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Heterologous Microarray Analysis of Transcriptome Alterations in Mus spretus Mice Living in an Industrial Settlement Nieves Abril,*,† Julia Ruiz-Laguna,† Miguel Á ngel García-Sevillano,‡ Ana M. Mata,§ José Luis Gómez-Ariza,‡ and Carmen Pueyo† †

Department of Biochemistry and Molecular Biology, Agrifood Campus of International Excellence (ceiA3), Severo Ochoa Building, University of Córdoba, Rabanales Campus, 14071 Córdoba, 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 application 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.

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INTRODUCTION

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

Industrial settlements are a major source of employment and economic development. Industrial activities, however, continuously 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 TiO2, NH3, 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 mortality.3,4 Evaluating the impact of an industrial environment on the health of resident organisms presents a toxicological challenge © 2014 American Chemical Society

Received: Revised: Accepted: Published: 2183

July 23, 2013 January 23, 2014 January 24, 2014 January 24, 2014 dx.doi.org/10.1021/es4053973 | Environ. Sci. Technol. 2014, 48, 2183−2192

Environmental Science & Technology

Article

amounts of individual RNA samples (nine per sampling site) were pooled for microarray analysis. Microarray Experiment. Labeled cRNA was synthesized by amplifying 1 μg of total RNA with the Low RNA Input Linear Amplification Kit PLUS in the presence of cyanine 3CTP, along with the One-Color RNA Spike-in Kit, according to the manufacturer’s protocols (Agilent). Amplified cRNA was purified and quantified. Each cRNA sample was required to yield more than 1.65 μg, with a cyanine 3-CTP-specific activity of more than 8 pmol/μg, for use in hybridization. Hybridization was performed on G4122F Whole Mouse Genome Oligonucleotide Microarrays (4 × 44K, 60 mer) for 17 h at 65 °C in a rotating oven, using the Agilent Gene Expression Hybridization Kit with the GEx Hybridization Buffer HI-RPM (Agilent). Four technical replicate hybridizations were conducted for each sample. After stringent washing, the slides were scanned with an Axon GenePix 4000B microarray scanner. Data were captured with GenePix Pro 4.1 software (Molecular Devices) at a 5-μm resolution. The optimum baseline photomultiplier tube voltages were established at 450 and 500 V for SOL and PS arrays, respectively. Spot intensities and other quality control features were extracted with Agilent Feature Extraction Software version 9.5.3.1, using default parameters. Data were deposited in the gene expression omnibus platform (GEO) with the accession number GSE33663. Microarray Data Analysis. GeneSpring GX v. 9.0.2 software (Agilent) was used to analyze the microarray data. To minimize the number of false-positive genes due to the nonspecific hybridization of M. spretus cDNA with M. musculus sequences in the microarray, the threshold was increased to 5fold of the average background (median blank spot density minus the local background). The median of all spot signals on each microarray was shifted to percentile 75 to reduce microarray-wide variations in intensity (microarray normalization). The median log of the signal strength of each gene across all microarrays was subtracted from its signal value (gene normalization). Data from spots designated by the software as “present” in at least 75% of the replicates in both the SOL and PS samples were transformed to the log2 base scale and used for subsequent analyses. Genes with statistically significant expression differences were identified in an unpaired t-test with the Bonferroni family wise error rate (FWER, a type of error-control that reduces the occurrence of false positives) as the multiple testing correction (P < 0.01; asymptotic p-value computation). A list of differentially expressed genes was generated with an arbitrary fold change cutoff of >2-fold (SI Table 2). A gene ontology analysis of the differentially expressed genes was performed with GeneSpring GX v. 9.0.2 software (Agilent). The cutoff threshold for statistical significance was set at a false discovery rate (FDR) < 0.01. A literature review was also used to categorize the differentially expressed genes into functional categories. Primer Design. Primers (SI Table 3) were designed as described.21 Primers based on known M. musculus gene sequences were designed to amplify synthesized cDNA from the pooled M. spretus RNAs used in the microarray experiment. The PCR products were sequenced and deposited in the GenBank database (accession numbers, SI Table 3). These sequences were used to design primers that exactly complemented the M. spretus genes for the absolute quantification of transcript levels by real-time RT-PCR (qRTPCR). All primer pairs produced amplicons of the predicted

