlorella vulgaris were cultivated at room temperature under the stress of cadmium (1 mg L⁻¹) to obtain a toxic food, and then the contaminated food were directly supplied to oyster (Saccostrea cucullata) in seawater. After feeding with C. vulgaris contaminated with Cd (C. vulgaris-Cd), the differential proteins in the oyster gonad (OG) were effectively separated and identified with proteomic approaches. Eleven protein spots were observed to be significantly changed in the OG feeding with C. vulgaris-Cd, which seven spots of these differential proteins were down-regulated while four spots were up-regulated. These altered spots were further excised in gels and identified by a combined technique of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and database searching. A portion of these differential proteins were further proofed by real-time PCR and Western blotting. The results indicate that the major functions of these differential proteins were described as follows: binding, protein translocation, catalysis, regulation of energy metabolism, reproductive function and skeleton structure. These differential proteins in part may effectively provide a few novel biomarkers for the evaluation of Cd pollution level via a food pathway for harming halobios, mammal and human health, and for understanding the complex mechanisms of Cd toxicity in vivo.     

Biochemical modifications in Pinus pinaster Ait. as a result of environmental pollution

Exposure to chemical pollution can cause significant damage to plants by imposing conditions of oxidative stress. Plants combat oxidative stress by inducing antioxidant metabolites, enzymatic scavengers of activated oxygen and heat shock proteins. The accumulation of these proteins, in particular heat shock protein 70 and heme oxygenase, is correlated with the acquisition of thermal and chemical adaptations and protection against oxidative stress. In this study, we used Pinus pinaster Ait. collected in the areas of Priolo and Aci Castello representing sites with elevated pollution and reference conditions, respectively. The presence of heavy metals and the levels of markers of oxidative stress (lipid hydroperoxide levels, thiol groups, superoxide dismutase activity and expression of heat shock protein 70, heme oxygenase and superoxide dismutase) were evaluated, and we measured in field-collected needles the response to environmental pollution. P. pinaster Ait. collected from a site characterized by industrial pollution including heavy metals had elevated stress response as indicated by significantly elevated lipid hydroperoxide levels and decreased thiol groups. In particular, we observed that following a chronic chemical exposure, P. pinaster Ait. showed significantly increased expression of heat shock protein 70, heme oxygenase and superoxide dismutase. This increased expression may have protective effects against oxidative stress and represents an adaptative cellular defence mechanism. These results suggest that evaluation of heme oxygenase, heat shock protein 70 and superoxide dismutase expression in P. pinaster Ait. could represent a useful tool for monitoring environmental contamination of a region and to better understand mechanisms involved in plant defence and stress tolerance.     

Does the Harvard/U.S. Environmental Protection Agency Ambient Particle Concentrator Change the Toxic Potential of Particles?

Abstract

Inhalation exposure to urban air particles is known to increase morbidity in humans and animals. Our group utilizes the Harvard/U.S. Environmental Protection Agency Ambient Particle Concentrator (HAPC) to generate concentrated aerosols of outdoor air particles for experimental exposures. We have reported increased pathologic responses to inhalation of concentrated urban air particles and identified silicon (as silicate) as an element associated with many of these responses. Using silicate-rich Mt. St. Helen’s volcanic ash (MSHA), we exposed three groups of Sprague-Dawley rats by inhalation for 6 hr to filtered air, MSHA, or MSHA passed though the HAPC. Twenty-four hours following exposure, bronchoalveolar lavage was performed to assess total cell count, differential cell count, protein, lactate dehydrogenase, and n-β[notdef]glucosaminidase levels. Peripheral blood was examined for packed cell volume, total protein, total white cells, and differential cell count. Morphologic studies localized particles in the lung and assessed pulmonary vasculature. No significant differences were observed among any of the groups in any parameter measured including morpho-metric analysis of pulmonary vasoconstriction. Scanning electron microscopy and X-ray analysis identified particles as silicates typical of MSHA throughout the lung. These findings suggest that particles passing through the HAPC have no change in their toxic potential in an exposure setting where particle deposition in the lung has occurred.     

Subcellular distribution and toxicity of cadmium in Potamogeton crispus L

The submerged macrophyte Potamogeton crispus L. was subjected to varying doses of cadmium (0, 20, 40, 60 and 80 μM) for 7 d, and the plants were analyzed for subcellular distribution of Cd, accumulation of mineral nutrients, photosynthesis, oxidative stress, protein content, and ultrastructural distribution of calcium (Ca). Leaf fractionation by differential centrifugation indicated that 48-69% of Cd was accumulated in the cell wall. At all doses of Cd, the levels of Ca and B rose and the level of Mn fell; the levels of Fe, Mg, Zn, Cu, Mo, and P rose initially only to decline later. Exposure to Cd caused oxidative stress as evident by increased content of malondialdehyde and decreased contents of chlorophyll and protein. Photosynthetic efficiency, as indicated by the quenching of chlorophyll a fluorescence (Fv/Fm, Fo and Fm), decreased significantly, the extent of decrease being directly proportional to the concentration of Cd. Increased amounts of precipitates of calcium were noticed in the treated plants, located either outside the cell membrane or in chloroplasts, mitochondria, the nucleus, and the cytoplasm whereas control plants showed small deposits of the precipitates around surface of the vacuole membrane and in the intercellular space but rarely in the cytoplasm. Photosynthetic efficiency and oxidative stress could be used as indicators of physiological end-points in determining the extent of Cd phytotoxicity.     

