Toxicology and Applied Pharmacology 273 (2013) 269–280
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Toxicology and Applied Pharmacology
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Toxicogenomic outcomes predictive of forestomach carcinogenesis followingexposure to benzo(a)pyrene: Relevance to human cancer risk☆
Sarah Labib, Charles H. Guo, Andrew Williams, Carole L. Yauk, Paul A. White, Sabina Halappanavar ⁎Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario K1A 0K9, Canada
Abbreviations: AhR, aryl hydrocarbon receptor;benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide; BW, bodrate; GO, Gene Ontology; GSH, glutathione; MHC, majoNKT, natural killer T cells; PAH, polycyclic aromatic hydrreal time PCR.☆ This is an open-access article distributed under the tAttribution-NonCommercial-No Derivative Works Licommercial use, distribution, and reproduction in any mauthor and source are credited.⁎ Corresponding author at: Environmental Health S
Article history:Received 3 April 2013Revised 17 May 2013Accepted 21 May 2013Available online 2 June 2013
Keywords:Polycyclic aromatic hydrocarbonsCancerMicroarraysHuman relevanceAntigen processing and presentationImmunoproteasome
Forestomach tumors are observed inmice exposed to environmental carcinogens. However, the relevance of thisdata to humans is controversial because humans lack a forestomach. We hypothesize that an understanding ofearlymolecular changes after exposure to a carcinogen in the forestomachwill providemode-of-action informa-tion to evaluate the applicability of forestomach cancers to human cancer risk assessment. In the present studywe exposed mice to benzo(a)pyrene (BaP), an environmental carcinogen commonly associated with tumors ofthe rodent forestomach. Toxicogenomic tools were used to profile gene expression response in the forestomach.Adult Muta™Mouse males were orally exposed to 25, 50, and 75 mg BaP/kg-body-weight/day for 28 consecutivedays. The forestomach was collected three days post-exposure. DNA microarrays, real-time RT-qPCR arrays, andprotein analyses were employed to characterize responses in the forestomach. Microarray results showed alteredexpression of 414 genes across all treatment groups (±1.5 fold; false discovery rate adjusted P ≤ 0.05). Significantdownregulation of genes associatedwith phase II xenobiotic metabolism and increased expression of genes impli-cated in antigen processing and presentation, immune response, chemotaxis, and keratinocyte differentiationwere observed in treated groups in a dose-dependent manner. A systematic comparison of the differentiallyexpressed genes in the forestomach from the present study to differentially expressed genes identified inhuman diseases including human gastrointestinal tract cancers using the NextBio Human Disease Atlas showedsignificant commonalities between the two models. Our results provide molecular evidence supporting the useof the mouse forestomach model to evaluate chemically-induced gastrointestinal carcinogenesis in humans.
A review of the Carcinogenic Potency Database (http://toxnet.nlm.nih.gov/cpdb/) and the USNational Cancer Institute/National ToxicologyProgram (http://ntp-server.niehs.nih.gov) databases reveals that expo-sure to approximately 120 different substances results in the develop-ment of cancerous lesions in the rodent forestomach (Proctor et al.,
2007). Indeed, the forestomach is a target for carcinogenesis in rodentsfollowing oral exposure to various environmental chemicals. However,the human health relevance of this type of cancer is unclear becausehumans lack a forestomach (Proctor et al., 2007).
Anatomically, the rodent forestomach exhibits similarities to boththe human esophagus and stomach. Detailed analyses of cancers ofthe forestomach reveal that these tumors are initiated by hyperplasiaof the forestomach squamous epithelial cells that form preneoplasticlesions (Fukushima et al., 1997). These lesions progress into benignpapillomas and metastatic carcinomas over time, which histologicallyresemble squamous cell carcinomas of the human esophagus (Nyrén andAdami, 2002), stomach (Callery et al., 1985), colon (Landau et al., 2007),and anal canal (Szmulowicz and Wu, 2012). However, forestomachtumors are strictly of squamous cell origin, whereas esophageal cancerscan also originate from glandular columnar epithelial cells in the formof adenocarcinomas, suggesting multiple mechanisms in the develop-ment of esophageal tumors in humans (Proctor et al., 2007). In addition,more than 95% of human stomach cancers are adenocarcinomas thatoriginate from glandular columnar epithelial cells or poorly differen-tiated cells (Tsukamoto et al., 2007), and are thus distinct from rodentforestomach tumors (Proctor et al., 2007). Although there appear tobe some fundamental differences between the aforementioned tumortypes, it seems reasonable to contend that the molecular initiating
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events leading to cancers in the rodent forestomach and the humanstomach share commonalities. A detailed and systematic characteri-zation of the carcinogenic modes of action in the rodent forestomachwill provide an improved context for evaluating the biological rele-vance of rodent forestomach tumor data for human health riskassessment.
