Heterologous Microarray Analysis of Transcriptome Alterations in Mus spretus Mice Living in an Industrial Settlement Nieves Abril,* ,† Julia Ruiz-Laguna, † Miguel A ́ ngel García-Sevillano, ‡ Ana M. Mata, § Jose ́ Luis Gó mez-Ariza, ‡ and Carmen Pueyo † † Department of Biochemistry and Molecular Biology, Agrifood Campus of International Excellence (ceiA3), Severo Ochoa Building, University of Có rdoba, Rabanales Campus, 14071 Có rdoba, Spain ‡ Department of Chemistry and Materials Science, Faculty of Experimental Science, University of Huelva, El Carmen Campus, 21007 Huelva, Spain § Department of Biochemistry and Molecular Biology and Genetics, Faculty of Sciencies, Biology Building, University of Extremadura, Avda. de Elvas s/n., 06006 Badajoz, Spain * S Supporting Information ABSTRACT: This work demonstrates the successful applica- tion of a commercial oligonucleotide microarray containing Mus musculus whole-genome probes to assess the biological effects of an industrial settlement on inhabitant Mus spretus mice. The transcriptomes of animals in the industrial settlement contrasted with those of specimens collected from a nearby protected ecosystem. Proteins encoded by the differentially expressed genes were broadly categorized into six main functional classes. Immune-associated genes were mostly induced and related to innate and acquired immunity and inflammation. Genes sorted into the stress-response category were mainly related to oxidative-stress tolerance and biotransformation. Metabolism-associated genes were mostly repressed and related to lipid metabolic pathways; these included genes that encoded 11 of the 20 cholesterol biosynthetic pathway enzymes. Crosstalk between members of different functional categories was also revealed, including the repression of serine-protease genes and the induction of protease-inhibitor genes to control the inflammatory response. Absolute quantification of selected transcripts was performed via RT-PCR to verify the microarray results and assess interindividual variability. Microarray data were further validated by immunoblotting and by cholesterol and protein-thiol oxidation level determinations. Reported data provide a broad impression of the biological consequences of residing in an industrial area. ■ INTRODUCTION Industrial settlements are a major source of employment and economic development. Industrial activities, however, contin- uously release a broad range of wastes and pollutants into the environment that might represent a health risk to people residing in the area. Huelva is a city in southwestern Spain (Supporting Information, SI, Figure 1) in which an extensive industrialization process began in the 1960s. Most of its current industrial activities are based on the petrochemical sector and the production of phosphate derivatives, although a wide variety of other industrial activities such as Cu beneficiation, power generation and the production of TiO 2 , NH 3 , Cl, NaOH, and cellulose paste, among other products, are also present. 1,2 Several recent studies suggest that residing in the vicinity of those industries might induce a variety of pathologies, including cancer, cardiovascular morbidity, allergic diseases, and mortal- ity. 3,4 Evaluating the impact of an industrial environment on the health of resident organisms presents a toxicological challenge because of the complexity of the ecosystem, as this affects contaminant speciation and bioavailability and the coexistence of multiple types of xenobiotics that can mask cause-effect relationships by intraorganism interaction effects. In the past decade, the method by which ecotoxicological problems are investigated has changed dramatically, given that global assessments of toxicant-affected molecular pathways can be achieved via “omics” technologies. 5,6 DNA microarray-based global gene expression profiling provides a broad impression of organisms’ responses to environmental stressors and can potentially identify novel ecotoxicological biomarkers and thus gain insight into the underlying mechanisms of toxicity. Commercial microarrays target a limited number of organisms and exclude most species Received: July 23, 2013 Revised: January 23, 2014 Accepted: January 24, 2014 Published: January 24, 2014 Article pubs.acs.org/est © 2014 American Chemical Society 2183 dx.doi.org/10.1021/es4053973 | Environ. Sci. Technol. 2014, 48, 2183-2192