Antagonistic Interaction of Selenium and Cadmium in Human Hepatic Cells Through Selenoproteins.

Cadmium (Cd) is a highly toxic heavy metal for humans and animals, which is associated with acute hepatotoxicity. Selenium (Se) confers protection against Cd-induced toxicity in cells, diminishing the levels of ROS and increasing the activity of antioxidant selenoproteins such as glutathione peroxidase (GPx). The aim of this study was to evaluate the antagonistic effect of selenomethionine (SeMet) against Cd toxicity in HepG2 cells, through the modulation of selenoproteins. To this end, the cells were cultured in the presence of 100 µM SeMet and 5 µM, 15 µM, and 25 µM CdCl2 and a combination of both species for 24 h. At the end of the experiment, cell viability was determined by MTT assay. The total metal content of Cd and Se was analyzed by triple-quadrupole inductively coupled plasma-mass spectrometry (ICP-QqQ-MS). To quantify the concentration of three selenoproteins [GPx, selenoprotein P (SELENOP), and selenoalbumin (SeAlb)] and selenometabolites, an analytical methodology based on column switching and a species-unspecific isotopic dilution approach using two-dimensional size exclusion and affinity chromatography coupled to ICP-QqQ-MS was applied. The co-exposure of SeMet and Cd in HepG2 cells enhanced the cell viability and diminished the Cd accumulation in cells. Se supplementation increased the levels of selenometabolites, GPx, SELENOP, and SeAlb; however, the presence of Cd resulted in a significant diminution of selenometabolites and SELENOP. These results suggested that SeMet may affect the accumulation of Cd in cells, as well as the suppression of selenoprotein synthesis induced by Cd.

Iodine deficiency disturbs the metabolic profile and elemental composition of human breast milk.

Human breast milk (HBM) has a beneficial impact on health programming, growth and neurodevelopment of newborns.Increase in iodine intake is recommended for pregnant women in order to produce enough thyroid hormones to meet foetal requirements.In this work, a combined analytical multiplatform based on gas chromatography coupled to mass spectrometry and ultra-high performance liquid chromatography coupled toquadrupole-time-of-flight mass spectrometryhas been appliedinthe first metabolomic study of HBM ofiodine-deficientwomen. In addition, the elemental composition of HBM has been determined by inductively coupled plasma triple quadrupole mass spectrometry. Remarkably,31 metaboliteswith important biological roles(e.g. glycerophospholipids for neurodevelopment)were seentobe alteredin the HBM of iodine-deficient women. The main metabolic pathwaysalteredinclude lipid metabolism, amino acid cycle, the tricarboxylic acid cycle and glycolysis.Additionally, the concentration of selenium, zinc and copperwere seento be significantlylowerin HBM of iodine-deficient women.

Targeted and untargeted metabolomic analysis of Procambarus clarkii exposed to a "chemical cocktail" of heavy metals and diclofenac.

Water pollution poses an important problem, but limited information is available about the joined effects of xenobiotics of different chemical groups to evaluate the real biological response. Procambarus clarkii (P. clarkii) has been demonstrated to be a good bioindicator for assessing the quality of aquatic ecosystems. In this work, we studied the bioaccumulation of cadmium (Cd), arsenic (As) and diclofenac (DCF) in different tissues of P. clarkii during 21 days after the exposure to a "chemical cocktail" of As, Cd and DCF, and until 28 days considering a depuration period. In addition, a combined untargeted and targeted metabolomic analysis was carried out to delve the metabolic impairments caused as well as the metabolization of DCF. Our results indicate that As and Cd were mainly accumulated in the hepatopancreas followed by gills and finally abdominal muscle. As and Cd show a general trend to increase the concentration throughout the exposure experience, while a decrease in the concentration of these elements is observed after 7 days of the depuration process. This is also the case in the abdominal muscle for Cd, but not for As and DCF, which increased the concentration in this tissue in the depuration phase. The hepatopancreas showed the greatest number of metabolic pathways affected. Thus, we observed a crucial bioaccumulation of xenobiotics and impairments of metabolites in different tissues. This is the first study combining the exposure to metals and pharmaceutically active compounds in P. clarkii by untargeted metabolomics including the biotransformation of DCF.

Optimization of hollow-fiber liquid phase microextraction for polychlorinated biphenyls in human breast milk.