Benzo(a)pyrene (BaP), a known human carcinogen (IARC, 2012), isa polycyclic aromatic hydrocarbon (PAH) that is produced during theincomplete combustion of organic materials from various sources in-cluding wood and tobacco smoke, vehicle exhaust, residential heating,electric power, and cooking. Human exposure to BaP occurs primarilythrough oral consumption of BaP-containing foods (Hettemer-Freyand Travis, 1991). BaP requires metabolic activation by members ofthe cytochrome P450 family of enzymes, which can generate severalDNA-reactive metabolites including BaP-7,8-dihydrodiol-9,10-epoxide(BPDE). These metabolites are capable of forming covalent adductswith proteins andDNA. If left unrepaired, DNA damage can causemuta-tions leading to impaired gene function. We and others have demon-strated the formation of DNA adducts following exposure to BaP in avariety of tissues, including the gastrointestinal tract (Lemieux et al.,2011), which is a primary site of contact following oral gavage, andalso in distant organs, such as the lungs and liver (Halappanavar et al.,2011; Labib et al., 2012; Malik et al., 2012). In experimental animals,exposure to BaP via feed or gavage predominantly and consistentlyleads to the development of squamous cell papillomas in the fore-stomach that primarily originate from the epithelial cell lining (Culpet al., 1998; Wester et al., 2012). Although genotoxicity is the primarymode of action of BaP in virtually all tissues, we previously usedtoxicogenomics to demonstrate that the nature and extent of molecularresponses at the gene and protein level are somewhat different betweentissues (Halappanavar et al., 2011; Labib et al., 2012; Malik et al., 2012;Yauk et al., 2011). For example, a comparison of gene expression profilesin mouse lung and liver following acute BaP exposure (i.e. 150 or300 mg/kg-body-weight/day by oral gavage for 3 days) revealedbroad commonalities, including the activation of pathways involved inoxidative stress, xenobiotic metabolism, AhR signaling, and the DNAdamage response (Halappanavar et al., 2011). However, significant in-hibition of B-cell receptor signaling was a predominant perturbationuniquely found in the lungs of BaP-exposed mice (Halappanavar et al.,2011). In addition, the magnitude of the overall transcriptionalresponse (fold changes and the total number of genes) is much greaterin the lungs than in the liver. We also recently demonstrated that DNAadduct levels are higher in the lungs of BaP treated mice as comparedwith the liver, but that mutation frequencies in these two tissues arequite similar (Labib et al., 2012). In alignment with our findings, Unoet al. (2004) showed tissue-specific roles of Cyp1a1 using Cyp1a1knockout mice. These authors showed that Cyp1a1 expression in theintestine plays an important role in detoxification, whereas Cyp1a1activation in the liver is associated with DNA damage-induced livercarcinogenesis (Uno et al., 2004). These studies collectively demonstratethat although genotoxicity is a primary component of themode of actionof BaP-mediated carcinogenesis, differences in the underlying molecularresponses to BaP likely contribute to the observed tissue-specificityduring tumorigenesis.
The present study employed genomic and bioinformatic tools toidentify early molecular initiating events that contribute to tumorigen-esis in the forestomach, with the overarching objective of exploring thebiological relevance of forestomach tumors for human health riskassessment. More specifically, we employed global gene expressionprofiling of forestomach tissue from mice exposed to BaP at dosesthat are known to induce tumors in the mouse forestomach (Culpet al., 1998). Adult male Muta™Mouse were exposed to 25, 50, and75 mg/kg-bw/day of BaP for 28 consecutive days via oral gavage,and the top dose has previously been shown to cause squamouscell carcinomas, papillomas, and hyperplasia in the forestomach(Hakura et al., 1998). Mice were sacrificed 3 days after the last
exposure. Global transcription response was analyzed in detail inorder to identify the expression changes in biological pathways associ-ated with cancer formation in the forestomach. BaP-induced gene ex-pression profiles were compared with transcriptomic profiles ofhuman diseases (e.g., human gastrointestinal tract cancer) using theNextBio Human Disease Atlas.
