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Cover -- Half Title -- Title Page -- Copyright Page -- Preface -- Table of Contents -- Abbreviations -- 1: Element Speciation in Seawater: From Free Metal Determination to Proteomic Analysis -- 2: The Decisive Role of ICPMS and Related Hyphenated Techniques in Multielement and Speciation Analysis of Marine Biota -- 3: Mercury Speciation in Marine Biota and its Influence on Human Health: Metallomic and Metabolomic Approaches -- 4: Selenium Speciation in Marine Environment -- 5: Arsenic Occurrence in Marine Biota: The Analytical Approach -- 6: Biological Effects of Organotins in the Marine Environment -- 7: Functional Genomics Approaches in Marine Pollution and Aquaculture -- 8: New Trends in Aquatic Pollution Monitoring: From Conventional Biomarkers to Environmental Proteomics -- 9: Protein Expression Profiles in Marine Organisms Exposed to Nanoparticles -- 10: Effects of Climate Change on Marine Organisms: A Proteomic Approach -- 11: Environmental Metabolomics and Toxicometabolomics in Marine Pollution Assessment -- 12: Fate and Toxicity of Inorganic Engineered Nanomaterials in the Marine Environment: Analytical Techniques and Methods -- 13: Pharmaceuticals in the Marine Environment: Analytical Techniques and Applications -- 14: Biological Effects of Pharmaceuticals in Marine Environment -- Index

The present paper describes a method based on the extraction of analytes by multiple hollow fibre liquid-phase microextraction and detection by ion-trap mass spectrometry and electron capture detectors after gas chromatographic separation. The limits of detection are in the range of 0.13–0.67 μg kg−1, five orders of magnitude lower than those reached with the European Commission Official method of analysis, with three orders of magnitude of linear range (from the quantification limits to 400 μg kg−1 for all the analytes) and recoveries in fortified olive oils in the range of 78–104 %. The main advantages of the analytical method are the absence of sample carryover (due to the disposable nature of the membranes), high enrichment factors in the range of 79–488, high throughput and low cost. The repeatability of the analytical method ranged from 8 to 15 % for all the analytes, showing a good performance. ; This work was supported by the projects P08-FQM-3554 and P09-FQM-4659 from "Consejeria de Innovacion, Ciencia y Empresa" (Andalusian Government) and the project CTM2009-12858-C02-01 from the "Ministerio de Ciencia e Innovacion". J. Manso thanks the Consejeria de Innovacion Ciencia y Empresa for a predoctoral scholarship.

