2D-DIGE as a proteomic biomarker discovery tool in environmental studies with Procambarus clarkii.

A 2D-DIGE/MS approach was used to assess protein abundance differences in the red swamp crayfish Procambarus clarkii from polluted aquatic ecosystems of Doñana National Park and surrounding areas with different pollution loads. Procambarus clarkii accumulated metals in the digestive glands and gills reflecting sediment concentrations. We first stated that, probably related to elements accumulation, pollution increased oxidative damage in P. clarkii tissues, as shown by the thiol oxidation status of proteins and MDA levels. In these animals, the altered redox status might be responsible for the deregulated abundance of proteins involved in cellular responses to oxidative stress including protein folding, mitochondrial imbalance and inflammatory processes. Interestingly, polluted P. clarkii crayfish also displayed a metabolic shift to enhanced aerobic glycolysis, most likely aimed at generating ATP and reduction equivalents in an oxidative stress situation that alters mitochondrial integrity. The deregulated proteins define the physiological processes affected by pollutants in DNP and its surrounding areas and may help us to unravel the molecular mechanisms underlying the toxicity of environmental pollutants. In addition, these proteins might be used as exposure biomarkers in environmental risk assessment. The results obtained might be extrapolated to many other locations all over the world and have the added value of providing information about the molecular responses of this environmentally and economically interesting animal. SIGNIFICANCE: Metal content in digestive gland and gills of P. clarkii crayfish reflects their contents in sediments at sites of Doñana National Park and its surroundings. Accumulation of essential and toxic transition metals is paralleled by clear signs of oxidative stress to lipids and proteins and by significant deregulation of many proteins involved in protein folding, mitochondrial respiratory imbalance and inflammatory response. These results indicate that P. clarkii is an excellent bioindicator to be used in aquatic ecosystems quality monitoring. Additionally, results evidence that the anthropogenic activities carried out around Doñana National Park represent an extremely serious threat to this unique Biosphere Reserve and pose a risk to the environment and their inhabitants health. The identified deregulated proteins provide information about the metabolic pathways and/or physiological processes affected by pollutant-elicited oxidative stress, may also be useful as biomarkers of environmental pollution and have the added value of providing information about the molecular responses of this environmentally and economically interesting animal.

The ß-glucosidase assay: a new diagnostic tool for necrotizing enterocolitis. Sensitivity, specificity, and predictive values.

UNLABELLED: We aimed to establish the utility of serum cytosolic ß-glycosidase (CBG) assay as a NEC diagnosis tool. CBG activity has been compared in 192 NEC-free (NEC(-)) and 13 NEC-affected (NEC(+)) neonates, with modified Bell's stages II/III, born at Reina Sofia University Hospital; additional blood hematology, microbiology, and biochemical parameters have been assayed. NEC(+) neonates have higher serum CBG activity, 26.4 ± 12.4 mU/mg; 95 % CI (18.8-33.9), than NEC(-) infants, 11.0 ± 6.6 mU/mg; 95 % CI (10.1-11.9) (p < 0.0001). The CBG cutoff value in the ROC curve, 15.6 mU/mg, discriminates NEC(+)/NEC(-) infants with 84.6 % sensitivity, 85.9 % specificity, 37.9 positive predictive value and 98.2 negative predictive value, 6.11 positive likelihood ratio and 0.18 negative likelihood ratio, 33.61 DOR, and 0.89 AUC. A combined panel [CBG + aspartate aminotransferase + C-reactive protein] shows a 0.90 AUC value in multiple linear regressions. CONCLUSIONS: The serum CBG level is a good NEC diagnosis test and a novel NEC biomarker which may become a screening tool. WHAT IS KNOWN: •NEC affects ∼2.5 % of infants at NICU, ∼90 % of them weighing <1500 g. •NEC requires a careful differential diagnosis, being lethal if not diagnosed and treated. What is new: •CBG assay will be useful to determine infants without NEC and preventing unnecessary treatment. •CBG assay could discriminate NEC better than other gut-specific sera protein biomarkers.