Methods
Animal treatment. Mouse exposures and sample collection proce-dures are described in detail previously (Labib et al., 2012; Lemieuxet al., 2011; Malik et al., 2012). The Muta™Mouse contains around29 +/− 4 copies of λgt10lacZ shuttle vector, a non-transcribed insert,stably integrated in the mouse genome (Shwed et al., 2010), thuspermitting in vivo lacZ mutant frequency analysis. The transgenic in-sert, which contains lacZ, is employed as an in vivo mutation target.Briefly, 25-week old male Muta™Mouse were individually housed inplastic film isolators, providedwithwater and food (2012 Teklad Globalstandard rodent diet) ad libitum, and were subjected to a 12 h light/12 h dark cycle. Mice were divided into four experimental groupsconsisting of 5 animals each: 0 (control), 25 (low dose), 50 (mediumdose) and 75 (high dose) mg/kg-bw/day of BaP (Sigma-Aldrich, Oakville,ON, Canada) dissolved in olive oil. Mice were exposed daily by oralgavage for 28 consecutive days. The control group received only oliveoil, the vehicle control. We acknowledge that the doses used in thepresent study are high compared to the expected daily intake ofBaP via food in the United States (Hattemer-Frey and Travis, 1991).However, these high doses permit a toxicogenomic investigation oftoxicological mechanisms underlying BaP-induced effects at muchearly post-exposure time points. The animals were sacrificed by cardiacpuncture under isofluorane anesthesia on day 3 following the last expo-sure. The forestomach was excised, flash-frozen in liquid nitrogen, andstored at−80 °C. Care and maintenance of themice in this experimentwere approved by the Health Canada Animal Care Committee.
Tissue RNA extraction and purification. Total RNA was isolated froma random section of the forestomach tissue as described in Labib et al.(2012). In brief, a small random section of the mouse forestomach washomogenized immediately in TRIzol reagent (Invitrogen, Carlsbad, CA,USA) using the Retsch Mixer MM 400. The RNA was isolated usingchloroform and precipitated using isopropyl alcohol. The RNA wassubsequently purified using RNeasyMini Plus kits (Qiagen,Mississauga,ON, Canada). All RNA samples showed anA260/280 ratio between 2.0 and2.2, and an A260/230 ratio between 1.7 and 2.3. The integrity of the RNAsampleswas analyzed using an Agilent 2100 Bioanalyzer (Agilent Tech-nologies, Mississauga, ON, Canada). All samples had an RNA integritynumber above 6.6 and were all used for microarray analysis.
Microarray hybridization and analysis. Total RNA (200 ng) fromeach individual mouse forestomach sample in each treatment groupand Universal Mouse Reference RNA (UMRR, Stratagene, Mississauga,ON, Canada) was used to synthesize cDNA and cyanine-labeled cRNAusing the Agilent Linear Amplification Kit (Agilent Technologies Inc.,Mississauga, ON, Canada). Cyanine-labeled cRNA was in vitro tran-scribed using T7 RNA polymerase and purified using RNeasy Mini Kits(Qiagen, Mississauga, ON, Canada); experimental samples were labeledwith Cyanine-5 and the UMRR was labeled with Cyanine-3. 300 ng oflabeled cRNA from each experimental sample was hybridized with thesame amount of labeled reference RNA to Agilent Sureprint G3 MouseGE 8x60K microarrays (Agilent Technologies Inc., Mississauga, ON,Canada) at 65 °C overnight (17 h) in the Agilent SureHyb hybridizationchamber. The arrays were washed and scanned on an Agilent G2505BScanner according to the manufacturer's recommendations. Data wereextracted using Feature Extraction 10.7.3.1 (Agilent Technologies, Inc.,Mississauga, ON, Canada).