Background: The apolipoprotein E (APOE) gene exists in three isoforms in humans: APOE2, APOE3 and APOE4. APOE4 causes structural and functional alterations in normal brains, and is the strongest genetic risk factor of the sporadic form of Alzheimer's disease (LOAD). Research on APOE4 has mainly focused on the neuronal damage caused by defective cholesterol transport and exacerbated amyloid-β and Tau pathology. The impact of APOE4 on non-neuronal cell functions has been overlooked. Astrocytes, the main producers of ApoE in the healthy brain, are building blocks of neural circuits, and Ca2+ signaling is the basis of their excitability. Because APOE4 modifies membrane-lipid composition, and lipids regulate Ca2+ channels, we determined whether APOE4 dysregulates Ca2+signaling in astrocytes. Methods: Ca2+ signals were recorded in astrocytes in hippocampal slices from APOE3 and APOE4 gene targeted replacement male and female mice using Ca2+ imaging. Mechanistic analyses were performed in immortalized astrocytes. Ca2+ fluxes were examined with pharmacological tools and Ca2+ probes. APOE3 and APOE4 expression was manipulated with GFP-APOE vectors and APOE siRNA. Lipidomics of lysosomal and whole-membranes were also performed. Results: We found potentiation of ATP-elicited Ca2+responses in APOE4 versus APOE3 astrocytes in male, but not female, mice. The immortalized astrocytes modeled the male response, and showed that Ca2+ hyperactivity associated with APOE4 is caused by dysregulation of Ca2+ handling in lysosomal-enriched acidic stores, and is reversed by the expression of APOE3, but not of APOE4, pointing to loss of function due to APOE4 malfunction. Moreover, immortalized APOE4 astrocytes are refractory to control of Ca2+ fluxes by extracellular lipids, and present distinct lipid composition in lysosomal and plasma membranes. Conclusions: Immortalized APOE4 versus APOE3 astrocytes present: increased Ca2+ excitability due to lysosome dysregulation, altered membrane lipidomes and intracellular cholesterol distribution, and impaired modulation of Ca2+ responses upon changes in extracellular lipids. Ca2+ hyperactivity associated with APOE4 is found in astrocytes from male, but not female, targeted replacement mice. The study suggests that, independently of Aβ and Tau pathologies, altered astrocyte excitability might contribute to neural-circuit hyperactivity depending on APOE allele, sex and lipids, and supports lysosome-targeted therapies to rescue APOE4 phenotypes in LOAD. ; This research was mainly funded by grants TV3 – 20141430, TV3 – 20141432 and TV3 – 20141431 from La Marató de Televisió de Catalunya (TV3) to EG, AG and JV respectively, and grants 2107 SGR1780 from AGAUR (Generalitat de Catalunya) to RM, 2017 SGR547 from AGAUR (Generalitat de Catalunya) to EG, BFU2016 – 75107-P from Ministerio de Economia, Industria y Competividad (Spanish Government) to GP, BFU2015 – 68149-R from Ministerio de Ciencia e Innovación (Spanish Government) and co-financed by European Regional Development Fund to MDG and PI18/01557 from Instituto de Salud Carlos III (ISCiii, Spanish Government) co-financed by FEDER funds from European Union to AG. CG-A was awarded a PhD fellow- ship BES-2017-080303 from Ministerio de Economía, Industria y Competivi- dad (Spanish Government).

This paper comparatively shows the influence of four water treatment processes on the formation of trihalomethanes (THMs) in a water distribution system. The study was performed from February 2005 to January 2012 with analytical data of 600 samples taken in Aljaraque water treatment plant (WTP) and 16 locations along the water distribution system (WDS) in the region of Andévalo and the coast of Huelva (southwest Spain), a region with significant seasonal and population changes. The comparison of results in the four different processes studied indicated a clear link of the treatment process with the formation of THM along the WDS. The most effective treatment process is preozonation and activated carbon filtration (P3), which is also the most stable under summer temperatures. Experiments also show low levels of THMs with the conventional process of preoxidation with potassium permanganate (P4), delaying the chlorination to the end of the WTP; however, this simple and economical treatment process is less effective and less stable than P3. In this study, strong seasonal variations were obtained (increase of THM from winter to summer of 1.17 to 1.85 times) and a strong spatial variation (1.1 to 1.7 times from WTP to end points of WDS) which largely depends on the treatment process applied. There was also a strong correlation between THM levels and water temperature, contact time and pH. On the other hand, it was found that THM formation is not proportional to the applied chlorine dose in the treatment process, but there is a direct relationship with the accumulated dose of chlorine. Finally, predictive models based on multiple linear regressions are proposed for each treatment process. ; The authors wish to express their apprecia- tion to the plant managers GIAHSA (Gestión Integral del Agua de Huelva S.A.) and employees for their help and cooperation in facilitating the collection of water samples during this study. This work was supported by the project CTM2012-38720-C03-01 from the Spanish Ministry of Economy and Competitiveness (MINECO) and by projects P009-FQM-4659 and P12-FQM- 0442 from the Regional Ministry of Economy, Innovation, Sci- ence and Employment (Andalusian Government, Spain). ; The authors wish to express their appreciation to the plant managers GIAHSA (Gestion Integral del Agua de Huelva S.A.) and employees for their help and cooperation in facilitating the collection of water samples during this study. This work was supported by the project CTM2012-38720-C03-01 from the Spanish Ministry of Economy and Competitiveness (MINECO) and by projects P009-FQM-4659 and P12-FQM-0442 from the Regional Ministry of Economy, Innovation, Science and Employment (Andalusian Government, Spain).