A reliable and sensitive analytical approach has been optimized for the extraction of seven polychlorinated biphenyls (PCBs) from human breast milk. Hollow fiber liquid phase microextraction (HF-LPME) was applied for the first time for the extraction and pre-concentration of the analytes. Analytes were separated by gas chromatography with electron capture detector (GC-µECD) for the sensitive detection and mass spectrometry for the unequivocal identification. A rotable central composite design (RCCD) was performed for the multivariate optimization of the method. The best results were obtained at 40 °C during 30 min and 600 rpm of stirring speed using a hollow fiber length of 5 cm and toluene as an extractant phase and salt addition was not required. The detection limits were in the range 7-14 ng L-1 for PCBs. The coefficients of determination of the calibration curves indicated good linearity (R2> 0.96) and the enrichment factors ranged from 74 to 143. This type of study is of great importance due to the deleterious effect that the presence of contaminants can produce in infants health related to the immature character of the defense system. Moreover, exclusive breastfeeding is recommended by neonatologists up to six months of life and as complementary food during the first two years.

Effervescence-assisted spiral hollow-fibre liquid-phase microextraction of trihalomethanes, halonitromethanes, haloacetonitriles, and haloketones in drinking water.

A new analytical method was optimized to determine 18 disinfection by-products (DBPs) in drinking water, including four different chemical groups. For this purpose, spiral-shaped hollow-fibre liquid phase microextraction with 1-octanol as the acceptor solvent assisted by effervescence was applied using a homemade supporting device that was specifically designed for this application. The device was printed in a 3D printer and allows for an increased fibre surface even with a low sample volume, which significantly facilitates the extraction. The samples were analysed by gas chromatography coupled to both an electron capture detector and a mass spectrometer for the quantification and unequivocal identification of the analytes, respectively. Effervescence was generated using citric acid and bicarbonate at a molar ratio 1:2, which significantly improves the extraction efficiency and reduces mechanical operations, since stirring and modifiers are not required. The results showed enrichment factors ranging from 13.1 to 140.1. Satisfactory recoveries (80-113 %) were obtained, with relative standard deviations from 3 to 15 % and good linearity. The detection limits (ng L-1) ranged from 10 to 35 (trihalomethanes), 12 to 220 (halonitromethanes), 17 to 79 (haloacetonitriles) and 10 to 16 (haloketones). The applicability of the method was assessed in 6 local water distribution systems.

A novel HPLC column switching method coupled to ICP-MS/QTOF for the first determination of selenoprotein P (SELENOP) in human breast milk.

In this work, we describe for the first time the presence of selenoprotein P in human breast milk. To this end, a novel analytical method has been developed based on a two-dimensional column switching system, which consisted of three size exclusion columns and one affinity column coupled to inductively coupled plasma mass spectrometry (ICP-MS). The method combines the accurate quantification of selenoproteins and selenometabolites by species unspecific isotopic dilution ICP-MS, with unequivocal identification by quadrupole-time-of-flight mass spectrometry. Several selenopeptides, which contain the amino acid selenocysteine (U, SeCys), were identified after tryptic digestion followed by their separation. The results reveal that the relative selenium concentration in colostrum follows the order: glutathione peroxidase (GPX) ≈ selenoprotein P (SELENOP) > selenocystamine (SeCA) > other selenometabolites (SeMB), in contrast with previously published papers (GPX > SeCA > selenocystine > selenomethionine). A mean concentration of 20.1 ± 1.0 ng Se g-1 as SELENOP (1.45 µg SELENOP/g) was determined in colostrum (31% of total selenium).

Study of the metabolomic relationship between lung cancer and chronic obstructive pulmonary disease based on direct infusion mass spectrometry.

The high prevalence of lung cancer (LC) has triggered the search of biomarkers for early diagnosis of this disease. For this purpose the study of metabolic changes related to the development of lung cancer could provide interesting information about its early diagnosis. In this sense, chronic obstructive pulmonary disease (COPD), a disease associated with tumor development, is a comorbidity that increases the risk of onset and progression of lung neoplasia and has also to be considered in the study of pathology related to lung cancer. This work develop a metabolomic approach based on direct infusion mass spectrometry using a hybrid triple quadrupole-time of flight mass spectrometer (DI-ESI-QqQ-TOF-MS) in order to identify altered metabolites from serum of LC and COPD patients and evaluate its relationship and implication in the progression of LC. This methodology has been applied to 30 serum samples from LC, 30 healthy patients used as controls (HC) and 30 serum samples from COPD to found altered metabolites from both LC and COPD diseases. In addition, some metabolic differences and similarities were found in Pulmonary Emphysema and Chronic Bronchitis patients. On the other hand, altered metabolites were studied in different stages of LC (II, III and IV) to evaluate the perturbation of them throughout the progression of disease. The sample treatment consisted of the extraction of polar and non-polar metabolites from serum that was later infused into the mass spectrometer using an electrospray ionization source in positive and negative mode. Partial least squares discriminant analysis (PLS-DA) allowed a classification between LC, HC and COPD groups in all acquisition modes. A total of 35 altered and common metabolites between LC and COPD, including amino acids, fatty acids, lysophospholipids, phospholipids and triacylglycerides were identified, being alanine, aspartate and glutamate metabolism the most altered. Finally, ROC curves were applied to the dataset and metabolites with AUC value higher than 0.70 were considered as relevant in the progression of LC.