with environmental relevance. The hybridization of nucleic acids from nonmodel organisms onto DNA microarrays designed for phylogenetically related model species has been used as an alternative to building genomic resources for each new organism of interest. Heterologous microarray hybridization has proven effective in studies that focused mainly on identifying the responses of aquatic organisms to model pollutants in laboratory-based experiments.5,7−9 The mouse is the best-known vertebrate model organism, but the classical Mus musculus laboratory strains have a serious limitation compared to human populations because of their reduced natural genetic polymorphisms.10 In contrast, the aboriginal species Mus spretus now has a promising future in the provision of a reservoir of novel allelic variants and phenotypes that are not observed in commonly used laboratory mice.11,12 Mus spretus is widely distributed in various regions of France, Spain, Portugal, Tunisia, and Morocco, where this small mammal species achieves high population densities while consuming plants, seeds, and insects around its burrows. Therefore, within the last ten years, several laboratories have begun to use M. spretus as a bioindicator species in environmental monitoring programs through the use of different biometric,13 cytogenetic,14,15 biochemical biomarkers,16,17 absolute transcript expression signatures of selected genes,18 and global proteomic analyses.19 This work aimed to use a M. musculus whole-genome oligonucleotide microarray to evaluate the potential risk of residing in an industrial settlement, using the aboriginal species M. spretus as a bioindicator. The rationale of this study rests on the paucity of microarray experiments in mammals from natural ecosystems and the relatively high gene sequence homology between the two mouse species, as suggested by our own previous studies, among others.18,20 Animals from the industrial settlement of Punta del Sebo (PS), 5.6 km southeast of the city of Huelva, were compared to those collected 52.1 km southeast of Huelva in the heart of the Doñana National Park (one of Europe’s most important protected areas). A microarray analysis showed that many genes were differentially expressed, and a gene ontological analysis of the expression microarray data revealed altered key biological processes in the PS specimens. Up and downregulation of selected genes was confirmed by real-time RT-PCR quantification and immunoblotting.

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EXPERIMENTAL SECTION Sampling Areas and Animal Trapping. Mice were collected at a reference site (Santa Olalla Lagoon, SOL) and at a polluted industrial settlement (Punta del Sebo, PS) in SW Spain (SI Figure 1). Animals were captured with live traps and taken alive to the nearest laboratory at Doñana, where their sex and weight were determined. Nine male mice (approximately 12 g) from each sampling site were sacrificed by cervical dislocation and dissected. Individual livers and kidneys were frozen in N2 and stored at −80 °C for subsequent analyses. The Córdoba University Animal Ethics Committee approved this investigation. RNA Sample Preparation. Total RNA from individual livers was isolated according to the TRIzol method (Invitrogen), coupled with the GenElute Mammalian Total Kit (Sigma). RNA integrity was determined by electrophoresis, and concentrations were determined by spectrophotometry. Genomic DNA contamination was tested by PCR amplifications of RNA samples without prior cDNA synthesis. Equal 2184

dx.doi.org/10.1021/es4053973 | Environ. Sci. Technol. 2014, 48, 2183−2192


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Figure 1. Comparison of relative gene expression levels determined by microarray and qRT-PCR. Microarray data are shown in SI Table 2. Absolute transcript levels determined by qRT-PCR are shown in SI Table 5. Six upregulated and eight downregulated genes were placed into a miscellanea category that is not shown.