Trihalomethanes (THMs) are regulated disinfection by-products (DBPs) most commonly analyzed in quality control water supply due to their harmful effects on health. However, few data exist about the content of emerging iodo-trihalomethanes (I-THMs) which are present in drinking water at very low concentrations (in the order of ng L−1). For this reason a two-phase hollow fiber liquid phase microextraction method for the simultaneous determination of four regulated trihalomethanes and six emerging iodo-trihalomethanes using GC-μECD and GC–MS with detection limits in the range of few ng L−1 has been developed. A central composite design was used to optimize conditions for simultaneous extraction. The best extraction recovery was obtained with 19.2 min at 27.1 °C and 900 rpm, without salt addition, using a supported hollow fiber membrane of 10.5 cm (0.6 mm id) and 1-octanol as acceptor phase. The limits of detection for the regulated THMs and I-THMs were 3–44 ng L−1 and 1–3 ng L−1, respectively. The calibration curves showed good linearity (R2 > 0.995) and good repeatibility (3–22%). The relative recoveries in water were between 96.5% and 105.2%. The method was applied for the simultaneous determination of trihalomethanes in supply water samples from seven water distribution systems (WDS) in the Huelva area, located at the southwest Spain, which use different water-treatment processes. The highest concentrations of I-THMs, particularly CHBrClI and CHCl2I, were detected in water treated with advanced treatment process using pre-ozonation, however these compounds were not detected or decreased along distribution system. In the samples of treated water with conventional treatment, using pre-oxidation by permanganate and distribution network, CHCl2I, CHBrClI, CHClI2, CHBrI2 and CHI3 were detected at very low concentrations (1–18 ng L−1). Finally, in water samples from underground origin without oxidation treatment, in which only disinfection with sodium hypochlorite was applied, I-THMs were not detected. ; The authors wish to express their appreciation to the plant managers GIAHSA and employees for their help and cooperation in facilitating the collection of water samples during this study. This work was supported by the project CTM2012-38720-C03- 01 from the Spanish Ministry of Economy and Competitiveness (MINECO), and by project P012-FQM-0442 from the Regional Min- istry of Economy, Innovation, Science and Employment (Andalusian Government, Spain) ; The authors wish to express their appreciation to the plant managers GIAHSA and employees for their help and cooperation in facilitating the collection of water samples during this study. This work was supported by the project CTM2012-38720-C03-01 from the Spanish Ministry of Economy and Competitiveness (MINECO), and by project P012-FQM-0442 from the Regional Ministry of Economy, Innovation, Science and Employment (Andalusian Government, Spain).