Using environmental proteomics to assess pollutant response of Carcinus maenas along the Tunisian coast.

Biochemical responses to pollutants were studied at four Tunisia littoral sites using Carcinus maenas as a bioindicator. Proteomic analysis was used to assess the global impact of complex pollution mixtures, and to provide new biomarkers and basic insights into pollutant toxicity. Metal contents and metallothionein levels followed a gradient based on sampling sites: Bizerte ≫ Teboulba > Gargour~Mahres. Approximately 900 and 700 spots were resolved in digestive glands and gills, respectively. Gills from Bizerte animals had the maximum number of altered spots, mostly upregulated. In other locations, the number of altered spots in gills decreased in parallel to total metals in in the following order: Teboulba > Gargour > Mahres (mostly downregulated). Out of the 39 spots excised, ten proteins were identified in digestive glands and eight in gills. Digestive glands of Bizerte crabs had higher levels of ferritin, three vitellogenin forms and mannose-binding protein, while Gargour crabs had higher levels of four cryptocyanin forms. Gills of Bizerte crabs had higher levels of ferritin, three vitellogenins forms, lectin 4C, actin, and collagenolytic serine protease. Proteins with altered expression in crabs from Tunisia littoral are related to molting, oxidative stress and inflammation, innate immune response, and proteolysis.

Proteomic analysis through larval development of Solea senegalensis flatfish.

The post-embryonic development of the Senegalese sole, Solea senegalensis, a flatfish of growing interest in fisheries and aquaculture, is associated with drastic morpho-physiological changes during metamorphosis. Although in the last two decades knowledge on sole culture has notably increased, especially in Southern Europe, its progress was restricted due to lack of methods to control reproduction, improve larval quality and increase juvenile disease resistance. A limited knowledge of the physiological, molecular and genetic mechanisms involved is at the base of such limitation. A proteomic study was carried out to explore the molecular events that occur during S. senegalensis ontogenesis. Protein expression changes were monitored in larvae from 5 to 21 dph by combining 2DE and protein identification with de novo MS/MS sequencing. An average of 6177 ± 282 spots was resolved in 2DE gels. Hierarchical cluster analysis of the 705 selected spots grouped them in eight patterns. Thirty-four proteins were identified and assigned biological functions including structure, metabolism highlighting energy metabolism, transport, protein folding, stress response, chromatin organization and regulation of gene expression. These changes provide a sequential description of the molecular events associated with the biochemical and biological transformations that occur during sole larval development.

iTRAQ analysis of hepatic proteins in free-living Mus spretus mice to assess the contamination status of areas surrounding Doñana National Park (SW Spain).

This work aims to develop and integrate new -omics tools that would be applicable to different ecosystem types for a technological updating of environmental evaluations. We used a 2nd-generation (iTRAQ-8plex) proteomic approach to identify/quantify proteins differentially expressed in the liver of free-living Mus spretus mice from Doñana National Park or its proximities. Mass spectrometry was performed in an LTQ Orbitrap system for iTRAQ reporter ion quantitation and protein identification using a Mus musculus database as reference. A prior IEF step improved the separation of the complex peptide mixture. Over 2000 identified proteins were altered, of which 118 changed by ≥2.5-fold in mice from at least two problem sites. Part of the results obtained with the iTRAQ analysis was confirmed by Western blot. Over 75% of the 118 proteins were upregulated in animals captured at polluted sites and only 16 proteins were downregulated. Upregulated proteins were involved in stress response; cell proliferation and apoptosis; signal transduction; metastasis or tumour suppression; xenobiotic export or vesicular trafficking; and metabolism. The downregulated proteins, all potentially harmful, were classified as oncoproteins and proteins favouring genome instability. The iTRAQ results presented here demonstrated that the survival of hepatic cells is compromised in animals living at polluted sites, which showed deep alterations in metabolism and the signalling pathways. The identified proteins may be useful as biomarkers of environmental pollution and provide insight about the metabolic pathways and/or physiological processes affected by pollutants in DNP and its surrounding areas.