major biological processes affected by living in an industrial settlement. A commercial oligonucleotide microarray platform that contained probes against the whole laboratory mouse M. musculus genome was used to overcome the lack of available genomic sequence information for the aboriginal species M. spretus. Study Areas and Mouse Element Concentrations. PS is a heavily polluted industrial settlement in the Huelva Industrial Area (SI Figure 1). Industrial activities in PS continually release significant amounts of a very complex set of contaminants into the environment.1,24−26 In contrast, Doñana National Park is one of Europe’s most important wetland reserves, occupying an immense area of marsh, shallow streams, and sand dunes with great ecological wealth. The Santa Olalla Lagoon (SOL) is the largest pond in the dune area at the core of Doñana (SI Figure 1). Mus spretus mice were collected in November 2006 from PS and SOL for this paired sampling study. Following the rationale of previous studies,18,19 the pollution load sustained by the mice from PS was evaluated, taking as a pollution indicator the concentration of certain elements. Measurements were conducted in individual mouse livers and kidneys (SI Table 1). Compared to SOL, seven and three of the ten analyzed elements were statistically significantly concentrated in the livers and kidneys of PS animals, respectively. The hepatic and renal bioaccumulation of highly toxic metals (e.g., 28- and 15fold for Cd concentrations in the liver and kidney, respectively) was higher than that of essential elements (e.g., Fe, Cu, and Zn), which are presumably regulated by homeostatic mechanisms. In short, the element concentration data herein suggest that, compared to SOL, animals living in PS have a higher risk of exposure to contaminants from the industrial activities that surround the sampling site, which agrees with data from mice captured in previous 2002 and 2004 campaigns.18,19

sizes, and all PCR products were further verified by nucleotide sequencing. qRT-PCR. Absolute quantification of the mRNA levels by qRT-PCR was performed as described.22 cDNA was generated from 2 μg of RNA, and real-time PCR reactions were performed in quadruplicate with 50 ng/well of cDNA. All targets amplified with the same optimal PCR efficiency (100%) in the range of 20−2 × 105 pg of total RNA input, with high linearity (r > 0.99). An absolute calibration curve was constructed, and the number of transcript molecules was calculated from the linear regression of the standard curve.22 Immunoblotting. Pooled liver protein extracts were obtained as described.19,23 Proteins (25 μg/sample) were loaded and separated on 12% (MMP7, GPX3, and RARRES1 and IDI1) or 4−15% (A2M and FASN) Mini-PROTEAN TGX Stain-Free precast gels (BioRad). Following electrophoresis, the gels were UV-activated, the proteins were transferred to PVDF membranes with the Trans-Blot Turbo Transfer System (BioRad), and the membranes were blocked with iBind Solution (Novex). The primary polyclonal antibodies antiA2M, anti-GPX3, and anti-RARRES1 were obtained from Bioss USA Antibiotics, anti-FASN and anti-MMP7 from Novus Biological, and anti-IDI1 from Santa Cruz Biotechnology. The secondary antirabbit antibody was obtained from Sigma (A9169). Primary (1:100) and secondary (1:2000) antibody incubations and wash steps were performed using the iBind Western System (Novex). Blots were developed using the Clarity Western ECL Detection System (BioRad). Image captures and densitometric analyses were performed with the ChemiDoc MP Imaging system and ImageLab 4.1 software (BioRad), respectively.

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RESULTS AND DISCUSSION This work focused on assessing the potential utility of genomewide expression profiling in M. spretus for characterizing the 2185



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Figure 2. Absolute transcript levels of selected genes in individual SOL and PS mice. Each bar represents the number of mRNA molecules/pg of total RNA from the liver of a single mouse (mean ± SEM of four real-time PCR reactions). On the x-axis, mice are numbered consecutively. Numbers over the bars are the means ± SEM of transcript molecules/pg of total RNA from the nine mice at each capture site (SOL, light; PS, dark). Statistical significances were determined with Student’s t-test or a nonparametric Mann−Whitney test. Differences between SOL and PS mice were all statistically significant (P < 0.05).

