ORIGINAL PAPER Biological response of free-living mouse Mus spretus from Doñana National Park under environmental stress based on assessment of metal-binding biomolecules by SEC-ICP-MS M. A. García-Sevillano & M. González-Fernández & R. Jara-Biedma & T. García-Barrera & J. López-Barea & C. Pueyo & J. L. Gómez-Ariza Received: 12 April 2012 / Revised: 29 June 2012 / Accepted: 16 July 2012 / Published online: 11 August 2012 # Springer-Verlag 2012 Abstract 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 contaminat- ed and non-contaminated areas from Doñana National Park (southwest Spain) and the surroundings. Several areas af- fected 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 pres- ence 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. Keywords Speciation . SEC-ICP-MS . Mus spretus . Environmental metallomics . Biological response . Doñana National Park Introduction The use of free-living organisms as bioindicators of contam- ination has been proposed in numerous studies [1–4] since they can reflect the effect of pollutants on cellular metabo- lism and global homeostasis. Generally, environmental stress situations are marked by different metabolites (biomarkers), such as cytochromes P450, glutathione-S- transferases, metallothioneins, superoxide dismutases (SODs), glutathione peroxidases, peroxiredoxins, and many others [5, 6]. However, the use of biomarkers requires a deep knowledge of their toxicity mechanisms and only Electronic supplementary material The online version of this article (doi:10.1007/s00216-012-6274-2) contains supplementary material, which is available to authorized users. M. A. García-Sevillano : M. González-Fernández : R. Jara-Biedma : T. García-Barrera (*) : J. L. Gómez-Ariza (*) Department of Chemistry and Material Science, Faculty of Experimental Science, University of Huelva, Campus de El Carmen, 21007 Huelva, Spain e-mail: tamara@dqcm.uhu.es e-mail: ariza@uhu.es J. López-Barea : C. Pueyo Department of Biochemistry and Molecular Biology, Severo Ochoa Building, University of Córdoba, Campus de Rabanales, 14071 Córdoba, Spain M. A. García-Sevillano : M. González-Fernández : R. Jara-Biedma : T. García-Barrera : J. L. Gómez-Ariza Research Center on Health and Environment (CYSMA), University of Huelva, 21007 Huelva, Spain M. A. García-Sevillano : M. González-Fernández : R. Jara-Biedma : T. García-Barrera : J. L. Gómez-Ariza International Campus of Excellence on Agrofood (ceiA3), University of Huelva, 21007 Huelva, Spain Anal Bioanal Chem (2012) 404:1967–1981 DOI 10.1007/s00216-012-6274-2