Mass spectrometry based analytical approaches and pitfalls for toxicometabolomics of arsenic in mammals: A tutorial review.

The present review focus on the analytical platforms and the workflow for toxicometabolomics with a special emphasis on their strengths and pitfalls presenting as a case study the toxicometabolomics of arsenic in mammals. Although powerful analytical methods and techniques are currently available for metabolomics, the main "bottleneck" is still the absence of unified protocols for sample preparation (e.g. quenching, solvents used) as well as several important factors in toxicometabolomics, which drastically affect the metabolism (e.g. selection of model organisms, xenobiotic doses, chemical form of the xenobiotic, exposure route, biological sample). In this context, the applicability to complex samples, higher sensitivity, specificity and the possibility to perform quantitative analysis offered by MS is crucial to probe xenobiotic induced metabolic changes to evaluate the stress responses. Nowadays, the use of different metabolomic platforms allowed determining important changes in the metabolism induced by arsenic in mammals such as alterations in the energy (e.g. Glycolysis, Kreb's cycle), amino acid, lipid, nucleotide and androgen metabolisms.

Seasonal and spatial evolution of trihalomethanes in a drinking water distribution system according to the treatment process.

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.

Application of hollow fiber liquid phase microextraction for simultaneous determination of regulated and emerging iodinated trihalomethanes in drinking water.

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 ngL(-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 ngL(-1) has been developed. A central composite design was used to optimize conditions for simultaneous extraction. The best extraction recovery was obtained with 19.2min at 27.1°C and 900rpm, without salt addition, using a supported hollow fiber membrane of 10.5cm (0.6mm id) and 1-octanol as acceptor phase. The limits of detection for the regulated THMs and I-THMs were 3-44ngL(-1) and 1-3ngL(-1), respectively. The calibration curves showed good linearity (R(2)>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 (1-18ngL(-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.

Shotgun metabolomic approach based on mass spectrometry for hepatic mitochondria of mice under arsenic exposure.

Mass spectrometry (MS)-based toxicometabolomics requires analytical approaches for obtaining unbiased metabolic profiles. The present work explores the general application of direct infusion MS using a high mass resolution analyzer (a hybrid systems triple quadrupole-time-of-flight) and a complementary gas chromatography-MS analysis to mitochondria extracts from mouse hepatic cells, emphasizing on mitochondria isolation from hepatic cells with a commercial kit, sample treatment after cell lysis, comprehensive metabolomic analysis and pattern recognition from metabolic profiles. Finally, the metabolomic platform was successfully checked on a case-study based on the exposure experiment of mice Mus musculus to inorganic arsenic during 12 days. Endogenous metabolites alterations were recognized by partial least squares-discriminant analysis. Subsequently, metabolites were identified by combining MS/MS analysis and metabolomics databases. This work reports for the first time the effects of As-exposure on hepatic mitochondria metabolic pathways based on MS, and reveals disturbances in Krebs cycle, ß-oxidation pathway, amino acids degradation and perturbations in creatine levels. This non-target analysis provides extensive metabolic information from mitochondrial organelle, which could be applied to toxicology, pharmacology and clinical studies.

Combination of direct infusion mass spectrometry and gas chromatography mass spectrometry for toxicometabolomic study of red blood cells and serum of mice Mus musculus after mercury exposure.