Microarray Gene Expression Profiling in Free-Living M. spretus. Heterologous hybridization to DNA-based microarrays has become the method of choice for comparative transcriptome studies when species-specific platforms are not available. Sequence mismatches between the probes and target sequences constitute the primary technical challenge in heterologous microarray experiments.8,27 Overall, this problem increases with the phylogenetic distance between the species for which the microarray was constructed and the species providing the sample to which the microarray will be hybridized. Here, a cost-effective commercial microarray with 60-mer oligonucleotide probes, based on the whole M. musculus genome sequence, was used for the transcriptome analysis of the nonmodel species M. spretus. This aboriginal mouse species separated from the closely related M. musculus spp. one to three million years ago, and recent estimates suggest an average of one sequence variant between these species every 50 to 130 bp.11 Although the magnitude of this sequence divergence is not expected to significantly affect the results,27 the extent to which the analytical specificity might be compromised was minimized by maintaining high stringency in the hybridization conditions and in the subsequent microarray data analysis. Under these experimental conditions, a total of 19 871 features were successfully detected, reducing the effective size of the M. musculus microarray by approximately one-half. Although numerically diminished, the microarray detection power remained considerable. Statistical analysis (FWER < 0.01) showed that 131 genes were differentially expressed (using a cutoff of >2-fold change) in the pooled livers from PS mice, compared to the SOL specimens (SI Table 2). Among the differentially expressed genes, 50 were upregulated and 81 were

downregulated. The proteins encoded by the differentially expressed genes display a variety of cellular activities, which were broadly categorized into six functional classes: immune (18%) and stress (12%) responses, metabolism (32%), proteases (12%), transport (9%), and cell signaling (5%). Differentially expressed genes that did not match any of the six functional classes were placed into a miscellaneous category (12%). Verification of Microarray Results by Absolute qRTPCR. To verify that the candidate genes identified in the heterologous microarrays were indeed differentially expressed, we quantified the transcript copy numbers of 13 selected genes by qRT-PCR. These genes were chosen because their reported functions made them interesting candidates, they represented a variety of functional classes and they were either up or downregulated in the microarray experiment. As part of designing the qRT-PCR assay, we sequenced coding region segments of the selected gene loci from M. spretus. Sequence comparisons between M. spretus and M. musculus at these 13 loci revealed, on average, 99.2% DNA sequence similarity (SI Table 4), in agreement with the recent estimates11 mentioned above. Validation was initially performed with the pooled samples that were used in the microarray experiment. The results are shown in SI Table 5 and are presented as conventional fold-variations in Figure 1 to facilitate a comparison of the methodologies. Pooled designs are attractive in microarray experiments because they reduce the costs.28 The use of pooled samples to characterize populations can also yield more precise and less biased parameter estimations, compared to the use of individual samples.29 A potential risk is that a single aberrant sample could 2186



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rates, protein translation efficiencies, and/or the steady-state mRNA and protein abundances. As will be discussed later in greater detail, 11 differentially expressed genes encoded enzymes in the cholesterol biosynthesis pathway. Therefore, in our effort to validate the microarray results, we next determined and compared the hepatic cholesterol contents in PS and SOL specimens. The data in SI Table 6 show that the PS specimens had lower hepatic cholesterol levels (average reduction, 23%), in agreement with the downregulation of cholesterol biosynthesis gene expression. Additionally, some of the differentially expressed genes (e.g., Gpx3) in PS-dwelling mice were related to oxidative stress tolerance, in agreement with the idea that many pollutants, including pro-oxidant metals (e.g., Cu, Fe, or Cd), stimulate ROS generation.16,31 To support this interpretation, we determined the protein-thiol oxidation levels as biochemical indicator of oxidative stress.23 As shown in SI Figure 2, the PS environment significantly increased (average increase, 2-fold) the oxidation levels of the protein-thiol groups. Overall, the immunoblotting results and the cholesterol and protein-thiol oxidation levels validate our microarray data; the latter will likely serve as a valuable source of information about the biological consequences of living in an industrial settlement. Functional Relevance of the Differentially Expressed Genes. Immune Response. Twenty-four of the 131 differentially expressed genes were assigned to the immune response functional category, making this the most prevalent class of upregulated transcripts in PS mice. Overall, these differentially expressed genes were related to both innate and acquired immunity and inflammation. Immunoglobulins (Igs) are key humoral components of acquired immunity. Ten of the 24 immune-function related genes encode the light and heavy chains of Ig proteins, such as Igh (upregulated mRNA, verified by qRT-PCR). Although much of the experimental work on the ability of pollution to alter Ig production has focused on IgE, given its central role in the mucosal allergic response and the induction of allergic inflammation,32 other Ig types (e.g., IgM and IgG) have also been implicated in immunoglobulin responses to environmental pollution.33,34 Inflammation is triggered when innate immune cells detect infection or tissue injury, leading to the activation of NF-κB and other transcription factors and the subsequent transcription of target genes such as those that encode cytokines, chemokines, interferons, adhesion molecules, and extracellular matrix regulators.35,36 Mice from PS had increased mRNA levels of four interferon-induced genes (Oas1f, If i27, Tgtp, and BC023105). The upregulation of Mmp7 (validated at the protein level) and Ptger3 and the downregulation of Lect2 might also be related to inflammation. MMP7, like other MMPs, contributes to the inflammatory process by regulating physical barriers, modulating inflammatory mediators such as cytokines and chemokines, and establishing chemokine gradients.37 Ptger3 encodes a G-protein-coupled prostanoid receptor (EP3) that has been implicated in the prostaglandin E2-mediated inflammatory response.38 Finally, LECT-2 downregulation is considered an indicator of liver inflammation, given the role of this multifunctional protein in suppressing the production of inflammatory cytokines, especially TNF-α.39,40 Although inflammation is essential to host defense and tissue repair processes, unregulated or excessive inflammation can contribute to ongoing tissue injury, organ dysfunction, and chronic disease. The liver acute phase response is of particular importance to the usual effective inflammatory outcome.35

