A metallomic approach based on the use of size-exclusion chromatography (Superdex-75) with inductively coupled plasma mass spectrometry (ICP-MS) detection is combined with anion or cation exchange chromatography to characterize the biological response of the free-living mouse Mus spretus. The approach has been applied to contaminated and non-contaminated areas from Doñana National Park (southwest Spain) and the surroundings. Several areas affected by differential contamination from mining, industrial, and agricultural activities have been considered. The high presence of Mn, Cu, and Zn in liver and As and Cd in kidney is remarkable, especially in contaminated areas. The size exclusion chromatograms traced by Mn in liver cytosolic extracts are more intense than in kidney; a Mn-peak matching with the standard of 32 kDa (superoxide dismutase) is present in these organs, and its intensity is correlated with the concentration of Mn in the extracts. High-intensity peaks traced by Cu, Zn, and Cd at 7 kDa (matching with metallothionein I standard) in liver extract are triggered by the presence of contaminants. Other peaks related with molecules of 32 and 67 kDa traced by Cu and Zn can also be observed, although their intensity is higher in sites with low contamination. In kidney extracts, the presence of a Cd-peak with Mr of 7 kDa (tentatively Cd-metallothionein) with high intensity under the action of contaminants was observed, but high biological responses are also proven in the protected area of the Park, which denotes a progressive increase of diffuse contamination.

In mammals, the transport of essential elements from the gastrointestinal tract to organs is orchestrated by biochemical mechanisms which have evolved over millions of years. The subsequent organ-based assembly of sufficient amounts of metalloproteins is a prerequisite to maintain mammalian health and well-being. The chronic exposure of various human populations to environmentally abundant toxic metals/metalloid compounds and/or the deliberate administration of medicinal drugs, however, can adversely affect these processes which may eventually result in disease. A better understanding of the perturbation of these processes has the potential to advance human health, but their visualization poses a major problem. Nonetheless, liquid chromatography-inductively coupled plasma-based 'metallomics' methods, however, can provide much needed insight. Size-exclusion chromatography-inductively coupled plasma atomic emission spectrometry, for example, can be used to visualize changes that toxic metals/medicinal drugs exert at the metalloprotein level when they are added to plasma in vitro. In addition, size-exclusion chromatography-inductively coupled plasma mass spectrometry can be employed to analyze organs from toxic metal/medicinal drug-exposed organisms for metalloproteins to gain insight into the biochemical changes that are associated with their acute or chronic toxicity. The execution of such studies-from the selection of an appropriate model organism to the generation of accurate analytical data-is littered with potential pitfalls that may result in artifacts. Drawing on recent lessons that were learned by two research groups, this tutorial review is intended to provide relevant information with regard to the experimental design and the practical application of these aforementioned metallomics tools in applied health research.

The 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.

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 Doñana 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.

A metallomic analytical approach based on the use of size exclusion chromatography coupled to ICP-MS has been used to obtain metal profiles related to overexpression or inhibition of metal-binding biomolecules, which is connected to exposure experiment of laboratory mice Mus musculus to toxic metals, such as Cd, Hg and As. Exposure to Cd induces the formation of Cd-metallothionein in liver that reveals the protective role of this organ; however, exposure to Hg reduces the intensity of the peak associated to Cu-superoxide dismutase (Cu-SOD) while Hg-SOD peak increases, which suggests the competence of Cu and Hg for the active sites of SOD in liver that causes mercury translocation to kidney, in which the concentration of Hg as Hg-metallothionein increases drastically to be excreted by urine. It has been also observed the protective effect of selenium on mercury toxicity in blood plasma, which produces decreasing of the intensity of Se-protein in plasma with Hg exposure and correlative increases of Hg-albumin that transport mercury to kidney for excretion. Finally, arsenic exposure provokes the accumulation of small metabolites of this element, such as dimethylarsenic and monomethylarsenic for excretion. The application of the metallomic approach to liver extracts from free-living mouse Mus spretus shows the overexpression of Cu, Zn and Cd-peaks at 7 kDa (related to metal-metallothionein) in environmental contaminated sites, as well as the increase of peaks related to Cu- and Zn-SOD and Zn-albumin. However, in kidney, can be checked the presence of high concentration of arsenic small metabolites in contaminated areas, similarly to results found in exposure experiments. In addition, the application of a metabolomic approach based on direct infusion mass spectrometry to organ extracts (liver, kidney and serum) from mice (M. musculus) exposed to arsenic reveals important metabolic changes related to antioxidative activity, membrane cell damage, energy metabolism and arsenic elimination. Similar results were obtained from free-living mouse (M. spretus) from areas contaminated with arsenic. The integration of metallomics and metabolomics results provides a more comprehensive evaluation about the biological response in exposure experiments to toxic metals as well as in environmental assessment of contamination.