M. González-Fernández

Biological responses related to agonistic, antagonistic and synergistic interactions of chemical species

AbstractThe fact that the essential or toxic character of elements is species specific has encouraged the development of analytical strategies for chemical speciation over the last twenty years; indeed, there are now a great number of them that provide very good performance. However, biological systems are exposed to a complex environment in which species of elements can interact in a synergistic/antagonistic fashion. Thus, the metabolism of trace elements cannot be considered in isolation. On the other hand, biological systems are dynamic, so it is necessary to study the trafficking of species of elements between organs, tissues or cell compartments in order to decipher the biochemical processes of the interactions in which they are involved. Although the application of liquid chromatography–inductively coupled plasma-based “metallomics” methods in combination with organic mass spectrometry can provide much-needed insight, new analytical strategies are required to really understand the role of species of elements in biological systems and the mechanisms of their interactions. In the present paper, the interactions of the most widely studied elements in this context (Se, Hg and As) are discussed, as well as other important interactions between different elements. FigureInteractions of chemical species in biology

Size characterization of metal species in liver and brain from free-living ( Mus spretus ) and laboratory ( Mus Musculus ) mice by SEC-ICP-MS : Application to environmental contamination assessment

The molecular mass distribution of various metals was evaluated in cell lysates obtained from liver and brain of mice using size-exclusion chromatography (Superdex-75) with ICP-MS detection. Free-living mice Mus spretus were collected in polluted and non-polluted sites from Donana National Park (southwest Spain) and SEC(HPLC)-ICP-MS was used to generate element specific chromatograms for essential metals (Cu and Zn) as well as toxic metals and metalloids (Cd, As, Pb). Different molecular mass fractions containing Cu are remarkably abundant in liver from the specimens captured in the polluted area. The fraction of about 7 kDa is especially important since it matches with a metallothionein I standard. Zn and Cd chromatograms also show peaks with similar molecular mass, but lower intensity. Analogous chromatograms from the non-contaminated site show a considerable depletion of these metal-containing biomolecules possibly due to low contamination. Chromatograms from the liver of laboratory mice Mus musculus (genetically close to Mus spretus) were also obtained for comparison revealing a great similarity with non contaminated samples. On the other hand, metal profiles from brain extracts do not reflect significant differences between polluted and clean areas in comparison with those obtained from liver of Mus spretus. Finally, the daily in vivo subcutaneous administration of Cd aqueous solution to Mus musculus during 10 days resulted in great rise of a Cd-peak of 7 KDa in the extract from the liver extract that matches with the Cd-methallothionein standard. Other Cd-binding molecules with higher molecular mass are also bioinduced by Cd exposure that probably constitutes a protection mechanism against this toxic element. The application for the first time of this metallomic approach to free-living mouse Mus spretus provides promising results for environmental stress assessment.

Metallomics integrated with proteomics in deciphering metal-related environmental issues.

The present work shows the possibilities of metallomics to characterize metal-linking proteins in Mus Musculus that could be used in environmental assessment. The laboratory mouse M. musculus is used as reference of gene/protein sequence databases to address methodological approaches based on changes in transcripts regulation, proteins expression and metalloproteins profiles in the environmental bioindicator Mus spretus that has been demonstrated to be genetically homologous to M. Musculus. A metallomic approach using size exclusion chromatography with inductively coupled plasma-mass spectrometry detection (SEC-ICP-MS) was applied to cytosolic extracts from different M. musculus organs: lung, liver, spleen, kidney, brain, testicle, hearth and muscle. The resulting profiles of metallobiomolecules revealed the presence of a Cu-binding fraction in the 7-10 kDa range which was not present in the other tissues, can be associated to low molecular mass metallothionein-like proteins. The application of reverse phase chromatography with ICP-MS detection to this fraction gives two peaks that have been isolated for later identification by tandem mass spectrometry. The mass balance of copper evaluated by ICP-MS analysis of the digested brain fractions isolated by SEC and RP chromatography reveals good recoveries of the separations. The application of 2-DE to both crude brain extract and SEC fraction (7-10 kDa) reveals the considerably reduction of the number of proteins confirming that a good purification has been attained by SEC. This integration of metallomics with proteomics and transcriptomics can be useful in further studies involving the free-living mouse M. spretus for assessment of environmental issues.

Integrated application of transcriptomics, proteomics, and metallomics in environmental studies*

Here we report a preliminary working scheme for the integrative application of transcriptomic, proteomic, and metallomic methodologies in environmental monitoring, by using as sentinel the wildlife species Mus spretus and as reference the gene/protein sequence databases from the key model species Mus musculus. We have demonstrated that the absolute transcript expression signatures quantified by reverse transcription (RT) and real-time polymerase chain reaction (PCR) of selected key genes (e.g., those coding for biotransformation enzymes) in M. spretus is a useful and reliable novel biomonitoring end-point. The suitability of commercial M. musculus oligonucleotide arrays for genome-wide transcriptional profiling in M. spretus has been also shown. Transcriptomic studies indicate considerable gene sequence similarities between both mouse species. Based on these similarities, we have demonstrated the applicability in free-living M. spretus of high-throughput proteomic methods, based on matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOFMS) analysis of tryptic 2D electrophoresis (2-DE) spot digest and peptide matching with M. musculus database. A metallomic approach based on size exclusion chromatography inductively coupled plasma-mass spectrometry (SEC-ICP-MS) was applied to trace metal-biomolecule profiles. A preliminary integration of these three -omics has been addressed to M. musculus/M. spretus couple, two rodent species that separated 3 million years ago. The integrated application of transcriptomic and proteomic data and the bidirectional use of metallomics and proteomics for selective isolation of metal-biomolecules are covered in the working scheme MEPROTRANS-triple-OMIC reported in this study.