negatively impact the quality of a particular pool and hence confound the results and their interpretations. Another pivotal point is that biological replicates are crucial when estimating natural variability and thereby when defining the “biological noise” beyond which differential expression can be definitively established. On the basis of these considerations, the 13 selected transcripts were also quantified at the individual level. The data shown in Figure 2 clearly indicate that the results obtained in samples from mixture of individuals are not particularly prone to misinterpretations due to the interindividual differences in transcript levels and interindividual susceptibilities to the effects of living in the PS industrial settlement. Data validation by Western blotting, and by cholesterol and protein-thiol oxidation level determinations. Given that proteins are direct executors of life processes and protein levels are major biomonitoring end points, we next investigated whether the changes in mRNA expression were reflected at the protein level. For this purpose, Western blot experiments were performed for a total of six proteins (Figure 3). Proteins were selected according to

Figure 3. Western blotting of six proteins for which the mRNA expression levels were altered in the microarray analysis. Numbers indicate the Western blotting signal intensities normalized to the total protein contents, using a novel Stain-free technology for total protein quantification.79 Proteins were extracted from pooled livers. SOL and PS pools were generated by mixing equal amounts of homogenized liver from each of the nine male mice/sampling site used in the microarray experiment. The SOL2 and PS2 pools comprised nine and six male mice, respectively, that were captured in a previous 2004 campaign.19

multiple criteria, including their reported biological functions (immune response, oxidative stress, signaling, or lipid metabolism) and the microarray results (up or downregulation of the corresponding mRNA and fold-change values). In all cases, the protein levels verified the trends (up or downregulation) observed in the mRNA expression levels. Moreover, in four cases (MMP7, RARRES1, FASN, and IDI1), we found a very good correlation between the fold-changes in the protein and mRNA levels (Figure 3 vs SI Table 2), in agreement with the idea that mRNA level control is crucial to regulating protein production in mammals. In the remaining two cases (A2M and GPX3), the mRNA level changes were greater than the protein level changes. As discussed elsewhere,30 there might be methodological and biological reasons for such discrepancies, including differences in the protein and mRNA degradation 2187



