Publications by authors named "José Alhama"

18 Publications

  • Page 1 of 1

Meta-omic evaluation of bacterial microbial community structure and activity for the environmental assessment of soils: overcoming protein extraction pitfalls.

Environ Microbiol 2021 Aug 30;23(8):4706-4725. Epub 2021 Jul 30.

Departamento de Bioquímica y Biología Molecular, Campus de Excelencia Internacional Agroalimentario CeiA3, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, Córdoba, E-14071, Spain.

Microorganisms play unique, essential and integral roles in the biosphere. This work aims to assess the utility of soil's metaomics for environmental diagnosis. Doñana National Park (DNP) was selected as a natural lab since it contains a strictly protected core that is surrounded by numerous threats of pollution. Culture-independent high-throughput molecular tools were used to evaluate the alterations of the global structure and metabolic activities of the microbiome. 16S rRNA sequencing shows lower bacterial abundance and diversity in areas historically exposed to contamination that surround DNP. For metaproteomics, an innovative post-alkaline protein extraction protocol was developed. After NaOH treatment, successive washing with Tris-HCl buffer supplemented with glycerol was essential to eliminate interferences. Starting from soils with different physicochemical characteristics, the method renders proteins with a remarkable resolution on SDS-PAGE gels. The proteins extracted were analysed by using an in-house database constructed from the rRNA data. LC-MS/MS analysis identified 2182 non-redundant proteins with 135 showing significant differences in relative abundance in the soils around DNP. Relevant global biological processes were altered in response to the environmental changes, such as protective and antioxidant mechanisms, translation, folding and homeostasis of proteins, membrane transport and aerobic respiratory metabolism.
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http://dx.doi.org/10.1111/1462-2920.15673DOI Listing
August 2021

Constructing a de novo transcriptome and a reference proteome for the bivalve Scrobicularia plana: Comparative analysis of different assembly strategies and proteomic analysis.

Genomics 2021 May 25;113(3):1543-1553. Epub 2021 Mar 25.

Departamento de Bioquímica y Biología Molecular, Campus de Excelencia Internacional Agroalimentario CeiA3, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, E-14071 Córdoba, Spain. Electronic address:

Scrobicularia plana is a coastal and estuarine bivalve widely used in ecotoxicological studies. However, the underlying molecular mechanisms for S. plana pollutant responses are hardly known due to the lack of molecular databases. Thus, in this study we present a holistic approach to assess a robust reference transcriptome and proteome of this clam. A mixture of control and metal-exposed individuals was used for mRNA isolation. Four sets of high quality filtered preprocessed reads were generated (two quality scores and two sequenced lengths) and assembled with Mira, Ray and Trinity algorithms. The sixty-four generated assemblies were refined, filtered and evaluated for their proteomic quality. Eight assemblies presented top Detonate scores but one was selected due to its compactness and biological representation, which was generated: (i) from the highest quality dataset (Q20L100), (ii) using Trinity algorithm with all k-mers (AtKa), (iii) removing redundancy by CD-HIT (RR80), and (iv) filtering out poor contigs (F), that was subsequently named Q20L100AtKaRR80F. S. plana proteomic analysis revealed 10,017 peptide groups that corresponded to 2066 proteins with a wide coverage of molecular functions and biological processes, confirming the strength of the database generated.
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http://dx.doi.org/10.1016/j.ygeno.2021.03.025DOI Listing
May 2021

High-throughput molecular analyses of microbiomes as a tool to monitor the wellbeing of aquatic environments.

Microb Biotechnol 2021 05 9;14(3):870-885. Epub 2021 Feb 9.

Departamento de Bioquímica y Biología Molecular, Campus de Excelencia Internacional Agroalimentario CeiA3, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, Córdoba, E-14071, Spain.