Alterations of protein expression in serum of infants with intrauterine growth restriction and different gestational ages.

Intrauterine growth restriction (IUGR) is associated with increased morbidity and metabolic anomalies in adults. The serum proteome of venous blood was compared in 43 IUGR and 45 adequate gestational age (AGA) infants, separated into three gestational age groups, "Very Preterm" 29-32weeks, "Moderate Preterm" 33-36w, and, "Term" ≥37weeks, in samples drawn three times from birth to 1month of life. After depleting the abundant serum proteins (ProteoMiner(TM)), expression changes were studied by 2-DE, image analysis (Proteomweaver 4.0(TM)), and identification by MALDI-TOF/TOF. Significant expression differences were found in thirty-four proteins, and thirty-three were identified. Lysophospholipid acyltransferase 7 (MBOAT7), was detected exclusively in IUGR of all gestational ages and sampling times; seven other proteins were found only in AGA. Another twenty-five proteins had intensity changes ≥2.5 folds in IUGR: twenty were upregulated and five downregulated. Western blots confirmed the identification of several proteins: MBOAT7 increased 20.5-fold in IUGR, while AGA had 11.2-fold higher levels of SUMO3 and sumoylated proteins and 13.7-fold higher levels of APOL1. Upregulation of MBOAT7 in IUGR neonates could be an adaptive response to protect the brain from an adverse environment. BIOLOGICAL SIGNIFICANCE: There are significant protein expression differences between IUGR and AGA at different gestational age groups and blood extraction times. The extensive upregulation of lysophospholipid acyltransferase 7 in all IUGR gestational ages and extraction times might be an adaptative response to an adverse fetal environment, reminiscent of Barker's fetal programming theory. Two serotransferrins were also upregulated in IUGR of all gestational ages. Just at birth "Very Preterm" IUGR showed nine upregulated proteins, including five albumins, apolipoprotein E, keratin type I cytoskeletal 10, solute carrier family member 2 fragment, and anaphase-promoting complex subunit 2.

Redox proteomics as biomarker for assessing the biological effects of contaminants in crayfish from Doñana National Park.

Despite its environmental relevance and sensitivity, Doñana National Park (DNP) is under high ecological pressure. In crayfish (Procambarus clarkii), the utility of redox proteomics as a novel biomarker was evaluated in the aquatic ecosystems of DNP and its surroundings, where agricultural activity is a serious concern. After fluorescence labeling of reversibly oxidized Cys and 2-DE separation, the total density of proteins with reversibly oxidized thiols was found to be much higher in animals from the Matochal (MAT) and Rocina (ROC) streams, while no difference was found in crayfish from Partido (PAR) stream compared to those from the DNP core at Lucio del Palacio (the negative control). The 2-DE analysis revealed 35 spots with significant differences in thiol oxidation, among which 19 proteins were identified via MALDI-TOF/TOF. While 3 spots, identified as ferritin, showed higher oxidation levels in ROC, other identified proteins were more intense at MAT than at ROC (superoxide dismutase, protein disulfide isomerase and actin) or were overoxidized only in MAT (nucleoside diphosphate kinase, fructose-biphosphate aldolase, fatty acid-binding protein, phosphopyruvate hydratase). For most of the identified proteins, spots corresponding to different Cys oxidized forms were detected, and the native forms, without oxidized thiol groups were also found in some of them. Evidence of reversible oxidation was found for specific Cys residues, including Cys13 in ferritin as well as Cys76 and Cys108 in nucleoside diphosphate kinase. The identified thiol-oxidized proteins provide information about the metabolic pathways and/or physiological processes affected by pollutant-elicited oxidative stress.