In the last years, the development of new methods for analyzing accurate and precise individual metalloproteins is of increasing importance, since numerous metalloproteins are excellent biomarkers of oxidative stress and diseases. In that way, methods based on the use of post column isotopic dilution analysis (IDA) or enriched protein standards are required to obtain a sufficient degree of accuracy, precision and high limits of detection. This paper reports the identification and absolute quantification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in cytosol and mitochondria from mice hepatic cells using a innovative column switching analytical approach. The method consisted of orthogonal chromatographic systems coupled to inductively coupling plasma-mass spectrometry equipped with a octopole reaction systems (ICP-ORS-MS) and UV detectors: size exclusion fractionation (SEC) of the cytosolic and mitochondrial extracts followed by online anion exchange chromatographic (AEC) separation of Cu/Zn containing species. After purification, Cu,Zn-SOD was identified after tryptic digestion by molecular mass spectrometry (MS). The MS/MS spectrum of a doubly charged peptide was used to obtain the sequence of the protein using the MASCOT searching engine. This optimized methodology reduces the time of analysis and avoids the use of sample preconcentration and clean-up procedures, such as cut-off centrifuged filters, solid phase extraction (SPE), precipitation procedures, off-line fractions insolates, etc. In this sense, the method is robust, reliable and fast with typical chromatographic run time less than 20 min. Precision in terms of relative standard deviation (n = 5) is of 3-5% and detection limits is 0.21 ngCug(-1). The application of the methodology to hepatic cells from mice exposed to inorganic mercury reveals decreased levels of Cu,Zn-SOD in cytosolic and mitochondrial extracts, as a consequence of the oxidative stress caused by this toxic metal. Additionally, the quantification of mitochondrial Cu,Zn-SOD in hepatic cells from Mus musculus has been carried out for the first time. ; This work was supported by the project CTM2012-38720-C03-01 from the Spanish Ministry of Economy and Competitiveness, and P12-FQM-0442 from the Regional Ministry of Economy, Innovation, Science and Employment (Andalusian Government, Spain). Miguel Ángel García Sevillano thanks Ministerio de Educación, Cultura y Deporte for a predoctoral scholarship. We also thank to the group of Prof. Sanz-Medel at the University of Oviedo, especially to Dr. M.L. Fernández-Sánchez and Dr. Nuevo-Ordóñez, for introducing and training in copper isotope dilution analysis.

Lung cancer (LC) is one of the most common causes of cancer-related deaths in the world and it is well known that trace elements play important roles in the carcinogenic process activating and inhibiting enzymatic reactions and metalloproteins, in which they usually participate as cofactors. A cross-sectional study was conducted on 48 lung cancer patients and 39 controls (56 men and 31 women), aged 44-76 years between March 2011 and June 2012. Eleven elements have been included in the study: V, Cr, Mn, Fe, Co, Cu, Zn, Se, Mo, Cd, and Pb, some of them considered toxic (V, Cd, Cr and Pb), while others are essential (Co, Mo, Se, Fe and Zn), and they have been analyzed by ICP-QQQ-MS in serum, urine and for the first time in bronchoalveolar lavage fluid (BALF). In order to understand the involvement of metals in this process, an analytical metallomic approach based on non-denaturing precipitation of proteins (NDPP) has been optimized for the fractionation of high molecular mass (HMM) and low molecular mass (LMM) metal species, in order to distinguish between metal species that affect the biological activity and toxicological potential of the elements. In this work, the NDPP followed by the analysis of metals by ICP-QQQ-MS has been applied for the first time to serum, urine and BALF samples from lung cancer patients and controls in order to get metal-size molecule profiles (MSMP), which can be used as metal-based biomarkers of altered metabolic processes such as oxidative stress and homeostasis. In this sense, we have demonstrated that several metals are good biomarkers when they are related to labile complexes, complexed with low molecular mass ligands, or in the form of metalloproteins (i.e. V and Cr in HMM and Cu in LMM), which has been described for the first time. On the other hand, metal dyshomeostasis biomarkers are proposed using metal ratios and correlations. Finally, the ratios between elements were shown to be important biomarkers for lung cancer in serum (V/Mn, V/Pb, V/Zn, Cr/Pb), urine (Cr/Cd, Mn/Cd, V/Cd, Co/Cd, Cd/Pb) and BALF (V/Cu), which reflects the dyshomeostasis of metals in lung cancer. In this sense, several metals are correlated to others suggesting also the existence of an interconnected homeostasis in lung cancer. ; The authors thank the Spanish Ministry of Economy and Competitiveness (CTM2015-67902-C2-1-P) and Regional Ministry of Economy, Innovation, Science and Employment (Andalusian Government. P12-FQM-0442). B. Callejon-Leblic thanks the Ministerio de Educacion for a predoctoral scholarship FPU13/03615. Finally, the authors are grateful to FEDER (European Community) (UNHU13-1E-1611 and UNHU15-CE-3140).