Aquatic environments are the recipients of many sources of environmental stress that trigger both local and global changes. To evaluate the associated risks to organisms and ecosystems more sensitive and accurate strategies are required. The analysis of the microbiome is one of the most promising candidates for environmental diagnosis of aquatic systems. Culture-independent interconnected meta-omic approaches are being increasing used to fill the gaps that classical microbial approaches cannot resolve. Here, we provide a prospective view of the increasing application of these high-throughput molecular technologies to evaluate the structure and functional activity of microbial communities in response to changes and disturbances in the environment, mostly of anthropogenic origin. Some relevant topics are reviewed, such as: (i) the use of microorganisms for water quality assessment, highlighting the incidence of antimicrobial resistance as an increasingly serious threat to global public health; (ii) the crucial role of microorganisms and their complex relationships with the ongoing climate change, and other stress threats; (iii) the responses of the environmental microbiome to extreme pollution conditions, such as acid mine drainage or oil spills. Moreover, protists and viruses, due to their huge impacts on the structure of microbial communities, are emerging candidates for the assessment of aquatic environmental health.
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http://dx.doi.org/10.1111/1751-7915.13763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085945PMC
May 2021

Metal body burden and tissue oxidative status in the bivalve Venerupis decussata from Tunisian coastal lagoons.

Mar Environ Res 2020 Jul 14;159:105000. Epub 2020 May 14.

Departamento de Bioquímica y Biología Molecular, Campus de Excelencia Internacional Agroalimentario CeiA3, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, E-14071, Córdoba, Spain. Electronic address:

Coastal transitional waters are exposed to many anthropogenic threats. This study aims to assess the trace metals' pollution status of transitional waters by evaluating its biological effects in the clam Venerupis decussata. Among the studied sites along the Tunisian littoral, South Tunis and Boughrara were the most impacted, since clams from these two lagoons presented significant differences in: (i) trace metal contents, (ii) in-cell hydrogen peroxide, (iii) enzymatic and non-enzymatic defenses, (iv) damage to lipids and proteins, and (v) protein post-translational modifications. These changes related to evident histopathological traits. PCA showed a clear separation between the digestive gland and gills tissues and illustrated an impact gradient in Tunisian coastal lagoons. Water temperature was revealed as an added natural stressor that, when concurring with high pollution, may jeopardize an ecosystem's health and contribute to the accumulation of hazardous metals in organisms.
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http://dx.doi.org/10.1016/j.marenvres.2020.105000DOI Listing
July 2020

Redox and global interconnected proteome changes in mice exposed to complex environmental hazards surrounding Doñana National Park.

Environ Pollut 2019 Sep 27;252(Pt A):427-439. Epub 2019 May 27.

Department of Biochemistry and Molecular Biology, University of Córdoba, Córdoba, Spain. Electronic address:

Natural environments are receiving an increasing number of contaminants. Therefore, the evaluation and identification of early responses to pollution in these complex habitats is an urgent and challenging task. Doñana National Park (DNP, SW Spain) has been widely used as a model area for environmental studies because, despite its strictly protected core, it is surrounded by numerous threat sources from agricultural, mining and industrial activities. Since many pollutants often induce oxidative stress, redox proteomics was used to detect redox-based variations within the proteome of Mus spretus mice captured in DNP and the surrounding areas. Functional analysis showed that most differentially oxidized proteins are involved in the maintenance of homeostasis, by eliciting mechanisms to respond to toxic substances and oxidative stress, such as antioxidant and biotransformation processes, immune and inflammatory responses, and blood coagulation. Furthermore, changes in the overall protein abundance were also analysed by label-free quantitative proteomics. The upregulation of phase I and II biotransformation enzymes in mice from Lucio del Palacio may be an alert for organic pollution in the area located at the heart of DNP. Metabolic processes involved in protein turnover (proteolysis, amino acid catabolism, new protein biosynthesis and folding) were activated in response to oxidative damage to these biomolecules. Consequently, aerobic respiratory metabolism increased to address the greater ATP demands. Alterations of cholesterol metabolism that could cause hepatic steatosis were also detected. The proteomic detection of globally altered metabolic and physiological processes offers a complete view of the main biological changes caused by environmental pollution in complex habitats.
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http://dx.doi.org/10.1016/j.envpol.2019.05.085DOI Listing
September 2019

Alterations in oxidative responses and post-translational modification caused by p,p´-DDE in Mus spretus testes reveal Cys oxidation status in proteins related to cell-redox homeostasis and male fertility.