Omics technologies and their applications to evaluate metal toxicity in mice M. spretus as a bioindicator.

Metals are important components of living organisms since many biological functions critically depend on their interaction with some metal in the cell. However, human activities have increased toxic metal levels in the terrestrial and aquatic ecosystems affecting living organisms. The impact of metals on cellular metabolism and global homeostasis has been traditionally assessed in free-living organisms by using conventional biomarkers; however, to obtain a global vision of metal toxicity mechanisms and the responses that metals elicit in the organisms, new analytical methodologies are needed. We review the use of omics approaches to assess the response of living organisms under metal stress illustrating the possibilities of different methodologies on the basis of our previous results. Most of this research has been based on free-living mice Mus spretus, a conventional bioindicator used to monitor metal pollution in Doñana National Park (DNP) (SW Spain), which is an important European biological reserve for migrating birds affected by agricultural, mining and industrial activities. The benefits of using omic techniques such as heterologous microarrays, proteomics methodologies (2-DE, iTRAQ®), metallomics, ionomics or metabolomics has been remarked; however, the complexity of these areas requires the integration of omics to achieve a comprehensive assessment of their environmental status. This article is part of a Special Issue entitled: Environmental and structural proteomics. BIOLOGICAL SIGNIFICANCE: This work presents new contributions in the study of environmental metal pollution in terrestrial ecosystems using Mus spretus mice as bioindicator in Doñana National Park (SW Spain) and surroundings. In addition, it has been demonstrated that the integration of omics multi-analytical approaches provides a very suitable approach for the study of the biological response and metal interactions in exposed and free-living mice (Mus musculus and Mus spretus, respectively) under metal pollution.

Proteomic analysis in caged Mediterranean crab (Carcinus maenas) and chemical contaminant exposure in Téboulba Harbour, Tunisia.

This study uses proteomics approach to assess the toxic effects of contaminants in the Mediterranean crab (Carcinus maenas) after transplantation into Téboulba fishing harbour. High levels of aliphatic and aromatic hydrocarbons were detected in sediments. Although their effects on vertebrates are well described, little is known about their early biological effects in marine invertebrates under realistic conditions. Protein expression profiles of crabs caged for 15, 30 and 60 days were compared to unexposed animals. Nineteen proteins with significant expression differences were identified by capLC-µESI-IT MS/MS and homology search on databases. Differentially expressed proteins were assigned to five different categories of biological function including: (1) chitin catabolism, (2) proteolysis, (3) exoskeleton biosynthesis, (4) protein folding and stress response, and (5) transport. The proteins showing major expression changes in C. maenas after different caging times may be considered as novel molecular biomarkers for effectively biomonitoring aquatic environment contamination.

Biochemical responses in seabream (Sparus aurata) caged in-field or exposed to benzo(a)pyrene and paraquat. Characterization of glutathione S-transferases.

Gilthead seabream (Sparus aurata) specimens were caged in-field at the Téboulba harbour or exposed to benzo(a)pyrene [B(a)P] or to paraquat [PQ] plus B(a)P, and several biochemical biomarker responses were investigated. Antioxidant enzymes, such as glutathione peroxidase, catalase and glutathione reductase, significantly increased in the in-field and B(a)P+PQ exposures, but were only moderately affected by B(a)P alone. Glucose-6-phosphate and 6-phosphogluconate dehydrogenases significantly diminished after in-field exposure. Different responses with biotransformation enzymes were observed: the P4501A-associated EROD activity was highly induced in response to B(a)P and B(a)P+PQ exposures, while total activity of the glutathione S-transferase (GST) was similar to control. However, after purification of the GST proteins by affinity chromatograpy and analysis by two-dimensional electrophoresis, nineteen highly reproducible isoforms were resolved. In addition, some of reproducible isoforms showed different and specific expression patterns in response to contaminants. Thus, proteomic analysis of the purified GST subunits is a reliable tool for ecotoxicological research, useful in polluted marine ecosystem as an effective biomarker of contamination.

