Estudio metalo-metabolómico de bioindicadores de áreas costeras y estuarinas del suroeste de la Península Ibérica

Los invertebrados acuáticos son muy buenos bioindicadores que resultan particularmente útiles para evaluar la calidad de los ecosistemas acuáticos; las especies indicadoras deben reunir una serie de características como poseer una amplia distribución y una gran abundancia; además presentar una relación entre el contenido de contaminante en sus tejidos y la concentración de éste en el ambiente. Por otro lado, son adaptables a estudios de laboratorio. Estos organismos han sido empleados en numerosos trabajos de investigación, en relación con la capacidad de acumulación de contaminantes y para determinar los cambios producidos en sus rutas metabólicas debido a exposición a xenobióticos, en la línea de nuestro estudio, metales y fármacos; lo que ha determinado su selección para el desarrollo de esta Tesis Doctoral. La problemática de la contaminación ambiental en ecosistemas acuáticos debido a actividades antropogénicas ha provocado que en los últimos años exista un especial interés en el estudio de biomarcadores, que son parámetros biológicos medidos en los organismos bioindicadores, que indican, al alterarse sus niveles, la exposición a estos contaminantes, sus efectos biológicos o los riesgos de su presencia en los ecosistemas. El análisis de biomarcadores convencionales es un instrumento clave para detectar el impacto de la contaminación sobre la salud de los ecosistemas, aunque se requiere un profundo conocimiento sobre los mecanismos toxicológicos en los que se ven envueltas las sustancias. Por esta razón las ómicas son ahora la nueva herramienta para evaluar la respuesta biológica global a la contaminación, en concreto la presencia y modificaciones que sufren las metalobiomoléculas y los metabolitos, utilizados como biomarcadores de estrés ambiental. Las nuevas tecnologías analíticas para el estudio de la respuesta biológica, en particular el empleo de procedimientos de especiación basados en acoplamientos HPLC-ICP-MS y las técnicas de extracción de especies químicas rápidas y eficaces junto a técnicas de masas en tandem (Q-TOF), están abriendo nuevas posibilidades en el estudio y caracterización de metalobiomoléculas y metabolitos. Asimismo, el desarrollo de técnicas metalómicas que utilizan cromatografía multidimensional (SEC-HPLC) y detección ICP-MS, y métodos metabolómicos mediante espectrometría de masas por infusión directa y análisis estadístico multivariante constituyen herramientas muy poderosas e innovadoras que pueden ser útiles en la biomonitorización de ecosistemas contaminados y en el estudio de la acción de sustancias contaminantes sobre organismos bioindicadores. El presente estudio se centra en la evaluación y seguimiento de la contaminación ambiental del suroeste de la Península Ibérica utilizando como bioindicadores tres invertebrados, los moluscos Scrobiculaña plana y Ruditapes philippinarum y el crustáceo Procambarus clarkii, en los que se han estudiado la presencia y modificaciones que sufren biomarcadores de estrés ambiental (metalobiomoléculas y metabolitos). Asimismo se ha lleva a cabo experiencias de exposición a fármacos en ambientes controlados para evaluar cambios producidos en las rutas metabólicas. El estudio de la contaminación ambiental en el suroeste de la Península Ibérica (Algarve-Cádiz- Parque Nacional de Doñana y alrededores) se ha llevado a cabo mediante el análisis de biomarcadores relacionados con el estrés ambiental, concretamente metalobiomoléculas, utilizando invertebrados Scrobicularia plana y Procambarus clarkii capturados en distintas zonas de estudio. Para ello se ha desarrollado un procedimiento metalómico basado en el acoplamiento de la cromatografía de exclusión de tamaño con detección elemental ICP-MS, el cual se ha aplicado a diferentes órganos con objeto de identificar posibles cambios de expresión de metalobiomoléculas como consecuencia de la contaminación ambiental. Además, en el caso de Procambarus clarkii, en base a los resultados obtenidos, se han estudiado otros biomarcadores relacionados con la contaminación por metales (metalotioneínas y peroxidación lipídica) y se ha desarrollado un método analítico para especiación de arsénico mediante cromatografía iónica acoplada a ICP-MS para comparar zonas contaminadas y no contaminadas. Por otro lado, se ha desarrollado un procedimiento metabolómico, en el contexto de la metabolómica medioambiental, mediante la extracción rápida de metabolitos y detección por espectrometría de masas en tándem por infusión directa (ESI-QqTOF) con la el objetivo de encontrar biomarcadores metabolómicos relacionados con la respuesta al estrés ambiental en distintos órganos del cangrejo Procambarus clarkii. En este sentido, se ha desarrollado en la presente Tesis un estudio de exposición a diferentes fármacos (ibuprofeno, carbamazepina y diclofenaco) en condiciones de laboratorio, en el