Estudio metalómico/metabolómico comparativo del ratón de laboratorio Mus musculus y del ratón de vida libre Mus spretus para evaluar el estrés ambiental de ecosistemas terrestres

Resum

Diagnosis of environmental stress requires the combination of chemical analysis of contaminants with biological responses. This response can be estimated by biological exposure experiments, which integrate synergism/antagonism process among contaminants and interactions with other compounds in the ecosystems. Throughout the period of this PhD thesis, we used the free-living mouse Mus spretus as bioindicator of terrestrial pollution caused by toxic metals in an ecological environment like Doñana National Park. These studies have focused on the use of omics technologies in this bioindicator in the reference area threatened by pollution from toxic metals. Studies have deepened in the knowledge related to the trafficking of metals, including interactions and homeostasis in these free-living organisms. Doñana National Park, despite it has been declared a Biosphere Reserve, especially for the presence of migratory birds, is threatened by various natural and anthropogenic activities taking place in the surroundings, such as industrial, mining and agricultural activities. For this purpose, we have developed and applied metallomic methodologies in order to study metal contamination in this area, which have been centered in the use of a detector inductively-coupled plasma mass spectrometry (ICP-MS). Furthermore, the application of metabolomic methodologies based on direct infusion into a high resolution mass spectrometer (DI-ESI-QTOF-MS) and gas chromatography with mass detector (GC- MS), followed by discriminant analysis of the results (PLS-DA) has been used to compare the biological response of Mus spretus from different areas of the Doñana National Park and surrounding areas. On the other hand, parallel studies have been developed throughout this thesis based on the evaluation of the toxicological effects of metals, such as As, Cd, Hg on the laboratory mouse Mus musculus under controlled exposure experiments. Furthermore, we have developed a metallomic approach based on the use of size exclusion chromatography (SEC) coupled to ICP-MS, in order to get a better understanding of functional changes suffered by metallo-biomolecules, the detoxification processes and regulation of metals in mice (Mus musculus) under these exposure experiences that can be compared to similar situations occurring in the environment. In this sense, we have conducted experiments exposure to undertake the study of the potential biological interactions Mus musculus, firstly to evaluate the protective effect of selenium against the toxicity of mercury and cadmium, and on the other side to deep insight into the antagonistic or synergistic effects when arsenic and cadmium are administered simultaneously. Furthermore, these studies have been complemented with innovative techniques based on the use of isotope dilution (IDA) to quantify selenium and copper associated with the cytosolic proteins and other molecules in the blood stream, using an ICP-MS system and multielement detector. This methodology has been developed and validated by multidimensional chromatography methods based on 2D/SEC-AF-HPLC-SUID-ORS-ICP-MS, 3D/SEC-AF-AEC-HPLC-SUID-ORS-ICP-MS for absolute quantification selenium species and 2D/SEC-AEC- HPLC-SUID-ORS-ICP-MS for speciation of metalloproteins containing Cu. In addition, an isotopic pattern deconvolution methos has been used to study the transport and fate of 77Se (selenite) in mice under cadmium exposure and the effect of this toxic element in plasma selenoproteins. In summary, environmental studies aimed at identifying biological responses to environmental pollutants present serious difficulties in their application, as they must deal with the complexity of ecosystems in which many xenobiotics affecting organisms coexist, but also interact with each other and they behave differently to the various biological filters. As a result, it is difficult to pinpoint the cause-effect by the multiplicity of possible answers. Despite these drawbacks, this type of study is very necessary as integrate all variables in a particular ecosystem and its interaction with the organisms studied. Therefore, to establish secure mechanisms of action of contaminants exposure studies in the laboratory are mandatory to allow adequate control of all the variables related to these compounds. Finally, the conclusions obtained can be extrapolated and validated in free-living animals in suitable sampling campaigns in the field. All these aspects considered in relation to the study of pollution and its effects, are the purpose of this thesis, to which the objectives proposed below are supported.