Hepatic proteome changes in Solea senegalensis exposed to contaminated estuarine sediments: a laboratory and in situ survey.

Assessing toxicity of contaminated estuarine sediments poses a challenge to ecotoxicologists due to the complex geochemical nature of sediments and to the combination of multiple classes of toxicants. Juvenile Senegalese soles were exposed for 14 days in the laboratory and in situ (field) to sediments from three sites (a reference plus two contaminated) of a Portuguese estuary. Sediment characterization confirmed the combination of metals, polycyclic aromatic hydrocarbons and organochlorines in the two contaminated sediments. Changes in liver cytosolic protein regulation patterns were determined by a combination of two-dimensional electrophoresis with de novo sequencing by tandem mass spectrometry. From the forty-one cytosolic proteins found to be deregulated, nineteen were able to be identified, taking part in multiple cellular processes such as anti-oxidative defence, energy production, proteolysis and contaminant catabolism (especially oxidoreductase enzymes). Besides a clear distinction between animals exposed to the reference and contaminated sediments, differences were also observed between laboratory- and in situ-tested fish. Soles exposed in the laboratory to the contaminated sediments failed to induce, or even markedly down-regulated, many proteins, with the exception of a peroxiredoxin (an anti-oxidant enzyme) and a few others, when compared to reference fish. In situ exposure to the contaminated sediments revealed significant up-regulation of basal metabolism-related enzymes, comparatively to the reference condition. Down-regulation of basal metabolism enzymes, related to energy production and gene transcription, in fish exposed in the laboratory to the contaminated sediments, may be linked to sediment-bound contaminants and likely compromised the organisms' ability to deploy adequate responses against insult.

Serum proteomic changes in adults with obstructive sleep apnoea.

To examine whether differentially expressed proteins are present in the serum of patients with obstructive sleep apnoea (OSA), iTRAQ techniques (isobaric tags for relative and absolute quantification) were employed in a prospective study. Individuals were assigned to either a non-OSA control group (apnoea-hypopnoea index, AHI <5) or an OSA group (AHI ≥5). Blood samples were collected, aliquoted and frozen at -80 °C. Protein digestion and tagging with iTRAQ4plex® and mass spectrometry analysis was then performed (MALDI TOF/TOF). Ten male subjects were included in the control group (age = 45 ± 9.7 years) and 30 male patients in the OSA group (age = 45 ± 10.7 years), the latter being then subdivided into three severity groups. A total of 103 proteins were identified with differential levels between patients with OSA and controls. Of these, 11 proteins were underexpressed and 19 were overexpressed in patients with OSA. C4BPA and thrombospondin were underexpressed in all three OSA severity groups. Among the overexpressed proteins, 13 were overexpressed in the mild OSA group, seven in the moderate group and five in the severe group. Analysis of interactions between the identified proteins revealed that protein alterations in OSA are primarily associated with derangements in lipid and vascular metabolic pathways. This study provides initial evidence that differential protein expression occurs in adults with OSA, and that such proteins change according to disease severity, and appear to primarily involve lipid and vascular metabolic pathways.

Omic approaches in environmental issues.

Biomonitoring requires the application of batteries of different biomarkers, as environmental contaminants induce multiple responses in organisms that are not necessarily correlated. Omic technologies were proposed as an alternative to conventional biomarkers since these techniques quantitatively monitor many biological molecules in a high-throughput manner and thus provide a general appraisal of biological responses altered by exposure to contaminants. As the studies using omic technologies increase, it is becoming clear that any single omic approach may not be sufficient to characterize the complexity of ecosystems. This work aims to provide a preliminary working scheme for the use of combined transcriptomic and proteomic methodologies in environmental biomonitoring. There are difficulties in working with nonmodel organisms as bioindicators when combining several omic approaches. As a whole, our results with heterologous microarrays in M. spretus and suppressive subtractive hybridization (SSH) in P. clarkii indicated that animals sustaining a heavy pollution burden exhibited an enhanced immune response and/or cell apoptosis. The proteomic studies, although preliminary, provide a holistic insight regarding the manner by which pollution shifts protein intensity in two-dimensional gel electrophoresis (2-DE), completing the transcriptomic approach. In our study, the sediment element concentration was in agreement with the intensity of protein expression changes in C. maenas crabs. In conclusion, omics are useful technologies in addressing environmental issues and the determination of contamination threats.