Although mercury (Hg) is an important environmental and occupational pollutant, its toxicological effects, especially in serum and red blood cells (RBCs), have been scarcely studied. A toxicometabolomics workflow based on high resolution mass spectrometry approaches has been applied to investigate the toxicological effects of Hg in Mus musculus mice after subcutaneous injection for 10 days, which produced inflammation and vacuolization, steatosis and karyolysis in the hepatic tissue. To this end, direct infusion mass spectrometry (DIMS) of polar and lipophilic extracts from serum and RBCs, using positive and negative mode of acquisition (ESI+/ESI-), and gas chromatography-mass spectrometry were used. A quantitative analysis of reversible oxidized thiols in serum proteins demonstrated a strong oxidative stress induction in the liver of Hg-exposed mice. Endogenous metabolites alterations were identified by partial least squares-discriminant analysis (PLS-DA). Mercury-exposed mice show perturbations in energy metabolism, amino acid metabolism, membrane phospholipid breakdown and oxidative stress-related metabolites in serum along the exposure. This work reports for the first time the effects of Hg-exposure on RBCs metabolic pathways, and reveals disturbances in glycolysis, membrane turnover, glutathione and ascorbate metabolisms.

Biological interactions between mercury and selenium in distribution and detoxification processes in mice under controlled exposure. Effects on selenoprotein.

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.

Using direct infusion mass spectrometry for serum metabolomics in Alzheimer's disease.

Currently, there is no cure for Alzheimer's disease and early diagnosis is very difficult, since no biomarkers have been established with the necessary reliability and specificity. For the discovery of new biomarkers, the application of omics is emerging, especially metabolomics based on the use of mass spectrometry. In this work, an analytical approach based on direct infusion electrospray mass spectrometry was applied for the first time to blood serum samples in order to elucidate discriminant metabolites. Complementary methodologies of extraction and mass spectrometry analysis were employed for comprehensive metabolic fingerprinting. Finally, the application of multivariate statistical tools allowed us to discriminate Alzheimer patients and healthy controls, and identify some compounds as potential markers of disease. This approach provided a global vision of disease, given that some important metabolic pathways could be studied, such as membrane destabilization processes, oxidative stress, hypometabolism, or neurotransmission alterations. Most remarkable results are the high levels of phospholipids containing saturated fatty acids, respectively, polyunsaturated ones and the high concentration of whole free fatty acids in Alzheimer's serum samples. Thus, these results represent an interesting approximation to understand the pathogenesis of disease and the identification of potential biomarkers.

Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach.

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.

Metabolomic study of lipids in serum for biomarker discovery in Alzheimer's disease using direct infusion mass spectrometry.

In this study, we demonstrated the potential of direct infusion mass spectrometry for the lipidomic characterization of Alzheimer's disease. Serum samples were extracted for lipids recovery, and directly analyzed using an electrospray source. Metabolomic fingerprints were subjected to multivariate analysis in order to discriminate between groups of patients and healthy controls, and then some key-compounds were identified as possible markers of Alzheimer's disease. Major differences were found in lipids, although some low molecular weight metabolites also showed significant changes. Thus, important metabolic pathways involved in neurodegeneration could be studied on the basis of these perturbations, such as membrane breakdown (phospholipids and diacylglycerols), oxidative stress (prostaglandins, imidazole and histidine), alterations in neurotransmission systems (oleamide and putrescine) and hyperammonaemia (guanidine and arginine). Moreover, it is noteworthy that some of these potential biomarkers have not been previously described for Alzheimer's disease.

Use of metallomics and metabolomics to assess metal pollution in Doñana National Park (SW Spain).

Monitoring organism exposure to heavy metals has acquired increased importance in the last decades. The mouse Mus spretus has been used to assess the biological response to contaminants in the relevant ecological area of Doñana National Park (DNP) and surrounding areas (SW Spain), where many migrating birds land for breeding and feeding every year. A metallomics approach, based on the characterization of metal biomolecules using size exclusion chromatography coupled with inductively coupled plasma-mass spectrometry (SEC-ICP-MS) and a metabolomics approach based on direct infusion to a mass spectrometer (DI-ESI-QTOF-MS) followed by a partial linear square-discriminant analysis (PLS-DA), were used to compare the biological responses of M. spretus living in three areas of DNP (the reference) and surrounding areas (El Partido and El Matochal). The activities of key antioxidant enzymes, such as Cu/Zn-SOD, Mn-SOD, CAT, GR, and guaiacol peroxidase, were also determined in connection with environmental contamination issues. The results show differences caused by the presence of metals in the ecosystem that affected to the levels of metals and metalloproteins, such as MT, Cu/Zn-SOD, or Mn-CA, the breakdown of membrane phospholipids, perturbations in metabolic pathways, related to energy metabolism, and oxidative stress.

Cadmium toxicity in Mus musculus mice based on a metallomic study. Antagonistic interaction between Se and Cd in the bloodstream.