Sci Total Environ 2018 Sep 1;636:656-669. Epub 2018 May 1.

Departamento de Bioquímica y Biología Molecular, Campus de Excelencia Internacional Agroalimentario CeiA3, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, E-14071 Córdoba, Spain. Electronic address:

The major derivate of DDT, 1,1-dichloro-2,2-bis (p-chlorophenyl) ethylene (p,p´-DDE), is a persistent pollutant previously associated with oxidative stress. Additionally, p,p´-DDE has been linked to several metabolic alterations related to sexual function in rodents. In this study, we analysed the effects of a non-lethal p,p´-DDE dose to Mus spretus mice in testes, focusing on oxidative damage to biomolecules, defence mechanisms against oxidative stress and post-translational protein modifications. No increase in lipid or DNA oxidation was observed, although antioxidative enzymatic defences and redox status of glutathione were altered in several ways. Global protein carbonylation and phosphorylation were significantly reduced in testes from p,p´-DDE-exposed mice; however, the total redox state of Cys thiols did not exhibit a defined pattern. We analysed the reversible redox state of specific Cys residues in detail with differential isotopic labelling and a shotgun labelling-based MS/MS proteomic approach for identification and quantification of altered peptides. Our results show that Cys residues are significantly affected by p,p´-DDE in several proteins related to oxidative stress and/or male fertility, particularly those participating in fertilization, sperm capacitation and blood coagulation. These molecular changes could explain the sexual abnormalities previously described in p,p´-DDE exposed organisms.
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http://dx.doi.org/10.1016/j.scitotenv.2018.04.305DOI Listing
September 2018

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

Sci Total Environ 2016 Jan 22;541:109-118. Epub 2015 Sep 22.

Department of Biochemistry and Molecular Biology, University of Córdoba, 14071 Córdoba, Spain.

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.
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http://dx.doi.org/10.1016/j.scitotenv.2015.09.032DOI Listing
January 2016

Proteomic analysis through larval development of Solea senegalensis flatfish.

Proteomics 2015 Dec 12;15(23-24):4105-19. Epub 2015 Oct 12.

Department of Biochemistry and Molecular Biology, University of Córdoba (UCO), Córdoba, Spain.

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.
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http://dx.doi.org/10.1002/pmic.201500176DOI Listing
December 2015

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

Sci Total Environ 2014 Aug 21;490:121-33. Epub 2014 May 21.

Department of Biochemistry and Molecular Biology, Agrifood Campus of International Excellence (ceiA3), University of Córdoba, Córdoba, Spain. Electronic address:

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.
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http://dx.doi.org/10.1016/j.scitotenv.2014.04.117DOI Listing
August 2014

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

Ecotoxicol Environ Saf 2014 Feb 20;100:15-26. Epub 2013 Dec 20.

Department of Biochemistry and Molecular Biology, University of Córdoba, Severo Ochoa Building, Rabanales Campus, Highway A4 Km 396a, 14071-Córdoba, Spain.

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.
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http://dx.doi.org/10.1016/j.ecoenv.2013.11.025DOI Listing
February 2014

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

Ecotoxicol Environ Saf 2013 Feb 11;88:169-77. Epub 2012 Dec 11.

Laboratory of Biochemical and Environmental Toxicology, Higher Institute of Agriculture, Chott-Mariem, 4042-Sousse, Tunisia.

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.
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http://dx.doi.org/10.1016/j.ecoenv.2012.11.013DOI Listing
February 2013

Omic approaches in environmental issues.

J Toxicol Environ Health A 2011 ;74(15-16):1001-19

Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Córdoba, Spain.

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.
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http://dx.doi.org/10.1080/15287394.2011.582259DOI Listing
September 2011

New metallothionein assay in Scrobicularia plana: heating effect and correlation with other biomarkers.

Environ Pollut 2008 Dec 10;156(3):1340-7. Epub 2008 Apr 10.

Department of Biochemistry and Molecular Biology, University of Córdoba, Córdoba, Spain.