Variación de la expresión proteica en función de la gravedad del síndrome de apnea-hipopnea del sueño / Variation in Protein Expression Depending on the Severity of Sleep Apnoea-Hypopnoea Syndrome

ObjetivoEstudio prospectivo con muestreo consecutivo y grupo control para determinar si la expresión proteica en pacientes con SAHS es diferente a la de un grupo control (IAH ≤5).Pacientes y métodosFueron incluidos 32 pacientes, entre 35 y 60 años, a los que se les realizó una polisomnografía. Fueron excluidos los sujetos con enfermedad aguda o crónica. La primera dimensión del estudio proteómico se realizó en tiras IPG (18cm, pH 4–7) y, la segunda, en geles SDS-PAGE por triplicado para cada grupo. Los geles se tiñeron con SYPRO-Ruby (Bio-Rad®), se obtuvieron las imágenes con un escáner láser FX-Imager, y el análisis de los spots se realizó con el software ProteomWeaver v4.0 (Bio-Rad®). Se analizaron los cambios significativos entre los geles agrupados por réplicas y por separado, considerándose un cambio significativo si la intensidad relativa en los spots fue superior o inferior en 3 veces a la del control y se observó en 2 de las 3 réplicas de cada grupo con un coeficiente de variación <20%.ResultadosLos pacientes fueron divididos en 8 sujetos por grupo (control, leve, moderado y grave). La comparación de los geles constató diferencias significativas entre el grupo control y los 3 grupos clínicos, observándose 3 spots con sobreexpresión significativa y 7 spots subexpresados respecto al grupo control.ConclusiónExisten cambios significativos en la expresión protéica entre un grupo control y pacientes en distintos estadios de enfermedad. El estudio proteómico puede identificar biomarcadores relacionados con el diagnóstico y gravedad del SAHS(AU)

ObjectiveA prospective study with a consecutive sample and a control group to determine whether protein expression in patients with sleep apnoea-hypopnoea syndrome (SAHS) is different from that of the control group (IAH ≤5).Patients and methodsA total of 32 patients aged between 35 and 60 years who had a polysomnograph performed were included. Patients with an acute or chronic were excluded. The first dimension of the proteomic study was carried out on IPG strips (18cm, pH 4–7) and the second on SDS-PAGE gels in triplicate for each group. The gels were stained with SYPRO-Ruby (Bio-Rad®), the images obtained with an FX-Imager laser scanner and the spots were analysed using ProteomWeaver v. 4.0 (Bio-Rad®) software. Significant changes between the gels were analysed by replicates and separately, being considered a significant change if the relative intensity of the spots was three times higher or lower than that of the control and if it was observed in 2 of the 3 replicates of each group, with a coefficient of variation of <20%.ResultsThe patients were divided into 8 subjects per group (control, mild, moderate and severe). The comparison of the gels showed significant differences between the control group and the 3 clinical groups, with significant over-expression being observed in 3 spots, and under-expression in 7 spots in the control group.ConclusionThere are significant changes in protein expression between a control group and patients in different stages of disease. The proteomic study can identify biomarkers associated with the diagnosis and severity of the SAHS(AU)

[Variation in protein expression depending on the severity of sleep apnoea-hypopnoea syndrome]. / Variación de la expresión proteica en función de la gravedad del síndrome de apnea-hipopnea del sueño.