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Acute phase (AP) proteins such as A2M exhibit multiple functions that are important for restoring homeostasis after disturbances from injury and inflammation. Traditionally, A2M has been viewed as a plasma and inflammatory fluid proteinase scavenger, but more recent evidence has revealed the abilities of A2M to bind a plethora of cytokines, growth factors and hormones and to modulate mitogen and antigen-driven T responses.35 Here, we found that mice from the PS industrial settlement displayed strong upregulation of both the A2Mencoding transcript and protein. The concomitant upregulation of Mug2 in PS mice agrees with the fact that murinoglobulin (another broad-spectrum protease inhibitor) belongs to the αmacroglobulin family. Another two upregulated genes (Serpin4ps1 and Il1r2) encode proteins with anti-inflammatory properties.41−43 Downregulated Tf f 2 expression (verified by qRT-PCR) in PS mice might also be consequent to the objective of reducing inflammation and restoring homeostasis, as TFF2 is necessary for the rapid production of the proinflammatory cytokine IL-33.44,45 Stress Response. The differentially expressed genes that were grouped into the stress response category were mainly related to oxidative stress tolerance and biotransformation, which are fundamental biological functions in contaminated environments. GPX3 (also named plasma or extracellular GPX) is a major Se-containing antioxidant enzyme that catalyzes the GSHdependent reduction of hydrogen peroxide and lipohydroperoxides. Gpx3 is expressed in various tissues, including kidney, lung, and liver, from which the protein is secreted into the plasma.46 Recently, it was shown that Gpx3 upregulation protects against ROS-induced hepatic injury in mice.47 Therefore, increased Gpx3 expression (verified by qRT-PCR and validated at the protein level) might protect PS animals from excessive oxidative damage, as indicated by the elevated oxidation of their protein-thiol groups. A growing body of evidence suggests that oxidative stress, the inflammatory response and endoplasmic reticulum (ER) stress are linked.48 DERL3 is a key component of the ER-associated degradation machinery. Derl3 expression is upregulated in cells that undergo ER stress, in which misfolded glycoprotein degradation is accelerated.49 Hence, the upregulation of Derl3 mRNA in PS mice might be important for ER homeostasis maintenance. CTH is a key transsulfuration enzyme and a critical factor in GSH biosynthesis. Accordingly, the concomitant Cth upregulation might also be important for protecting PS specimens against oxidative stress and GSH depletion caused by ROS generators and protein misfolding during ER stress.50,51 HSPB1 is a ubiquitously expressed small heat shock protein which expression is induced in response to a wide variety of unfavorable physiological and environmental conditions. HSPB1-mediated protection includes redox homeostatic maintenance, protein homeostatic restoration, cell survival promotion and actin-cytoskeleton stabilization (reviewed in52,53). As expected from these protective roles, the Hspb1 mRNA level was upregulated in PS mice. In mammals, iron uptake by TFR is tightly regulated to maintain the cellular labile iron pool at the lowest acceptable level, thereby preventing excessive oxidative damage from the excess generation of free radicals via Fenton/Haber-Weiss reactions.54 Accordingly, the downregulation of Tfrc mRNA in PS mice is consistent with previous findings showing that TFR expression was reduced under sustained ROS stress conditions.55