The present paper reports for the first time the bioaccessibility of arsenic species in Anemonia sulcata and its metabolization in the human body after the ingestion of this seafood, a delicacy highly appreciated in the south of Spain. Speciation in tissue extracts and human fluids was carried out by anion/cation exchange chromatography with inductively coupled plasma mass spectrometry ((IC)-ICP-MS). Results obtained show that about 54% of the arsenic present in the anemones is lost after cooking, around 95% of total arsenic in cooked anemones is bioaccessible and 85% of the amount ingested is eliminated from the body by urine in 90 h. The relative abundance of As in urine varies along the time, but after the first 10 h follows the order arsenobetaine (AB) ˜ DMAV (dimethylarsinate) > arsenocholine (AsC) > tetra-methyl-arsonium (TETRA) > trimethyl-arsine oxide (TMAO) > MAV (monomethylarsonate) > iAsV > dimethylarsenoethanol (DMAE), while the major species in cooked anemones is AB followed by DMAV > dimethylmonothioarsinic acid (DMASV) > AsC > TETRA > TMAO > iAsV > glycerylphosphorylarsenocholine(GPAsC) > MAV > DMA. After 30 min from the ingestion, DMAV is the most abundant in human serum, followed by AB, MAV and AsC. Finally, the use of organic mass spectrometry allowed the standardless identification of AsC, GPAsC, DMASV and DMAE. ; The authors thank the Spanish Ministry of Science and Innovation project CTM2009-12858-C02-01 and the projects P08-FQM-03554 and P09-FQM-4659 from Regional Ministry of Economy, Innovation, Science and Employment (Andalusian Government) for funding. The authors also thank Dr. Vicenta Devesa and Dinoraz Velez (Instituto de Agroquimica y Tecnologia de Alimentos (IATA-CSIC), Valencia, Spain) and Dr. Riansares Munoz (Universidad Complutense de Madrid) for the kind donation of TMAO, TETRA and DMAS. M.A. Garcia Sevillano thanks the Ministerio de Educacion for a PhD scholarship.

Antagonistic interactions between mercury (Hg) and selenium (Se), were evaluated in mouse (Mus musculus), as a mammalian model, in a series of controlled exposure experiments. The beneficial effect of Se against Hg toxicity involves a variety of biochemical and toxicological processes that have not been clarified yet. For this purpose, a metallomic workflow based on the use of size-exclusion chromatography (SEC) with inductively coupled plasma mass spectrometry (ICP-MS) detection was complemented with the speciation of selenoproteins and low molecular mass selenium species in serum and liver cytosolic extracts using a multidimensional approach based on SEC-AF-HPLC-ICPMS, using species-unspecific isotope dilution (SUID)-ICP-MS for selenium quantification. The results showed potential interactions between Hg/Se in organs and serum related to accumulation and detoxification processes, in addition to the effects of mercury on selenoproteins in hepatic cytosolic extracts and bloodstream when both elements are administrated at the same time. These results provide information about elements distribution, interactions and homeostasis and reveal the potential of metallomic approaches in exposure experiments. ; This work was supported by the project CTM2012-38720-C03-01 from the Spanish Ministry of Economy and Competitiveness, and by projects P08-FQM-3554 and P09-FQM-4659 from the Regional Ministry of Economy, Innovation, Science and Employment (Andalusian Government, Spain). M.A. Garcia Sevillano thanks the Spanish Ministry of Education for a PhD scholarship. G. Rodriguez-Moro thanks the Ministry of Innovation and Science for a Ph.D. grant.