OBJECTIVE: A prospective study with a consecutive sample and a control group to determine whether protein expression in patients with sleep apnoea-hypopnoea syndrome (SAHS) is different from that of the control group (IAH < or =5). PATIENTS AND METHODS: A total of 32 patients aged between 35 and 60 years who had a polysomnograph performed were included. Patients with an acute or chronic were excluded. The first dimension of the proteomic study was carried out on IPG strips (18cm, pH 4-7) and the second on SDS-PAGE gels in triplicate for each group. The gels were stained with SYPRO-Ruby (Bio-Rad((R))), the images obtained with an FX-Imager laser scanner and the spots were analysed using ProteomWeaver v. 4.0 (Bio-Rad((R))) software. Significant changes between the gels were analysed by replicates and separately, being considered a significant change if the relative intensity of the spots was three times higher or lower than that of the control and if it was observed in 2 of the 3 replicates of each group, with a coefficient of variation of <20%. RESULTS: The patients were divided into 8 subjects per group (control, mild, moderate and severe). The comparison of the gels showed significant differences between the control group and the 3 clinical groups, with significant over-expression being observed in 3 spots, and under-expression in 7 spots in the control group. CONCLUSION: There are significant changes in protein expression between a control group and patients in different stages of disease. The proteomic study can identify biomarkers associated with the diagnosis and severity of the SAHS.

Environmental monitoring of Domingo Rubio stream (Huelva Estuary, SW Spain) by combining conventional biomarkers and proteomic analysis in Carcinus maenas.

Element load, conventional biomarkers and altered protein expression profiles were studied in Carcinus maenas crabs, to assess contamination of "Domingo Rubio" stream, an aquatic ecosystem that receives pyritic metals, industrial contaminants, and pesticides. Lower antioxidative activities - glucose-6-phosphate and 6-phosphogluconate dehydrogenases, catalase - were found in parallel to higher levels of damaged biomolecules - malondialdehyde, oxidized glutathione -, due to oxidative lesions promoted by contaminants, as the increased levels of essential - Zn, Cu, Co - and nonessential - Cr, Ni, Cd - elements. Utility of Proteomics to assess environmental quality was confirmed, especially after considering the six proteins identified by de novo sequencing through capLC-muESI-ITMS/MS and homology search on databases. They include tripartite motif-containing protein 11 and ATF7 transcription factor (upregulated), plus CBR-NHR-218 nuclear hormone receptor, two components of the ABC transporters and aldehyde dehydrogenase (downregulated). These proteins could be used as novel potential biomarkers of the deleterious effects of pollutants present in the area.

Metallomics integrated with proteomics in deciphering metal-related environmental issues.

The present work shows the possibilities of metallomics to characterize metal-linking proteins in Mus Musculus that could be used in environmental assessment. The laboratory mouse M. musculus is used as reference of gene/protein sequence databases to address methodological approaches based on changes in transcripts regulation, proteins expression and metalloproteins profiles in the environmental bioindicator Mus spretus that has been demonstrated to be genetically homologous to M. Musculus. A metallomic approach using size exclusion chromatography with inductively coupled plasma-mass spectrometry detection (SEC-ICP-MS) was applied to cytosolic extracts from different M. musculus organs: lung, liver, spleen, kidney, brain, testicle, hearth and muscle. The resulting profiles of metallobiomolecules revealed the presence of a Cu-binding fraction in the 7-10 kDa range which was not present in the other tissues, can be associated to low molecular mass metallothionein-like proteins. The application of reverse phase chromatography with ICP-MS detection to this fraction gives two peaks that have been isolated for later identification by tandem mass spectrometry. The mass balance of copper evaluated by ICP-MS analysis of the digested brain fractions isolated by SEC and RP chromatography reveals good recoveries of the separations. The application of 2-DE to both crude brain extract and SEC fraction (7-10 kDa) reveals the considerably reduction of the number of proteins confirming that a good purification has been attained by SEC. This integration of metallomics with proteomics and transcriptomics can be useful in further studies involving the free-living mouse M. spretus for assessment of environmental issues.