Aldo-keto reductases (AKRs) are NADPH-dependent oxidoreductases that catalyze the reduction of a wide variety of carbonyls. The AKR1B subfamily members are generally considered detoxification enzymes that share the ability to reduce many redundant substrates, including aldehydes derived from lipid peroxidation and xenobiotics.56 In agreement with these biological functions, the Akr1b7 and Akr1b8 transcripts were both upregulated in PS mice. Among the AKRs, human AKR1B10 (the ortholog of mouse AKR1B8) is of special interest because of its high catalytic efficiency in the reduction of all-trans-retinaldehyde. Through this activity, AKR1B10 participates in the regulation of retinoic acid (RA) biosynthesis and is therefore involved in RA signaling role. Accordingly, the upregulated expression of AKR1B10/AKR1B8 (verified by qRT-PCR) under oxidative stress conditions might switch its beneficial role in protecting cells against lipid peroxidation to a deleterious role that promotes cell proliferation and fosters tumorigenesis through RA deprivation.56 This dual biological role might explain the concomitant downregulation of mRdh11 mRNA in PS mice. Mouse liver RDH11 catalyzes the reduction of all-trans-retinaldehyde but does not catalyze the reverse reaction, in contrast to its human counterpart.57 Consequently, the hepatic downregulation of mRdh11 might be a compensatory mechanism to maintain the RA-signaling pathway in a cellular environment where Akr1b8 upregulation could trigger reduced RA biosynthesis. Cytosolic sulfotransferases (SULTs) are phase II conjugation enzymes that catalyze the sulfonation of a broad range of substrates and thus play an important role in xenobiotics metabolism and the homeostasis of potent endobiotics such as catecholamine neurotransmitters. Sult1d1 is a novel glucocorticoid-responsive gene in the mouse liver, where it acts to attenuate elevated catecholamine activity during the stress response.58 Accordingly, Sult1d1 upregulation (verified by qRTPCR) might be critical for maintaining metabolic homeostasis in animals that dwell in the PS industrial settlement. Although sulfonation is generally considered a detoxification reaction, some sulfonate conjugates are reactive electrophilic metabolites that can lead to mutagenesis and carcinogenesis. In this context, we can hypothesize that Sult2a2 and Sult2a5 downregulation in PS mice might be a mechanism to combat the genotoxicity of hydroxymethyl polycyclic aromatic hydrocarbons, given the involvement of SULT2 sulfotransferases in the bioactivation of these compounds.59 Metabolism. Genes grouped into the broad functional metabolism category were mostly downregulated and associated with lipid metabolic pathways. Liver disease is a major health issue that is characterized by several pathological changes, with steatosis (fatty liver) representing a common initial pathogenic step. Recent studies have indicated that exposure to fine ambient particulate matter60 and environmentally relevant chemicals61 can cause fatty liver. Some of the differentially expressed genes in PS animals encoded proteins which altered expression and might compromise the delicate balance between lipid influx and fat clearance, the hallmark of hepatic steatosis.62 For instance, the monoacylglycerol acyltransferase pathway is one of two convergent pathways for triacylglycerol (TAG) biosynthesis. Mogat1 expression was strongly upregulated (15 to 48-fold) in mouse models of hepatic steatosis.63,64 A similar but less dramatic upregulation (10-fold by qRT-PCR) of Mogat1 was quantified in the PS mouse livers. Hepatic TAG accumulation could also result from a relative reduction in very low-density lipoprotein (VLDL) 2188



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mediating chemical signaling. MUP1 was recently identified to regulate liver metabolism by inhibiting the expression of key gluconeogenic and lipogenic genes such as those that encode glucose-6-phosphatase and fatty acid synthase.74 Accordingly, in PS animals, the upregulation of MUP transcripts (7-fold for Mup5 by qRT-PCR) was associated with a concomitant downregulation of G6pc and Fasn transcripts. RARRES1 (a novel retinoid-responsive gene) was recently recognized as a core regulator of liver fibrosis, the hallmark of all chronic inflammatory liver pathologies. Accordingly, Rarres1 mRNA and protein expression is readily upregulated in carbon tetrachloride-challenged mice.75 In agreement with these previous observations, we quantified elevated RARRES1encoding transcript and protein levels (3.4- and 2.2-fold, respectively) in PS mice. The concomitant upregulation of Rarres1 and Cth transcripts in PS specimens agrees with the identification of Cth as a downstream target of RARRES1 in hepatic cells.75 Activins and inhibins are members of the TGF-β superfamily of growth factors that participate in diverse biological processes, including reproduction, development, hematopoiesis, inflammation, and tumor development. Recently, dramatic Inhbb mRNA induction (encodes the activin/ inhibin subunit βB) was demonstrated in the livers of lipopolysaccharide-challenged mice (an agent commonly used for its immunostimulatory properties).76 Accordingly, Inhbb transcript upregulation (3.5-fold in qRT-PCR verification assays) and the immune response stimulation discussed above might be interrelated in PS mice. Also of interest is the finding that PS mice display downregulated levels of transcripts (8-fold for Try5 by qRT-PCR) that encode proteases, including twelve different serine proteases. We speculate that these transcriptional changes might be related to the induction of genes that encode protease inhibitors (such as A2m, Mug2, and Serpin4ps1) to control the inflammatory response.77,78

