Gut-gonad crosstalk in mice exposed to a "chemical cocktail" combining metabolomics and microbial profile by amplicon sequencing.

Testes are very prone to be damaged by environmental pollutants, but there is a lack of information about the impact of "chemical cocktails" (CC) on the testicular metabolome and the possible influence in the gut-gonad crosstalk. For this, BALB/c mice were given flumequine and diclofenac orally in food and potentially toxic trace elements (Cd, Hg, As) in drinking water. A mice group was supplemented with selenium, a well-known antagonist against many pollutants. Our results revealed that the steroid 5-alpha-androstan-17-beta-ol propionate, suggested as a parameter of androgenicity independent of testosterone levels, proline that improves reproductive indicators in male rabbits affected by environmental stress) among others metabolites are only present after CC exposure with rodent and selenium supplemented diet. Selenium also antagonized the up-or down-regulation of anandamide (20:l, n-9) (p < 0.001 and FC 0.54 of CC vs C but p > 0,05 and FC 0.74 of CC-Se vs C), that regulates gonadotropin-releasing hormones in mammals, 2,3-dinor-11b-PGF2a (p < 0.001 and FC 0.12 of CC vs C but p > 0,05 and FC 0.34 of CC-Se vs C), which has been related with reproductive hormones, besides others testicular metabolites altered by the exposure to the CC and reversed the levels to control. Moreover, numerous significant associations between gut microbes and testicular metabolites indicated a possible impact of pollutants in the testes mediated by gut microbiota due to a gut-gonad crosstalk.

Impact of "chemical cocktails" exposure in shaping mice gut microbiota and the role of selenium supplementation combining metallomics, metabolomics, and metataxonomics.

Biological systems are exposed to a complex environment in which pollutants can interact through synergistic or antagonistic mechanisms, but limited information is available on the combined effects. To this end, conventional and antibiotic-treated (Abx) mice models were fed regular rodent or selenium (Se) supplemented diets and exposed to a "chemical cocktail" (CC) including metals and pharmaceuticals. Metallomics, metabolomics, and metataxomics were combined to delve into the impact on gut microbiota, plasma selenoproteome, metabolome, and arsenic metabolization. At the molecular level, Se decreased the concentration of the antioxidant glutathione peroxidase in plasma and increased the arsenic methylation rate, possibly favoring its excretion, but not in the Abx and also plasma metabolomes of Abx, and Abx-Se were not differentiated. Moreover, numerous associations were obtained between plasma selenoproteins and gut microbes. Se-supplementation partially antagonizes the gut microbiota alteration caused by Abx, and slightly by CC, but strongly altered profiles were observed in CC-Abx-Se, suggesting synergistic deleterious effects between pollutants, Abx and Se. Moreover, although CC and Abx changed gut microbiota, several common taxa were enriched in CC-Abx and control mice, indicating possible synergistic effects. Our results suggest a potential beneficial impact of supplementation, but mediated by gut microbes being reversed in their absence.

Mice brain metabolomics after the exposure to a "chemical cocktail" and selenium supplementation through the gut-brain axis.

Several environmental pollutants have been shown to damage brain and affect gut microbiota. Limited evidence is available about the impact of "chemical cocktails" (CC) of xenobiotics on brain metabolome and their possible influence in the gut-brain crosstalk. To this end, BALB/c mice were exposed to heavy metals (As, Hg, Cd) and pharmaceuticals (diclofenac and flumequine) under regular rodent diet or supplemented with selenium (Se). Selenium, an antioxidant well-known for its antagonism against the neurotoxicity of several pollutants, modulated several brain metabolic impairments caused by CC (e.g., brain levels of the excitatory amino acid N-acetyl aspartic acid) by influencing mainly the metabolisms of purine, glycosylate and dicarboxylate, glutamate, glycerophospholipid, alanine and aspartate. Numerous associations were obtained between brain metabolites and gut microbes and they changed after Se-supplementation (e.g., Lactobacillus was positively associated with a brain ceramide, phosphoserine, phosphocholine, vitamin D3 derivative, fatty acids, malic acid, amino acids, and urea after the exposure, but not after Se-supplementation). Our results showed numerous evidences about the impact of CC on brain metabolome, the potential role of Se as an antagonist and their impact on the gut-brain axis. Further research is needed to understand the complex mechanism of action implied on CC-brain-microbiota interactions.