Identification of potential biomarkers for diazinon exposure to Japanese Medaka (Oryzias latipes) using annealing control primers

A new differential display-polymerase chain reaction (PCR) method based on annealing control primers was used to screen and identify potential biomarkers from differentially expressed genes (DEGs) in medaka exposed to sub-lethal concentration of diazinon (100 ppb). Among the differentially expressed genes identified, the majority were in functional categories of protein biosynthesis, transport and metabolism according to the gene ontology classification. The differential expression of ribosomal protein genes was quantified by real time PCR. The genes encoding ribosomal proteins including L3 and S17 were selected as potential biomarkers for diazinon exposure in medaka fish.     

Identification of potential biomarkers for diazinon exposure to Japanese Medaka (Oryzias latipes) using annealing control primers

A new differential display-polymerase chain reaction (PCR) method based on annealing control primers was used to screen and identify potential biomarkers from differentially expressed genes (DEGs) in medaka exposed to sub-lethal concentration of diazinon (100 ppb). Among the differentially expressed genes identified, the majority were in functional categories of protein biosynthesis, transport and metabolism according to the gene ontology classification. The differential expression of ribosomal protein genes was quantified by real time PCR. The genes encoding ribosomal proteins including L3 and S17 were selected as potential biomarkers for diazinon exposure in medaka fish.     

Biochemical and cellular effects of electrophiles present in ambient air samples

Ambient vapor-phase samples collected in Riverside, California had shown that both redox and electrophilic activity were present, with the vapor phase containing higher levels of electrophiles than the particle phase. In this study, the biochemical effects of the vapor-phase electrophiles were examined using the purified thiol proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), protein tyrosine phosphatase 1B (PTP1B) and KELCH-1 like ECH-associated protein 1 (Keap1). The results demonstrated that the thiol proteins were inactivated by the vapor-phase samples through covalent modifications. Next, two cellular responses, epidermal growth factor receptor (EGFR)/mitogen-activated protein (MAP) kinase and NF-E2-related factor 2 (Nrf2), to the ambient vapor-phase samples were assessed in A549 and RAW 264.7 cell lines, respectively. The vapor-phase samples, at non-oxidative concentrations, increased phosphorylation of EGFR, which is negatively regulated by PTP1B, and its downstream MAP kinase, extracellular signal-regulated kinase (ERK)1/2. Activation of Nrf2, which requires Keap1 alkylation, and expression of its downstream proteins were also observed. The electrophilic compounds present in ambient vapor-phase were shown to modify cellular proteins through covalent modification and to activate diverse cellular responses that can lead to inflammatory and adaptive responses.     

Evaluation of phenanthrene toxicity on earthworm (Eisenia fetida): An ecotoxicoproteomics approach

The goal of this study was to identify promising new biomarkers of phenanthrene by identifying differentially expressed proteins in Eisenia fetida after exposure to phenanthrene. Extracts of earthworm epithelium collected at days 2, 7, 14, and 28 after phenanthrene exposure were analyzed by two dimensional electrophoresis (2-DE) and quantitative image analysis. Comparing the intensity of protein spots, 36 upregulated proteins and 45 downregulated proteins were found. Some of the downregulated and upregulated proteins were verified by MALDI-TOF/TOF-MS and database searching. Downregulated proteins in response to phenanthrene exposure were involved in glycolysis, energy metabolism, chaperones, proteolysis, protein folding and electron transport. In contrast, oxidation reduction, oxygen transport, defense systems response to pollutant, protein biosynthesis and fatty acid biosynthesis were upregulated in phenanthrene-treated E. fetida. In addition, ATP synthase b subunit, lysenin-related protein 2, lombricine kinase, glyceraldehyde 3-phosphate dehydrogenase, actinbinding protein, and extracellular globin-4 seem to be potential biomarkers since these biomarker were able to low levels (2.5 mg kg⁻¹) of phenanthrene. Our study provides a functional profile of the phenanthrene-responsive proteins in earthworms. The variable levels and trends in these spots could play a potential role as novel biomarkers for monitoring the levels of phenanthrene contamination in soil ecosystems.     