Cadmium (Cd) is an important inorganic toxicant in the environment which impacts on human health. A metallomic approach based on size-exclusion chromatography (SEC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) and multidimensional chromatography separation based on SEC coupled to affinity chromatography 2D-SEC-AF-ICP-MS have been applied to achieve a better understanding of the function, detoxification processes and regulation of metals in mice (Mus musculus) under controlled exposure to both Cd and Cd plus (77)Se. Isotopic dilution analysis (IDA) was performed to quantify selenium containing proteins in mice plasma with ICP-qMS as a multielemental detector. Additionally, isotope pattern deconvolution (IPD) was applied to study the fate of enriched (77)selenite in mice subjected to cadmium exposure and the effect of selenoprotein production in plasma. Moreover, the affinity of Cd for SeP in plasma of mice was corroborated using anion exchange chromatography (AEC) after AF separation and identified by organic mass spectrometry. This work illustrates the high reliability of the integrated use of inorganic and organic mass spectrometry to get a metallomic approximation, which provides a good alternative to gain deep insight into the fate of elements in exposed organisms, providing information about metal trafficking, interactions and homeostasis.

Simultaneous speciation of selenoproteins and selenometabolites in plasma and serum by dual size exclusion-affinity chromatography with online isotope dilution inductively coupled plasma mass spectrometry.

A method for the simultaneous speciation of selenoproteins and selenometabolites in mouse plasma has been developed based on in series two-dimensional size exclusion and affinity high-performance liquid chromatography (2D/SE-AF-HPLC), using two columns of each type, and hyphenation to inductively coupled plasma-(quadrupole) mass spectrometry (ICP-QMS). The method allows the quantitative determination of selenoprotein P (SeP), extracellular glutathione peroxidase (eGPx), selenoalbumin (SeAlb), and selenometabolites in mouse plasma using species-unspecific isotope dilution (SUID). The 2D chromatographic separation is proposed to remove typical spectral interferences in plasma from chloride and bromide on (77)Se ((40)Ar(37)Cl) and (82)Se ((81)Br(1)H). In addition, the approach increases chromatographic resolution allowing the separation of eGPx from Se metabolites of low molecular mass. The method is robust, reliable, and fast with a typical chromatographic runtime less than 20 min. Precision in terms of relative standard deviation (n = 5) is in the order of 4 %, and detection limits are in the range of 0.2 to 1.0 ng Se g(-1). Method accuracy for determination of total protein bound to Se was assessed by analyzing human serum reference material (BCR-637) certified for total Se content, and latterly applied to mouse plasma (Mus musculus). In summary, a reliable speciation method for the analysis of eGPx, selenometabolites, SeP, and SeAlb in plasma/serum samples is proposed for the first time and is applicable to the evaluation of Se status in human in clinical studies and other mammals for environmental or toxicological assessment.

Application of metallomic and metabolomic approaches in exposure experiments on laboratory mice for environmental metal toxicity assessment.

Metals have a central role in biological systems, regulating numerous cellular processes, and in other cases having toxic or deleterious effects on the metabolism. Hence, the study of metal-induced changes in cellular metabolic pathways is crucial to understanding the biological response associated with environmental issues. In this context, the finding of biomarkers has great interest, representing -omics techniques, such as metallomics and metabolomics, powerful tools for this purpose. The present work evaluates the exposure of mice Mus musculus to toxic metals (As, Cd and Hg), considering the changes induced in both the metallome and metabolome as a consequence of the high genetic homology between Mus musculus/Mus spretus mice, which allows the use of the database from M. musculus to identify the proteins and metabolites expressed by M. spretus. For this purpose a metallomic approach based on size exclusion chromatography (SEC) in combination with other complementary orthogonal separation techniques and heteroelement monitoring by ICP-ORS-qMS was performed, followed by identification of metallobiomolecules by organic mass spectrometry. In addition, simultaneous speciation of selenoproteins and selenometabolites in mouse plasma was accomplished by tandem (double) SEC-(dual) affinity chromatography (AF)-HPLC and online isotope dilution analysis (IDA)-ICP-ORS-qMS. Finally, the simultaneous changes in metabolic expression in mice caused by metal exposure (metabolome) were considered, using direct infusion mass spectrometry (DI-ESI-QqQ-TOF-MS) of extracts from mice plasma. Subsequently altered metabolites were identified using MS/MS experiments. The results obtained under controlled conditions were extrapolated to homologous free-living mice captured in Doñana National Park (DNP) and surroundings (southwest Spain) affected by As, Cd and Hg pollution. In summary, such studies are needed to understand the effect of heavy metal exposure and cope with heavy metal toxicity.