Selenium is a well-known essential element with important roles in human reproductive health mainly due to its antioxidant character. This study aimed to investigate the potential role of selenoproteins on gut microbiota and male reproductive health. A new assay for the absolute quantification of selenoproteins in testicular tissue based on two dimensional chromatography with inductively coupled plasma mass spectrometry was performed for the first time. The gut microbiota profile was obtained by 16S rRNA gene sequencing. Numerous associations were found between testicular selenoproteins and gut microbiota (e.g. Mucispirillum, related with sperm activity and testosterone, was associated with glutathione peroxidase (GPx) and selenoalbumin (SeAlb), while Escherichia/Shigella, related to sex hormones, correlated with GPx, selenoprotein P (SelP) and SeAlb). The effects of Se-supplementation on testicular selenoproteins only occur in conventional mice, suggesting a potential selenoproteins-microbiota interplay that underlies testicular function. The selenoproteins GPx and SelP have been quantified for the first time in the testicles, and the novel identification of SeAlb, a protein with nonspecifically incorporated Se, is also reported. These findings demonstrate the significant impact of Se-supplementation on gut microbiota and male reproductive health. In addition, the analytical methodology applied here in selenoprotein quantification in testicular tissue opens new possibilities to evaluate their role in gut microbiota and reproductive health axis. ; This article was funded by the projects PGC 2018- 096608-B-C21 from the Spanish Ministry of Science and innovation (MCI) (Generación del Conocimiento. MCI/AEI/FEDER, EU "Una manera de hacer Europa") and UHU-1256905 from the FEDER Andalusian Operative Program 2014-2020 (Ministry of Economy, Knowledge, Business and Universities, Regional Government of Andalusia, Spain). S.R.A. thanks the Spanish Ministry of Economy and Competitiveness for a PhD scholarship (BES-2016-076364). The authors are grateful to FEDER (European Community) for financial support, Grant UNHU13-1E-1611. The authors would like to acknowledge the support from The Ramón Areces Foundation (ref. CIVP19A5918). ; Peer reviewed

Carcinogenesis is a very complex process in which metals have been found to be critically involved. In this sense, a disturbed redox status and metal dyshomeostasis take place during the onset and progression of cancer, and it is well-known that trace elements participate in the activation or inhibition of enzymatic reactions and metalloproteins, in which they usually participate as cofactors. Until now, the role of metals in cancer have been studied as an effect, establishing that cancer onset and progression affects the disturbance of the natural chemical form of the essential elements in the metabolism. However, it has also been studied as a cause, giving insights related to the high exposure of metals giving a place to the carcinogenic process. On the other hand, the chemical species of the metal or metallobiomolecule is very important, since it finally affects the biological activity or the toxicological potential of the element and their mobility across different biological compartments. Moreover, the importance of metal homeostasis and metals interactions in biology has also been demonstrated, and the ratios between some elements were found to be different in cancer patients; however, the interplay of elements is rarely reported. This review focuses on the critical role of metals in lung cancer, which is one of the most insidious forms of cancer, with special attention to the analytical approaches and pitfalls to extract metals and their species from tissues and biofluids, determining the ratios of metals, obtaining classification profiles, and finally defining the metallome of lung cancer. ; This research was funded by the projects CTM2015-67902-C-1-P from the Spanish Ministry of Economy and Competitiveness, and P12-FQM-0442 from the Regional Ministry of Economy, Innovation, Science and Employment (Andalusian Government, Spain). Projects Neumosur (8/2012 and 9/2015) and SEPAR (124/2012 and 091/2016). Belén Callejón Leblic thanks the Ministry of Education, Culture and Sport for a predoctoral scholarship. Finally, authors are grateful to FEDER (European Community) for financial support, grants number UNHU13-1E-1611 and UNHU15-CE-3140.