Lipophilic organic pollutants induce changes in phospholipid and membrane protein composition leading to Vero cell morphological change

Membrane damage related to morphological change in Vero cells is a sensitive index of the composite biotoxicity of trace lipophilic chemicals. However, judging whether the morphological change in Vero cells happens and its ratio are difficult because it is not a quantitative characteristic. To find biomarkers of cell morphological change for quantitatively representing the ratio of morphological changed cell, the mechanism of cell membrane damage driven by typical lipophilic chemicals, such as trichlorophenol (TCP) and perfluorooctanesulphonate (PFOS), was explored. The ratio of morphologically changed cells generally increased with increased TCP or PFOS concentrations, and the level of four major components of phospholipids varied with concentrations of TCP or PFOS, but only the ratio of phosphatidylcholine (PC)/phosphatidylethanolamine (PE) decreased regularly as TCP or PFOS concentrations increased. Analysis of membrane proteins showed that the level of vimentin in normal cell membranes is high, while it decreases or vanishes after TCP exposure. These variations in phospholipid and membrane protein components may result in membrane leakage and variation in rigid structure, which leads to changes in cell morphology. Therefore, the ratio of PC/PE and amount of vimentin may be potential biomarkers for representing the ratio of morphological changed Vero cell introduced by trace lipophilic compounds, thus their composite bio-toxicity.     

Does the Harvard/U.S. Environmental Protection Agency Ambient Particle Concentrator change the toxic potential of particles?

Inhalation exposure to urban air particles is known to increase morbidity in humans and animals. Our group utilizes the Harvard/U.S. Environmental Protection Agency Ambient Particle Concentrator (HAPC) to generate concentrated aerosols of outdoor air particles for experimental exposures. We have reported increased pathologic responses to inhalation of concentrated urban air particles and identified silicon (as silicate) as an element associated with many of these responses. Using silicate-rich Mt. St. Helen's volcanic ash (MSHA), we exposed three groups of Sprague-Dawley rats by inhalation for 6 hr to filtered air, MSHA, or MSHA passed though the HAPC. Twenty-four hours following exposure, bronchoalveolar lavage was performed to assess total cell count, differential cell count, protein, lactate dehydrogenase, and n-beta-glucosaminidase levels. Peripheral blood was examined for packed cell volume, total protein, total white cells, and differential cell count. Morphologic studies localized particles in the lung and assessed pulmonary vasculature. No significant differences were observed among any of the groups in any parameter measured including morphometric analysis of pulmonary vasoconstriction. Scanning electron microscopy and X-ray analysis identified particles as silicates typical of MSHA throughout the lung. These findings suggest that particles passing through the HAPC have no change in their toxic potential in an exposure setting where particle deposition in the lung has occurred.     

Impact of four pesticides on the growth and metabolic activities of two photosynthetic algae

The acute toxicity was determined for soil algae Chlorella kesslerei and Anabaena inaequalis, exposed to pesticides lindane, pentachlorophenol (PCP), isoproturon (IPU), and methyl parathion (MP). Toxicity markers included growth inhibition, chlorophyll biosynthesis, and total carbohydrate content, as a function of dose and time. Concentration response functions (EC50) were estimated by probit data transformation and weighted linear regression analyses. Lindane's toxicity to Chlorella increased sharply with time (EC50 = 7490, 10.3, 0.09 mg L(-1); 24, 48, 72 h), but remained nearly constant through 72 h with Anabaena (8.7-6.7 mg L(-1); 24-72 h). PCP at low concentrations stimulated algal growth and chlorophyll a production, an effect reversed at higher doses. Anabaena was less tolerant of PCP and MP than was Chlorella. The 96-h static EC50 values for Chlorella were: 0.003, 34, 0.05, and 291 mg L(-1) for lindane, PCP, isoproturon, and MP, respectively; for Anabaena, these were 4.2, 0.13, 0.21, and 19 mg L(-1). Carbohydrate production responses were similar to those of cell density (growth) and chlorophyll biosynthesis, with MP having the lowest adverse impact. The overall relative toxicity among the four tested pesticides was: for Chlorella, lindane > IPU > PCP > MP; and for Anabaena, PCP > IPU > lindane > MP. The results confirm that toxicants such as these pesticides may affect individual (though related) species to significantly different degrees.     

Proteomic analysis in the lichen Physcia adscendens exposed to cadmium stress

This work was undertaken to explore the potential of proteomics to dissect parallel and consecutive events of cadmium stress response in the lichen Physcia adscendens (Fr.) H. Olivier. Thalli were exposed to 0 (control) and 36 μM Cd for 6, 18, 24 and 48 h. Two-dimensional electrophoresis and mass spectrometry analyses showed an 80-85% spot identity between 6 and 18 h vs. 24 and 48 h of Cd exposure. Putative heat-shock proteins and glutathione S-transferase generally increased their expression all over the Cd treatments. By contrast, ABC transporters were underexpressed after 6-18 h, but in some cases induced after 24-48 h of Cd exposure. The cytochrome P450 appeared to have a variable expression pattern over time. Overall these data suggest that a considerable importance in the response of P. adscendens thalli to Cd stress can be assumed by differential expression of various protein families.