Biochemical and proteomic effects in Procambarus clarkii after chlorpyrifos or carbaryl exposure under sublethal conditions.

In vivo effects of two sublethal doses of chlorpyrifos and carbaryl were studied in Procambarus clarkii after 2 and 7 days of exposure, and after pesticide removal. Chlorpyrifos inhibited carboxylesterase activity in a concentration-dependent manner, but acetylcholinesterase was less sensitive. Compared with chlorpyrifos, carbaryl had a less marked effect on esterase activity. The effects of selected pesticides on biotransformation or oxidative stress biomarkers were contradictory. Chlorpyrifos lowered ethoxyresorufin-O-deethylase (EROD), catalase and oxidized glutathione (GSSG) levels but raised glutathione-S-transferase activity, while carbaryl raised EROD, catalase and glutathione-S-transferase, but lowered glutathione peroxidase and reduced glutathione (GSH) levels. The effects on protein expression patterns depending on pesticide type and the tissue used for analysis were studied in parallel by 2-DE. In gill and nervous tissue about 2000 spots (pI 4-7) were resolved, with quite different expression patterns. Chlorpyrifos altered 72 proteins, mostly in nervous tissue, and carbaryl 35, distributed evenly between organs. Several specific spots were selected as specific protein expression signatures for chlorpyrifos or carbaryl exposure in gills and nervous tissue, respectively.

Multivariate discriminant analysis distinguishes metal- from non metal-related biomarker responses in the clam Chamaelea gallina.

Molecular biomarkers are among the most sensitive and earliest responses to pollutants. However, lack of detailed knowledge on variability of responses and their possible seasonal variation limit their use. In addition, the seasonality of biological processes modulates the response of organisms to pollutant stressors. Using multivariate statistics, we have studied the influence of environmental and biological factors on the response of a battery of molecular biomarkers in the clam Chamaelea gallina collected along the South-Spanish littoral. Multivariate discriminant analysis clearly distinguished biomarker response between clean and polluted areas, using heavy metals as indicator of pollution. Such differences disappeared when the dataset was normalised for metal content, thus indicating that pollution was the main significant cause of the changes observed between clean and polluted sites. In conclusion, this work shows that, when applying a complete biomarker panel, multivariate statistical tools can be used to discern pollutant- from non pollutant-related responses.

Assessment of Doñana National Park contamination in Procambarus clarkii: integration of conventional biomarkers and proteomic approaches.

Aquatic ecosystems of Doñana National Park (DNP) were monitored using Procambarus clarkii as bioindicator in four campaigns carried out between 2003 and 2004 to assess environmental quality possibly threatened by agrochemicals used in nearby areas. An integrated approach was carried out, by combining the responses of well-established biomarkers and the massive analysis of biological effects at the proteomic level. In sites potentially polluted, lower catalase, glucose-6-P dehydrogenase, and esterase activities, and higher malondialdehyde, metallothionein and glutathione levels were found. Two-dimensional gel electrophoresis resolved >2500 gill spots, and image analysis detected that 35 showed significant intensity differences between the reference site and the other seven sites studied. The superiority of proteomic approaches was clearly recognized in our study since four different protein expression patterns were established based in the fold-number of up-/down-regulation of the 35 differentially expressed proteins. Sites located within Doñana Biological Reserve were essentially free of contaminants and those near the DNP limits were only slightly polluted. The higher proteomic responses found at the upper "Rocina" and "Partido" courses indicate that non-persistent agrochemicals are mainly used in Doñana surroundings. The highest responses corresponded to rice growing areas placed between the Guadiamar stream and the Guadalquivir River, according to the extended and intensive use of agrochemicals in such areas.