secretion. Hepatic microsomal triglyceride transfer protein (MTP) plays an essential role in VLDL production, and the livers of MTP-deficient mice have increased TAG levels.65 Protein disulfide isomerase (PDI) is a key structural subunit of MTP. PDIA2 has been detected in several mouse tissues other than the pancreas, including the liver.66 Although the biological role of PDIA2 in the mouse liver remains elusive, we can speculate that Pdia2 downregulation might promote TAG accumulation by reducing MTP activity. MTP inhibition/ deficiency is also known to lead to liver cholesterol accumulation,65 an effect that might be intensified in PS mice by the concomitant downregulation of Pcsk9, a gene that encodes a serine protease that destroys the LDL receptor, thereby affecting cholesterol metabolism.67 Pcsk9 is positively regulated by SREBP-2, a transcription factor that remains inactive in cholesterol-replete cells.67 Hmgcr encodes the enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis. Hmgcr, along with more than 20 other genes encoding enzymes that mediate cholesterol uptake (such as PCSK9) and synthesis, is also positively regulated by SREBP2.68,69 In addition to Hmgcr, 10 other genes encoding enzymes in the cholesterol biosynthesis pathway were downregulated in PS mice, with transcript level reductions ranging from 3.5 to 13.8-fold (4.4 to 14.2-fold for Hmgcr and Idi1, respectively, by qRT-PCR). These findings were further validated by the reduced cholesterol and IDI1 protein levels (23% and 12-fold, respectively, on average) in the livers of PS mice, compared to SOL specimens. Concomitantly with the downregulated expression of cholesterol biosynthesis genes, PS mice displayed reduced levels of both the FASN-encoding transcript and protein. While SREBP-2 activity is more restricted to regulating genes involved in cholesterol homeostasis, SREBP-1 preferentially controls genes (such as Fasn) involved in fatty acid (FA) biosynthesis. Despite differences in their transcriptional targets, the proteolytic activation of these two SREBP isoforms is regulated by cholesterol through a common mechanism.70 This common post-transcriptional regulation blocks their activation upon cholesterol accumulation. Although our study did not address the possible differences in SREBP activation between PS and SOL animals, these differences might be a plausible explanation for the differential expression of the abovementioned SREBP-dependent genes. Interestingly, a recent study has firmly established a role for the aryl hydrocarbon receptor (AhR) as a regulator of the cholesterol biosynthetic pathway.71 Accordingly, TCDD, a prototypical potent AhR ligand, is known to downregulate the expression of genes (e.g., Hmgcr, Sqle, Sc4 mol, Fdf t1, Lss, Fdps, and Idi1) involved in cholesterol biosynthesis.71,72 Because mice that express a DRE (dioxin response element)-binding mutant AhR remain capable of modulating the expression of cholesterol-synthesis genes upon ligand activation, it has been hypothesized that AhR might interact with SREBP-2 to attenuate the hepatic transcription of cholesterol biosynthetic genes. This hypothesis might also apply to SREBP-1, 71 thus explaining the concomitant downregulation of critical FA synthesis genes (such as Fasn) in experimental animals upon exposure to TCDD.72,73 Representative Members of Other Functional Categories and Crosstalk. Transcripts encoding major urinary proteins (MUPs) were all upregulated in PS mice. MUP1 (the beststudied MUP) is a lipocalin family member that is abundantly secreted into the bloodstream by the liver. Circulatory MUP1 binds to lipophilic molecules, including pheromones, thereby

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ASSOCIATED CONTENT

S Supporting Information *

Location of PS and SOL sampling areas (Table S1); statistically significant (FWER < 0.01) differentially expressed genes with >2-fold change (Table S2); primers used in this work (Table S3); sequence comparison of selected Mus spretus and Mus musculus genes (Table S4); absolute transcript levels of selected genes in pooled RNA samples (Table S5); and cholesterol contents in pooled livers (Table S6). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +34 957 218 082; fax: +34 957 218 592; e-mail: [email protected]. Author Contributions

N.A.D. and J.R.-L. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financed by the Junta de Andaluciá (CVI-3829) and the Spanish Government (CTM2012-38720-C03-02). REFERENCES

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