Speciation of arsenic metabolites in the free-living mouse Mus spretus from Doñana National Park used as a bio-indicator for environmental pollution monitoring

A speciation approach based on orthogonal chromatographic systems coupled to inductively coupled plasma mass spectrometry (ICP-MS) was used to characterise the biological response of free-living mice Mus spretus to environmental pollution caused by arsenic in different areas of the Doñana National Park (south-west Spain). The relative presence of inorganic and organic forms of arsenic was studied in cytosolic extracts from high metabolic activity organs of Mus spretus mice: kidneys, liver, and brain. An instrumental coupling of size-exclusion chromatography with UV and collision/reaction cell-ICP-MS detectors (SEC-UV-ICP-ORC-MS) both in analytical and preparative scale was used for this purpose. The results showed the presence of low molecular mass (LMM) molecules linked to arsenic in these tissues especially in the kidneys, where the presence of these arsenic metabolites was higher. On the other hand, the presence of these arsenicals varied from one area to the other, which can be related to a different occurrence of contaminants. These low molecular mass fractions were collected by preparative SEC chromatography for later study with ion exchange chromatography and detection by ICP-ORC-MS, using both anionic and cationic columns. The results showed the higher presence of MMA and DMA in kidneys of mice caught in contaminated areas and the existence of small amounts of unidentified arsenicals when cation-exchange chromatography was used, which could be related to the presence of dimethylarsinoylethanol (DMAE), thioarsenic species, or arsenocholine (AsC).

Molecular mass spectrometric identification of superoxide dismutase in the liver of mice Mus musculus and Mus spretus using a metallomics analytical approach

This paper reports the identification and quantification of superoxide dismutase in the liver of Mus musculus and Mus spretus mice using a metallomics analytical approach. The approach consisted of using orthogonal chromatographic systems coupled to ICP–MS and UV detectors. Size-exclusion fractionation of the cytosolic extracts was followed by anion-exchange chromatographic separation of Cu- and Zn-containing species. After purification then tryptic digestion, Cu- and Zn-containing superoxide dismutase was identified by nESI-QqTOF. The MS–MS spectra of doubly charged peptides, with the Mascot searching engine, were used to obtain the sequence of the protein.

Metallomics and Metabolomics of Plants Under Environmental Stress Caused by Metals

The role of metals in living organisms is considered on the basis of abundance of metalloproteins and metallometabolites and the occurrence of environmental hazards caused by metals lixiviation and mobility from soils, industrial and mining wastes, which contribute to plant uptake, and can finally get to man from seed and vegetal foods. Intake by plants of toxic metals such as mercury, arsenic, and cadmium, and the transformation that suffers in the organism, as well as the alteration of metabolism represents a valuable appraisal of organism’s behavior under the presence of deleterious metals as well as their traffic along the components of cell and tissues, and the interaction with essential elements. Recent analytical approaches to obtain massive information from complex living organisms, such as metallomics to characterize the entirety metal biomolecules in an organism (metallome) and metabolomics to decipher the whole molecules with mass less than 1,000 Da, are the new generation of analytical techniques for assessment plant and other organisms’ metal stress, as well as the study of metal pollution remediation driven by plants (hyperaccumulators), preparation of plant-based essential enriched food, and other useful applications. Metallomics techniques are based on hyphenated analytical units combining chromatographic components, high sensitivity element detectors (mainly ICP-MS) for metal species detection, and tandem mass spectrometry for chemical species identification, integrating a three-dimensional analytical platform. Metabolomics mainly uses high resolution mass spectrometry as QqQ-TOF-MS or Orbitrap. Therefore, Integration of these omics provides results with high-added value representing a new angle to study overall response of plants under the action of metals. A great variety of examples can be pointed out in relation to plant exposure experiments to metals, use of plant as bioindicator for environmental monitoring of metal pollution, preparation of essential elements of functional foods based on microalgae under biotechnological production, behavior of heavy metal hyperaccumulator plants, and many other cases.

CHAPTER 6:Current Analytical Strategies and Techniques Related to Selenoproteins in Foods

The biological importance of selenium to ensure human health depends on the chemical forms of this element, as well as the key role of selenoproteins on the basic functions of life. Speciation of heteroelement-containing proteins is a rapidly developing area of detection, identification and quantification of these biomolecules in biological samples, and particularly in food. Hyphenated techniques based on the coupling of chromatographic separation with inductively coupled plasma spectrometry (ICP-MS) and its combination with organic mass spectrometry allows characterization and quantification of selenium species and selenoproteins. Sample preparation is a crucial step in Se speciation in food, in which cell and tissue disaggregation, analyte extraction, protein disulfide bridges prevention and proteases inhibition are key points of these treatments. In addition, some specific sample-preparation procedures based on enzymatic extraction, protein precipitation, ultrafiltration and sonication have been recently proposed. New analytical methodologies have been developed for the characterization of unknown selenium species and selenoproteins in food and other matrices, which exhibit essential or harmful functions, and play important biological roles depending on their bioavailability, mobility, interactions with other species and molecules and transport processes.