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Data normalization and analyses were conducted in the R environ-ment (R-Development-Core-Team, 2010). Briefly, the background fluo-rescence was measured using the negative control 3xSLv1 probes;probes with median signal intensities less than the trimmed mean(trim = 5%) plus three trimmed standard deviations of 3xSLv1 probewere flagged as absent (i.e. within the background signal). Probes wereconsidered present if at least four of the five samples within a conditionhad signal intensities greater than three trimmed standard deviationsabove the trimmed mean of the 3xSLv1 probes (background signal).Data were normalized using LOWESS (Yang et al., 2002), and ratiointensity plots and heat maps for the raw and normalized datawere constructed to identify outliers. Three samples were removedfrom the analysis based on clustering (one sample in each of the25, 50, and 75 mg/kg-bw/day dose groups). The final sample sizeconsisted of n = 5 in control, n = 4 in 25 mg/kg-bw/day, n = 4 in50 mg/kg-bw/day, n = 4 in 75 mg/kg-bw/day groups. Differentiallyexpressed transcripts (upregulated or downregulated relative to theolive oil treated control mouse forestomach) were determined usingthe MAANOVA library in R. The statistical model included the fixedeffects slide and treatment condition, and was applied to the log2of the relative intensities. The Fs statistic (Cui et al., 2005) wasused to test for treatment effects. The P values for all statisticaltests were estimated by the permutation method using residualshuffling, followed by adjustment for multiple comparisons usingthe false discovery rate (FDR) approach (Benjamini and Hochberg,1995). The fold change calculations were estimated as described previ-ously (Malik et al., 2012). Significant genes were selected based onFDR-adjusted P value (FDR P) ≤ 0.05 for any BaP exposed versus controlcontrast. All microarray data have been deposited in the NCBI GeneExpressionOmnibus database and can be accessed through the accessionnumber GSE43438.
Bioinformatic and pathway analysis. Following microarray normali-zation, all genes that passed the FDR P ≤ 0.05 and fold change ≥ 1.5cut-off were considered for further analysis using various bioinfor-matic and pathway analysis tools to identify the biological functionsor processes in the forestomach perturbed in response to BaP. TheDatabase for Annotation, Visualization, and Integrated Discovery(DAVID) (Huang da et al., 2009a, 2009b) Functional AnnotationTools (clustering and charts) were used to identify Gene Ontology(GO) terms associated with the differentially expressed gene list.Genes significantly differentially expressed in each of the significantclusters were plotted on polar plots using SigmaPlot for Windowsversion 12.0 build 12.0.0.182. In addition to the functional classification,DAVID was used to visualize the differentially expressed genes in theKyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Thebiological and molecular functions of the significantly differentiallyexpressed genes following BaP treatment in the forestomach werefurther analyzed and explored in Ingenuity Pathway Analysis (IPA,Ingenuity Systems, Redwood City, CA, USA) and MetaCore (ThomsonReuters, http://www.genego.com/metacore.php). The significance ofthe association between the dataset and the canonical pathways andfunctions in IPA was measured using the Fisher's exact test. IPAUpstream Regulator Analysis was used to identify the cascade of up-stream transcriptional regulators that explain the observed gene ex-pression changes in the forestomach. Only regulators with a Z-scoreabove 2.0 were considered in this analysis. MetaCore process networksidentified the networks associated with GO processes that were mostsignificant in this dataset.
Mining of public databases for human disease correlation. All statisti-cally significant and differentially expressed genes from all dosegroups were mined against the genomic data repositories in NextBio(http://nextbio.com). Disease prediction was performed using the75 mg/kg-bw/day dose group. Data were compared to curateddatasets available in NextBio to identify published studies of diseases
with similar gene profiles, gene ranking, and consistency in fold changedirectionality. Pairwise gene signature correlations and rank-basedenrichment statistics were employed to calculate the NextBio scoresfor each disease. The disease that ranked the highest in comparisonwithBaP exposurewas given a score of 100 and the restwere normalizedaccordingly. The meta-analysis function was used to compare the signif-icantly differentially expressed genes associated with the most enrichedgene ontologies from the BaP exposed forestomach to 59 gastrointestinaltract cancer biosets from curated databases available in NextBio (Supple-mentary Table 1), including esophageal cancers, gastric cancers, intestinalcancers, and colon cancers.
Quantitative real time (qRT)-PCR array validation. Eighty-four geneswere selected for further validation by quantitative real-time RT-qPCRusing custom RT2 Profiler PCR Arrays and a BioRad CFX96 real-timePCR detection system. Genes were selected based on their statisticalsignificance, and their relevance to biological phenomena of interestand relevance to the project. A custom RT2 Profiler PCR Array plate, theRT2 First Strand Kit and RT2 SYBR® Green qPCR Mastermix (QIAGENSciences, Maryland, USA) were used. Reference genes for normalizationwere selected based on their stable expression levels in the treated andcontrol samples. Ct values for each gene were normalized to actin β(Actb) and glyceraldehyde 3-phosphate dehydrogenase (Gapdh), themost stable reference genes. A threshold value was set to 102. Onlysamples that were included in the microarray analysis were used forvalidation. Furthermore, an additional 2 samples (one in each of the50 and75 mg/kg-bw/daydose groups)were removedbased on improperclustering. Given this criteria, the final validation group consisted ofsample size of n = 5 in control, n = 4 in 25 mg/kg-bw/day, n = 3 in50 mg/kg-bw/day, n = 3 in 75 mg/kg-bw/day groups.