que se realiza este mismo procedimiento metabolómico para el estudio de la respuesta biológica del bioindicador bivalvo Ruditapes philippinarum, con objeto de identificar posibles cambios en rutas metabólicas como consecuencia de la exposición a estos contaminantes emergentes. Al tratarse de crustáceos y moluscos bivalvos, poseen un metabolismo más sencillo que permite realizar de forma simplificada ensayos de exposición a elementos tóxicos, extrapolables, posteriormente a organismos más complejos. En general, esto convierte a estos organismos en bioindicadores muy adecuados para estudios de toxicidad y contaminación ambiental, mediante la comparación de la respuesta biológica al estrés y los diferentes niveles de expresión de metalobiomoléculas y metabolitos relacionadas con exposición a elementos tóxicos.Aquatic invertebrates are very good biomarkers that are particularly useful for assessing the quality of aquatic ecosystems, these bioindicator species have to meet a number of features such as having a wide distribution and abundance, as well as to present a relationship between pollutant content in their tissues and its concentration in the environment. On the other hand, they have to be suitable for laboratory studies. These organisms have been used in numerous studies related to bioaccumulation of pollutants to determine changes in their metabolic pathways caused by xenobiotics exposure. In connection to this our study considers the toxicological mode of action of metals and drugs on theses bioindicators, which has been the more important topic of this doctoral Thesis. Environmental pollution in aquatic ecosystems due to human activities has led to the existence of a special interest in the study of biomarkers. However, the conventional analysis of biomarkers although provide information about the impact of pollution on the health of ecosystems, they do not allow to deep insight into the toxicological mechanisms that are involved in the pollutant action. Therefore, the omics are now the new tool to assess the overall biological response to pollution, in particular the presence and modifications suffered by metal-biomolecules and metabolites used as biomarkers of environmental stress. The new analytical coupling based on HPLC-ICP-MS to characterized metal-containing biomolecules (metallomics) together to rapid an effective extraction technique of biological fluids and new high resolution mass spectrometric techniques for species identification are opening new possibilities in environmental studies. Besides the combination of omics, such as metallomics and metabolomics in the present study are opening a new frontier in biomonitoring the action of pollutants in the ecosystems. This study focuses on the evaluation and monitoring of environmental pollution in the southwest of the Iberian Peninsula using as biomarkers three invertebrates, the molluscs Scrobicularia plana and Ruditapes philippinarum molluscs and the crustacean Procambarus clarkii, studying the presence and modifications suffered by these biomarkers under environmental stress (considering the changes in metal-biomolecules and metabolites). In addition, controlled exposure experiments involving severl drugs have been performed and changes in metabolic pathways considered. The study was centered on the environmental pollution in the southwest of the Iberian Peninsula (Algarve-Cádiz-Donana National Park and surroundings) by analyzing metal-biomolecules related to environmental stress. For this purpose Scrobicularia plana and Procambarus clarkii collected in the different areas of study were studied. A metallomics workflow based on the use of size exclusion chromatography with elemental detection by ICP-MS was developed, which has been applied to different organs of the bioindicators in order to identify potential metal-containing biomolecules changes as consequence of environmental pollution. Furthermore, in the case of Procambarus clarkii, other biomarkers related to metal contamination (metallothioneins and lipid peroxidation) have been considered and an analytical approach for arsenic speciation using ion chromatography-ICP -MS was used to compare polluted and unpolluted areas. Furthermore, a metabolomic approach based on fast liquid-liquid extraction of metabolites followed by metabolites detection by direct infusion tandem mass spectrometry (ESI-QqTOF) has been performed in order to establish the metabolites related to the response of different organs from the crab Procambarus clarkii to environmental stress. In addition exposure experiments of bivalve Ruditapes phillipinarum to different drugs (ibuprofen, diclofenac and carbamazepine) with the purpose of identify possible changes in metabolic pathways under the action of these emerging contaminants. The use of crustaceans and bivalve molluscs, with a simpler metabolism allows easier interpretation of contaminant that can be extrapolated later to more complex organisms

