Uso combinado de metodologías -ómicas en organismos modelo para el estudio de la contaminación ambiental

Abstract

The increasing levels of a growing variety of chemical pollutants in the environment represent a health risk for the exposed organisms as well as for the ecosystems in which they live. Traditionally, the environmental monitoring studies of the ecosystems have been carried out by chemical analysis of conventional pollutants such as toxic trace elements (i.e. Cd, As, Pb, Hg.), organic contaminants (i.e. pesticides, PCBs, PAHs), emerging pollutants (i.e. endocrine disruptors, pharmaceuticals, health care products and detergents) and, more recently, nanoparticles. However, the biological response of an organism against pollutants can not be evaluated with these traditional chemical analyses. For all these reasons, different methodologies have been developed in recent years that allow evaluating the biological response of certain model organisms used as bioindicators against environmental contamination, since they reflect the effect on cellular metabolism and global homeostasis. Among these methodologies we can be highlighted the use of biomarkers, which can be defined as biochemical parameters measured in exposed organisms (bioindicators) that allow identifying, due to their altered levels, the presence of external risk factors, such as the presence of toxic substances. For this purpose free-living organisms are used, which integrate the synergism/antagonism between pollutants and the interaction with other substances present in the ecosystem, alternatively laboratory exposure experiments can be applied, which integrates the effect of one or more selected contaminants. In this Doctoral Thesis, different -omics methodologies have been applied in the search of biomarkers of environmental stress, both in organisms from estuarine ecosystems, such as the bivalve Scrobicularia plana; or terrestrial bioindicators, as the free-living mice Mus Spretus and the laboratory mice Mus Musculus. Among the exposure experiments performed under controlled laboratory conditions can be cited those with Mus musculus and Mus spretus mice with some toxics, such as Cd and DDE, administered together with Se to evaluate its possible antagonistic effect. In addition, exposure experiments to metal cocktails, such as As, Cd and Hg, have been carried out in Mus musculus mice, in order to assess the toxic effects of these metals together and the protective effect of selenium. Alterations in the levels of arsenic biomolecules have also been evaluated after the exposure of inorganic As in the bivalve Scrobicularia plana; and finally, free living bivalves were studied in the estuary of Guadalquivir river to evaluate the environmental stress in this area. Metallomic, proteomic and metabolomic approaches are the analytical techniques used in these studies to look for biomarkers of environmental pollution using the above mentioned bioindicators. All analytical methods developed in this Thesis are based on the use of mass spectrometry as a detection technique, due to its wide range of applicability, sensitivity and specificity. The metallomic studies have been carried out using liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) to determine altered profiles of some biomolecules after exposure to pollutants. On the other hand, nano liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS / MS), has become an essential tool in proteomics due to its greater sensitivity with respect to conventional LC-MS/MS as it allows the analysis of mixtures of peptides in small amounts of samples. For this reason, it was used to evaluate changes in the proteome of bioindicators after exposure to several contaminants. Finally, in order to get a comprehensive analysis of biomarkers of environmental pollution, metabolomic analysis was carried out. To this end, complementary metabolomic techniques were used, including screening procedures based on direct infusion mass spectrometry (DI-ESI-MS) and gas chromatography mass spectrometry (GC-MS), for the determination of low molecular mass metabolites. Direct infusion analysis showed a great potential for metabolomic studies, due to its reduced analysis time and its instrumental simplicity, but the combination with GC-MS allowed the identification of new biomarkers of environmental pollution. The analytical methodologies applied in this PhD Thesis allow us to deep insight into the biological response of these model organisms against contamination, that is, to understand the changes in the expression of certain biomolecules as a consequence of the exposure to certain pollutants. In addition, the studies conducted clarify several biological processes such as metals traffic, interactions between them and homeostasis, which take place in these organisms.