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.

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.

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.

A review of the impact of xenobiotics from dietary sources on infant health: Early life exposures and the role of the microbiota.

Xenobiotics are worldwide distributed and humans are unavoidably exposed to multiple chemical compounds during life, from preconception to adulthood. The human microbiota is mainly settled during early life and modulate host health and fitness. One of the main routes for chemical exposure is by intake of contaminated food and water. Thus, the interplay between diet-xenobiotics-microbiota during pregnancy and perinatal period may have relevant consequences for infant and adult health. Maternal exposure to metal(oid)s, persistent organic pollutants, and some food additives can modify the infant's microbiota with unknown consequences for child or adult health. Toxicants' exposure may also modulate the maternal transfer of microorganisms to the progeny during birth and breastfeeding; however, scarce information is available. The rapid increase in releasing novel chemicals to the environment, the exposure to chemical mixtures, the chronic/low dose scenario, and the delay in science-stakeholders action call for novel and groundbreaking approaches to improve a comprehensive risk assessment in sensitive population groups like pregnant women and neonates, with emphasis on microbiota as modulating factor and target-organ of xenobiotic's toxicity.

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.

Diversos metales pueden ser biomarcadores precoces de cáncer de pulmón / Different metals can be early biomarkers for lung cancer x

FUNDAMENTO Y OBJETIVOS: el cáncer de pulmón (CP) es el que provoca mayor mortalidad, especialmente por su frecuente diagnóstico tardío, con menos posibilidades de curación. En el inicio del proceso carcinogénico, previo al diagnóstico clínico, los oligoelementos (metales o metaloides), desempeñan un papel importante al activar o inhibir las reacciones enzimáticas y las metaloproteínas. El objetivo de nuestro estudio es analizar la utilidad de diversos metales como biomarcadores (BM) precoces de CP, obtenidos en muestras de suero, orina, y lavado broncoalveolar (LBA)MATERIAL Y MÉTODOS: hemos analizado las concentraciones totales, incluyendo fracciones de alto y bajo peso molecular, de 11 metales en muestras de suero, orina y LBA de pacientes CP, controles sanos (CS) y pacientes con patología respiratoria no cáncer (NCP) empleando una técnica de análisis basada en un plasma de acoplamiento inductivo-espectrometría de masas (ICP-QQQ-MS). RESULTADOS: obtuvimos una clara discriminación entre los grupos en las tres muestras analizadas. Hemos obtenido metales sobreexpresados o reducidos en el CP que podrían utilizarse como BM. La concentración de vanadio (V) y cromo (Cr) en suero es claramente mayor en pacientes con CP. Hemos demostrado que varios metales (V, Cr y cobre), relacionados con procesos metabólicos alterados en CP como estrés oxidativo y homeostasis, y/o sus relaciones podrían ser buenos BM de CP. CONCLUSIONES: diversos metales, y sus relaciones y correlaciones, en la población estudiada diferencian claramente a los pacientes con cáncer de pulmón de los CS y NCP y parecen ser buenos biomarcadores en el diagnóstico precoz del cáncer de pulmón

BACKGROUND AND OBJECTIVES: Lung cancer (LC) has the highest mortality rate, especially due to its late diagnosis, with a lower chance of recovery. At the start of the carcinogenic process, before a clinical diagnosis, trace elements (metals or metalloids) play an important role by activating or inhibiting enzymatic reactions and metalloproteins. The objective of our study is to analyze the utility of different metals as early biomarkers (BM) for LC which are obtained in serum, urine and bronchoalveolar lavage (BAL) samples. MATERIAL AND METHODS: We analyzed the total concentrations, including fractions of high and low molecular weight, of 11 metals in serum, urine and BAL samples from patients with LC, healthy controls (HC) and patients with non-cancerous respiratory pathology (NCP) using an analysis technique based on inductively coupled plasma mass spectrometry (ICP-QQQ-MS).RESULTS: We obtained a clear discrimination between groups for the three samples analyzed. We obtained overexpressed or reduced metals in LC that could be used as BM. The concentration of vanadium (V) and chromium (Cr) in serum is clearly higher in patients with LC. We have shown that several metals (V, Cr and copper) related to the altered metabolic processes in LC such as oxidative stress and homeostasis and/or their connections could be good BM for LC. CONCLUSIONS: in the population studied, several metals and their connections and correlations were clearly differentiated in the patients with lung cancer compared to the HC and NCP groups and they appear to be good biomarkers for the early diagnosis of lung cancer

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.