Assessment of pollution issues in Doñana National Park by the biological response of P. clarkii

Póster presentado en el 17th Symposium on Pollution Effects on Marine Organisms (PRIMO17) celebrado en Faro (Portugal) del 5 al 8 de mayo de 2013.The present study considers potential environmental problems in Doñana National Park using bioindicators that reflect the biological response to the presence of contaminants. Invertebrates have been frequently proposed for this aim [1]. The red swamp crayf ish Procambarus clarkii lives in a wide range of environmental conditions, even in highly polluted waters and it was introduced into the marshes of the lower Guadalquivir River (SW, Spain) in 1974, colonizing quickly all the aquatic systems around in the a bsence of natural predators, reaching the Doñana National Park. Therefore, the crab can be used as bioindicator to measure the effects of pollutants in this important ecological reserve. In this context, the omics techniques are suitable tools to evaluate the global biological response to contamination. Metabolomics studies the complete set of low molecular weight compounds in living organisms, which may be considered as the final product of interactions between gene and protein expression and the cellular environment. Thus, metabolomics aims to determining a sample’s profile at a specified time under specific environmental conditions for a better compression of biological systems [2]. Metallomics is focused on the study of metal -­‐ species related to cells, or gans and tissues, using multidimensional analytical approaches in which inductively coupled plasma mass spectrometry plays a fundamental role. Due to the importance of metals in biological systems, metallomics provides complementary information about the b iochemical changes associated with pollution [3]. In this study, crab specimens were sampled in different areas of Doñana National Park, for the dissection of digestive glands, gills and abdominal muscles, extraction and analysis. To assess heavy metal co ntamination, total metal content was performed, since it is one of the most important pollutants in marine origin organisms in this area. In addition, to evaluate the effects of these pollutants in the metabolism of the crab a supplementary analysis of met abolites was performed. Metabolomic analysis was carried out by direct infusion into a high resolution mass spectrometer with electrospray source. Finally, metabolomic profiles of different sampling points were compared using partial least squares discrimi nant analysis (PLS -­‐ DA), in order to obtain a relationship between pollution levels and the metabolic response of thePeer Reviewe

Metabolomic and Metallomic Analysis of crab Procambarus clarkii to assess the pollution in Doñana National Park

Trabajo presentado en el 7th International Franco-Spanish Workshop on Bioinorganic Analytical Chemistry, celebrado en Gijón del 1 al 3 de julio de 2012.Peer Reviewe

Use of a metallomics approach to assess the biological response of the clam Scrobiculariaplana in several environmental areas of South West Iberian Peninsula

Trabajo presentado en el XVI Seminario Ibérico de Química Marina, celebrado en Cádiz del 24 al 26 de enero de 2012.The assessment of environmental issues is generally performed by the use of bioindicators that provide the biological response to contaminants. Mammals and invertebrates have been frequently proposed for this aim, and the study of conventional biomarkers supplied a deeper knowledge about the toxicological mechanisms. The great number of substances and metabolites involved in these complex mechanisms has driven the use of omics, which are the new tool to evaluate the global biological response to contamination. Metallomics is the most recent omic focused in the study of metal-binding biomolecules using instrumental couplings, generally based on the use of SEC-ICP-MS and organic mass spectrometry. In the present study we apply this approach to compare the biological response of Scrobicularia plana collected from polluted and non-polluted areas from the south-west of Spain and Portugal. Digestive gland of the clam was extracted to obtain the cytosolic fraction and the chromatographic profiles of the extracts reveal the presence of low molecular mass Cu and Zn molecules about 300 Da that can be related to the Cu- and Zn-glutathiones and As metabolites such as arsenobetaine and arsenocholine. The up-regulation of Cu-biomolecules is higher in Cadiz area against Portugal zone while Zn-biomolecules present an opposite behaviour. In several samples has been observed the presence of a remarkable Cu-peak with molecular mass of about 67 kDa. Metal biomolecules up- or down-regulated under the action of contaminants are being identified.Peer Reviewe

Environmental metallomics and metabolomics in Marine coastal research

Trabajo presentado en el seminario Helmholtz Zentrum Geesthacth, celebrado en Hamburgo el 20 de septiembre de 2012.Projects: CTM 2006-08960-C02-01, CTM 2009-12858-C02-01, P08-RNM-00523, P08-FQM-03554 y P08-FQM-04659.Peer Reviewe

Metabolic signatures associated with environmental pollution by metals in Doñana National Park using P. clarkii as bioindicator

Bioindicators can reflect the effects of pollutants on their metabolism, being widely used to assess environmental stress. In this sense, the crab Procambarus clarkii has been previously proposed to monitor the contamination in Doñana National Park (southwest Spain) using conventional biomarkers. In this work, a metabolomic approach based on direct infusion mass spectrometry, which allows an easy and quick study of a large number of metabolites in a single run, was used for pollution assessment of this area, considering the biological response of this organism to contamination. In addition, metal accumulation in crab tissues was determined. Thus, the integrated analysis of metabolomic and metallomic data enabled the study of metabolic response of the organism against pollution. Several metabolites were discovered as potential biomarkers of pollution, such as decreased levels of carnosine, alanine, niacinamide, acetoacetate, pantothenic acid, ascorbate, glucose-6-phosphate, arginine, glucose, lactate, phospholipids, and tryglicerides, as well as elevated levels of acetyl carnitine, phosphocholine, choline, and uric acid. In this way, metal-induced toxicity could be related to metabolic impairments, principally oxidative stress, metabolic dysfunction, and dyslipidemia.CTM2012-38720-C03-01, P008-FQM-3554, P009-FQM-4659info:eu-repo/semantics/publishedVersio