Western blot analysis. Total protein from the frozen mouse fore-stomach experimental and control tissues was extracted usingBio-Plex Cell Lysis Kits (BioRad Laboratories, Mississauga, ON, Canada)containing a protease inhibitor cocktail (1:200) (Sigma-Aldrich,Oakville, ON, Canada) and a phosphatase inhibitor cocktail (PhosSTOP,ROCHE, Laval, QC, Canada). Total protein was quantified using a Bradfordprotein assay kit (BioRad Laboratories, Mississauga, ON, Canada).30–50 μg of total protein was run on 4–15% SDS/PAGE gels (BioRadLaboratories, Mississauga, ON, Canada) and was transferred onto apolyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) bywet transfer for 1 h at 100 V. Anti-proteasome 20S LMP2 (1 μg/ml;Abcam Inc., Toronto, ON, Canada) and anti-MECL-1 (E20) (1:200;Santa Cruz Biotechnology, Dallas, TX, USA) antibodies were used totest for immunoproteasome activation. Anti-20S proteasome β1(FL-241) (1:200; Santa Cruz Biotechnology) and anti-proteasome 20Sβ2 subunit (MCP165) (1:1000; Enzo Life Sciences, Brockville, ON,Canada) were used to monitor changes in proteasome composition.All expression values were normalized relative to β-actin (1:2500;Cell Signaling Technologies, Danvers, MA, USA) in Image Lab softwareversion 4.1 build 16 (BioRad Laboratories, Mississauga, ON, Canada).
Results
Daily exposure to 25, 50, and 75 mg/kg-bw/day BaP for 28 consecu-tive days elicited no overt signs of toxicity and there was no significantloss in body weight for any of the exposed mice compared tovehicle-treated controls (Lemieux et al., 2011).
General overview of forestomach gene expression profiles
Repeated oral gavage with BaP induced significant changes in theexpression of many genes in the forestomach. MAANOVA analysis re-vealed a total of 497 probes associatedwith 414 specific genes differen-tially expressed (up- or down-regulated) with a fold change ≥ 1.5 ineither direction, and an FDR P ≤ 0.05 in at least one treatment group
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(Supplementary Table 2). Compared to the vehicle controls, 12, 158,and 487 unique probes associated with 9, 135, and 408 uniquegenes were significantly differentially expressed in the 25, 50, and75 mg/kg-bw/day dose groups, respectively (Supplementary Fig. 1).Hierarchical cluster analysis of all significant, differentially expressedgenes (FDR P ≤ 0.05 and fold change ≥ 1.5) revealed that the treatmentgroups clustered separately from controls, thus a clear treatment effectwas observed as a result of exposure to BaP (Supplementary Fig. 2).
Biological function and pathway analysis of significantly differentiallyexpressed genes
GO and pathway analyses were employed to assign biological pro-cesses and functional categories to genes whose expression was signifi-cantly affected by BaP in DAVID, KEGG, IPA, and MetaCore.
Using the functional annotation tool in DAVID, significantly differen-tially expressed geneswere categorized into specific biological processes,cellular compartments, and molecular functions. The resulting clustersconsisting of GO terms were sorted based on DAVID enrichment scoreand were considered significant only if they exhibited Benjamini-corrected P ≤ 0.05 (Fig. 1). With this cut-off, four main GO clusterswere identified: antigen processing and presentation [GO:0019882],immune response [GO:0006955], chemotaxis [GO:0006935], andepithelial cell differentiation [GO:0030855]. Each of these biologicalprocesses was further expanded to identify the extent of enrichmentand coverage.