Perfiles metabolómicos de sujetos sanos y con cáncer de pulmón. Influencia de la carga tabáquica / The metabolomics of healthy subjects and those with lung cancer: the influence of smoking

INTRODUCCIÓN: la aplicación de técnicas metabolómicas presenta un gran potencial para la búsqueda de posibles biomarcadores de diagnóstico precoz en numerosas enfermedades. El empleo de procedimientos metabolómicos basados en espectrometría de masas permite estudiar las modificaciones metabólicas subyacentes al cáncer de pulmón (CP) y la influencia de la carga tabáquica (CT), medida en paquetes-año, en los correspondientes perfiles metabólicos. METODOLOGÍA: se estudió el suero de 9 controles sanos (no fumadores), 6 enfermos de CP con carga tabáquica moderada (CT 70). Se analizaron los metabolitos mediante técnicas de espectrometría de masas de alta resolución (DI-ESI-QTOFMS). Los perfiles metabólicos obtenidos se sometieron a análisis estadístico multivariante (PCA, PLS-DA). RESULTADOS: los tres grupos de estudio mostraron perfiles metabólicos claramente diferentes, lo que permitió identificar algunos posibles biomarcadores. Los niveles de glutatión se encontraron disminuidos en muestras de suero de pacientes con CP, mientras que se incrementó el contenido de distintos fosfolípidos de membrana (PL). La mayoría de estas anormalidades metabólicas se acentuó en pacientes con una CT elevada. CONCLUSIONES: el análisis metabolómico en muestras de suero permitió diferenciar claramente entre sujetos sanos y pacientes con CP. Así mismo, se identificaron posibles biomarcadores para el diagnóstico del CP (relacionados con rutas previamente conocidas en el proceso del cáncer). Además, estas alteraciones se vieron influenciadas por la carga tabáquica, confirmando la importancia del tabaquismo como factor de riesgo primario en el desarrollo del CP

By using high resolution metabolomics, we obtained the metabolomic profiles for patients who smoked and had lung cancer (LC) and a healthy, non-smoker control (HC) group; we assessed the influence of smoking on said profiles. The results show a clear discrimination between the metabolomic profiles of both groups studied; the metabolites causing said difference could be determined. Moreover, differences were encountered between the metabolomic profiles of heavy smokers with lung cancer compared to moderate smokers. INTRODUCTION: theapplicationof metabolomic techniques offers extensive potential to search for possible biomarkers in the early detection of several diseases. Using metabolomic procedures based on mass spectrometry allow us to study underlying metabolic changes in lung cancer and the influence of smoking, measured as packets/year, in the corresponding metabolic profiles. METHOD: the serum from 9 healthy control subjects (nonsmokers) was studied, as well as that of 6 patients with lung cancer who were moderate smokers (CT 70). The metabolites were analyzed using high resolution mass spectrometer techniques (DI-ESI-QTOF-MS). The metabolic profiles obtained were subject to multivariate statistical analysis (PCA, PLS-DA). RESULTS: the three groups studied showed clearly differentiated metabolic profiles, which facilitated the identification of certain biomarkers. Glutathione levels were found to be decreased in the sample from patients with lung cancer, while the content of various membrane phospholipids increased. Most of these metabolic abnormalities were heightened in patients who were heavy smokers. CONCLUSIONS: the metabolomics analysis in serum samples clearly differentiated healthy subjects from patients with lung cancer. Also, possible biomarkers were identified to diagnose lung cancer (linked to previously known routes in the cancer process. Moreover, these alterations were influenced by the amount smoked, thus confirming the importance of smoking as a primary risk factor in developing lung cáncer

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.

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.

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.

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.