Metallothionein (MT) and other biomarker levels were measured in Scrobicularia plana clams to assess pollution of the Guadalquivir Estuary possibly affected by metals released from Aznalcóllar pyrite mine in 1998. After optimizing reagent concentrations for monobromobimane derivatization, MT levels were quantified by reversed-phase high-performance liquid chromatography coupled to fluorescence detection (RP-HPLC-FD) in heated or unheated digestive gland extracts and compared to those obtained by differential pulse polarography (DPP). MT content assayed by RP-HPLC-FD in unheated samples was higher than that obtained by DPP and correlated better with metals and anti-oxidant activities. MT assay by RP-HPLC-FD in unheated extracts would be preferable for assessing metal pollution, due to its greater sensitivity and specificity. In addition to MT induction, glyoxalase II inhibition was well correlated with metal contents. Our results suggest that metals at the estuary do not originate from Aznalcóllar spill, but from those carried along by the river and deposited at its concave bank.
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http://dx.doi.org/10.1016/j.envpol.2008.02.022DOI Listing
December 2008

Metallothionein induction by Cu, Cd and Hg in Dicentrarchus labrax liver: assessment by RP-HPLC with fluorescence detection and spectrophotometry.

Mar Environ Res 2008 May 20;65(4):358-63. Epub 2008 Jan 20.

Laboratoire de Biochimie et de Toxicologie Environmentale, Institut Supérieur d'Agronomie de Chott-Mariem, 4042 Sousse, Tunisia.

Metallothionein was quantified in sea bass Dicentrarchus labrax intraperitoneally (i.p.) injected with different Cu, Cd and Hg doses (50-250 microg kg(-1) wet wt) after 48 h exposure. A distinct peak with 16.8 min retention time was obtained by reversed-phase high performance liquid chromatography coupled to fluorescence detection (RP-HPLC-FD) with the three metals. Total metallothionein levels assayed in unheated liver extracts by RP-HPLC-FD were significantly higher (1.3-1.95-fold) than those obtained by the well-established spectrophotometric method. In the RP-HPLC-FD method, metallothionein increased linearly with Cu and Hg doses, being saturated beyond 100 mug kg(-1) Cd. Maximum induction was obtained at 100 microg kg(-1) Cd (5.3-fold), and 250 microg kg(-1) Cu or Hg (8- and 5.1-fold, respectively). At low doses no metallothionein induction was shown by the less sensitive spectrophotometric assay.
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http://dx.doi.org/10.1016/j.marenvres.2008.01.002DOI Listing
May 2008

Utility of proteomics to assess pollutant response of clams from the Doñana bank of Guadalquivir Estuary (SW Spain).

Proteomics 2006 Apr;6 Suppl 1:S245-55

Department Biochemistry & Molecular Biology, University of Córdoba, Córdoba, Spain.

The utility of proteomics to assess pollutant response of Scrobicularia plana clams from three sites of Guadalquivir Estuary at the southern end of the National Park of Doñana (SW Spain) has been studied. Protein expression profiles were analyzed by 2-DE in soluble fractions of S. plana gills. Nearly 2000 well-resolved spots were detected in silver-stained gels, with focused areas in the 4-6.5 pH range. Different protein expression signatures were found at each site, with the highest number of more intense spots in animals with the highest metal content. Nineteen more intense protein spots were analyzed out by nanospray-ion trap tandem mass spectrometry, de novo sequencing and a bioinformatics search for their possible identification. While sequence tags of 16 more intense protein spots were obtained, including several proteins induced by pollutant exposure of model organisms, only 2 proteins were unambiguously identified: hypoxanthine-guanine phosphoribosyltransferase (HPRT) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Both enzymes were significantly higher in animals with the highest metal contents. Thus, we propose these two proteins, HPRT and G3PDH, as novel pollution biomarkers.
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http://dx.doi.org/10.1002/pmic.200500444DOI Listing
April 2006

Metallothionein quantification in clams by reversed-phase high-performance liquid chromatography coupled to fluorescence detection after monobromobimane derivatization.

J Chromatogr A 2006 Feb 15;1107(1-2):52-8. Epub 2005 Dec 15.

Department of Biochemistry and Molecular Biology, University of Córdoba, Severo Ochoa Building, 2nd floor, Campus de Rabanales, Highway A-4, Km 396a, 14071 Córdoba, Spain.