Selenium (Se) is an essential trace element with important health roles due to the antioxidant properties of selenoproteins. To analyze the interplay between Se and gut microbiota, gut metabolomic profiles were determined in conventional (C) and microbiota depleted mice (Abx) after Se-supplementation (Abx-Se) by untargeted metabolomics, using an analytical multiplatform based on GC-MS and UHPLC-QTOF-MS (MassIVE ID MSV000087829). Gut microbiota profiling was performed by 16S rRNA gene amplicon sequencing. Significant differences in the levels of about 70% of the gut metabolites determined, including fatty acyls, glycerolipids, glycerophospholipids, and steroids, were found in Abx-Se compared to Abx, and only 30% were different between Abx-Se and C, suggesting an important effect of Se-supplementation on Abx mice metabolism. At genus level, the correlation analysis showed strong associations between metabolites and gut bacterial profiles. Likewise, higher abundance of Lactobacillus spp., a potentially beneficial genus enriched after Se-supplementation, was associated with higher levels of prenol lipids, phosphatidylglycerols (C-Se), steroids and diterpenoids (Abx-Se), and also with lower levels of fatty acids (Abx-Se). Thus, we observed a crucial interaction between Se intake-microbiota-metabolites, although further studies to clarify the specific mechanisms are needed. This is the first study about untargeted gut metabolomics after microbiota depletion and Se-supplementation. ; This work was supported by the projects PG2018-096608-B-C21 f from the Spanish Ministry of Science and innovation (MCIN). Generación del Conocimiento. MCIN/ AEI /10.13039/501100011033/ FEDER "Una manera de hacer Europa" and UHU-1256905 from the FEDER Andalusian Operative Program 2014–2020 (Ministry of Economy, Knowledge, Business and Universities, Regional Government of Andalusia, Spain). Authors would like to acknowledge the support from The Ramón Areces Foundation (ref CIVP19A5918). Authors are grateful to FEDER (European Community) for financial support, Grant UNHU13-1X10-1611. Funding for open access charge: Universidad de Huelva / CBUA. ; Peer reviewed

Among organic contaminants, pesticides are one of the most important groups of chemicals due to their persistent character and toxicity. However, the biological systems are exposed to a complex environment in which the contaminants can interact in a synergistic/antagonistic fashion, and for this reason, the study of "chemical cocktails" is of great interest to fully understand the final biological effect. In this way, selenium is known for its antagonistic action against several toxicants. In this paper, metabolic impairments caused by the joint exposure of p,p′-dichloro diphenyl trichloroethane (DDE) and selenium (Se) have been issued for the first time. A metabolomic workflow was applied to mice fed DDE and DDE with Se diet, on the basis of the complementary use of two organic mass spectrometric techniques, combining direct infusion mass spectrometry (DI-ESI-QqQ-TOF MS) and gas chromatography–mass spectrometry (GC–MS). The results show a good classification between the studied groups caused by about 70 altered metabolites in the liver, kidney, or brain, including the pathways of energy metabolism, degradation of phospholipidic membrane, β-oxidation, and oxidative stress, which confirm the potential of combined metabolomic platforms in environmental studies. ; This work has been supported by projects CTM2015-67902-C2-1-P and PG2018-096608 B-C21 from the Spanish Ministry of Economy and Competitiveness and P12-FQM-0442 and B10-1657 from the Regional Ministry of Economy, Innovation, Science and Employment (Andalusian Government, Spain). G.R.-M. thanks the Spanish Ministry of Economy and Competitiveness for a PhD scholarship (BES-2013-064501). S.R.-A. thanks the Spanish Ministry of Economy and Competitiveness for a PhD scholarship (BES-2016-076364). Finally, the authors are grateful to FEDER (European Community) for financial support, Grant Nos. UNHU13-1E-1611 and UNHU15-CE-3140.

Metals are important components of living organisms since many biological functions critically depend on their interaction with some meta l 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 res ponses that metals elicit in the organisms, new analytical methodologies are needed. We re view 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 re sults. 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 a n important European biological reserve for migrating birds affected by agricu ltural, mining and industrial ac tivities. 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 Speci al Issue entitled: Enviro nmental and structural proteomics. Biological Significance This work presents new contributions in the study of environmental metal pollution in terrestrial ecosystems using Mus spretus mi ce as bioindicator in Doñana National Park ; This work was supported by the project CTM2012-38720-C03-01 from the Spanish Ministry of Economy and Competitiveness, and by projects P08-FQM-3554, P09-FQM-4659, P08-CVI-03829, and P08-RNM-00523 from the Regional Ministry of Economy, Innovation, Science and Employment (Andalusian Government, Spain). M.A. Garcia Sevillano thanks the Ministerio de Educacion (Spain) for a PhD scholarship.