In this paper, we describe a highly specific, sensitive and reliable method for total metallothionein (MT) quantification by RP-HPLC coupled to fluorescence detection following reaction with monobromobimane of thiols from metal-depleted MT after heat-denaturation of extracts in the presence of sodium dodecyl sulphate (SDS). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the identity of the peak resolved (t(R)=16.44) with MT: a highly fluorescent protein of approximately 8.3 kDa, in agreement with the high thiol content and low MT size. Other heat-resistant and Cys-containing proteins of 35 kDa were efficiently separated. The new method was successfully used to quantify MT content in digestive gland of clams from southern Spanish coastal sites with different metal levels, and is proposed as a tool for using MTs as biomarker in monitoring programmes.
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http://dx.doi.org/10.1016/j.chroma.2005.11.057DOI Listing
February 2006

Oxidative stress biomarkers in bivalves transplanted to the Guadalquivir estuary after Aznalcóllar spill.

Environ Toxicol Chem 2003 Jan;22(1):92-100

Department of Biochemistry and Molecular Biology, University of Córdoba, Campus of Rabanales, Severo Ochoa Building, 14071 Córdoba, Spain.

Biological effects of metals were studied in clams (Scrobicularia plana) transplanted to Guadalquivir estuary (Spain) at several times after the spill of toxic metals from Aznalcóllar pyrite mine (southwest Spain) (April 1998) using biochemical biomarkers responsive to reactive oxygen species. Significant As, Cd, Fe, Mn, Ni, and Pb uptake was found in clams living for seven months at the estuary (from July 1999). Increased activity of antioxidant (catalase, glucose-6-phosphate, and 6-phosphogluconate dehydrogenase) and glutathione-related (glutathione reductase and glyoxalase I and II) enzymes was also found after short exposures; the levels of malondialdehyde and metallothionein increased also, particularly with long exposures. Clams living four weeks at the estuary (from March 2000) but not at a reference site also accumulated metals. The higher malondialdehyde and lower reduced-glutathione levels and the more oxidized glutathione status confirmed the oxidative stress of clams living at the estuary, while no marked increase of antioxidant activities was found this time. Lower metal availability along the second transplant could explain the limited responses in this shorter experiment. Although the status of Guadalquivir estuary has recovered since Aznalcóllar spill, continuous monitoring is needed to confirm its progress and to be alert to possible deterioration after heavy rains.
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January 2003

Biochemical biomarkers of pollution in the clam Chamaelea gallina from south-Spanish littoral.

Environ Toxicol Chem 2002 Mar;21(3):542-9

Department of Biochemistry and Molecular Biology, University of Córdoba, Spain.

Fourteen biochemical pollution biomarkers were analyzed in the clam Chamaelea gallina sampled at seven South-Spanish littoral sites at different times in 1999. They included enzymes that regenerate reduced nicotinamide adenine dinucleotide phosphate (NADPH) or maintain the cytosol-reduced (glucose-6-P- and isocitrate dehydrogenases, glutathione reductase), that decompose reactive oxygen species (catalase, superoxide dismutase, glutathione peroxidase), or detoxify glutathione-reactive electrophiles (soluble and microsomal glutathione transferases, glyoxalases II and I). The levels of reduced glutathione, malondialdehyde, and metallothionein and the glutathione redox status were also determined. Clams from Huelva sites with higher contaminant loads and metallothionein levels showed higher antioxidant and glutathione-related activities and a decreased glutathione level, lower malondialdehyde content, and a less oxidized glutathione status. This suggests that clams chronically exposed to contaminants released by Huelva and Guadalquivir Estuaries are better protected from oxidative stress than reference animals. Most biomarkers showed six-month cyclicity with up to threefold amplitudes, further supporting the inverse relationship between antioxidant defenses and oxidative damages. Thus the lower antioxidant defenses in June fit to maximum oxidative damages, whereas the high antioxidant defenses found in March and September through October agree with lower lipid damages and less oxidized glutathione status. The effect of environmental or endogenous factors on this cyclic response is discussed.
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March 2002
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