The immune response classification consisted of 27 and 45 genesin the 50 and 75 mg/kg-bw/day dose groups, respectively, and wasrepresented by defense response [GO:0006952], immune response[GO:0006955], and response to virus [GO:0009615]. The antigenprocessing and presentation classification was enriched with a totalof 3, 14, and 40 genes in the 25, 50, and 75 mg/kg-bw/day dose groups,respectively. Within this classification, processes such as antigen pro-cessing and presentation via major histocompatibility complex (MHC)
Fig. 1. Step-wise organization of the most significant Gene Ontology processes perturbed inantigen processing and presentation to epithelial cell differentiation. Polar plots show the foldcategory (red circle) subdivided into individual pathways/networks associated with the genlines and circles indicate response in 25 mg/kg-bw/day, blue in 50 mg/kg-bw/day, and red in
class I [GO:0002474], response to virus [GO:0009615], cell surface[GO:0009986], antigen processing and presentation [GO:0019882],MHC protein complex [GO:0042611], MHC class I protein complex[GO:0042612], antigen processing and presentation of peptide antigen[GO:0048002] were over represented. The epithelial cell differentiationconsisted of 10 and 19 differentially expressed genes in the 50 and75 mg/kg-bw/day dose groups, respectively, and was enrichedwith GO terms such as cornified envelope [GO:0001533], ectodermdevelopment [GO:0007398], epidermis development [GO:0008544],epidermal cell differentiation [GO:0009913], keratinocyte differenti-ation [GO:0030216], epithelial cell differentiation [GO:0030855],keratinization [GO:0031424], intermediate filament-based process[GO:0045103], intermediate filament cytoskeleton organization[GO:0045104], and epithelial development [GO:0060429]. Lastly,the chemotaxis classification was associated with 2, 5, and 24 genes inthe 25, 50, and 75 mg/kg-bw/day dose groups, respectively, and wasrepresented by chemotaxis [GO:0006935], inflammatory response[GO:0006954], chemokine activity [GO:0008009], response towounding[GO:0009611], taxis [GO:0042330], and chemokine receptor binding[GO:0042379]. Detailed analysis of the entire list of differentiallyexpressed genes implicated in these processes revealed a toxic responsegradient involving activation of immune response and inflammation,thus reflecting a host defense response to BaP exposure. Induction ofthe keratinization process was observed primarily in the high dosegroup and is suggestive of activation of cellular transformation processesnormally observed during carcinogenic transformation. Fig. 1 demon-strates this gradual transition. GO clusters in Fig. 1 display genes associ-ated with all child GO terms. Genes common to more than one GOcluster are displayed with the most biologically relevant cluster.
Pathway analysis of all the differentially expressed genes in alldose groups was conducted using KEGG pathways in DAVID, IPACanonical Pathways, and MetaCore Network Processes (SupplementaryFig. 3). A total of seven KEGG pathways showing a significant (Benjaminicorrected P ≤ 0.05) enrichment according to DAVID, ten IPA canonical
the BaP-exposed forestomach showing dose-dependent transition in the response fromchanges associated with the significantly differentially expressed genes in each ontologyes (orange rectangles). Arrows indicate causal relationships between categories. Green75 mg/kg-bw/day dose groups.
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pathways (P ≤ 0.05), and nine MetaCore network processes (P ≤ 0.05),were significantly enriched in at least two treatment groups (Sup-plementary Fig. 3). Unique pathways in each dose group includedinflammation-related signaling at the 25 mg/kg-bw/day dose group, nat-ural killer T (NKT) cell mediated cytotoxicity at the 50 mg/kg-bw/daydose group, and p53 signaling and glutathione metabolism at the75 mg/kg-bw/day dose group. Supplementary Fig. 3 shows all of thepathways identified using each analysis tool.
Detailed analysis in IPA revealed that all of the differentiallyexpressed genes and the associated biological processes are regulatedby a finite number of upstream regulators, including cytokines, tran-scriptional factors, kinases, and receptors (Supplementary Table 3).The primary upstream regulator was interferon γ (IFNγ), which is acytokine with immunoregulatory properties. IFNγ was associated with69 differentially expressed genes in our dataset, and the expressionprofiles of these genes reflect IFNγ activation (Fig. 2). The analysisalso revealed the inhibition of the transcriptional regulator tripartitemotif containing 24 (Trim24), which is associated with 28 differentiallyexpressed genes, and p53, which is associated with 21 genes.
Disease prediction
In order to identify the expression patterns in the BaP-exposedforestomach that may be relevant to human diseases, differentiallyexpressed genes from the 75 mg/kg-bw/day BaP dose group wereanalyzed using the NextBio Human Disease Atlas. This analysisrevealed bacterial infectious diseases, respiratory disorders, parasiticdiseases, and viral infectious diseases as the top four human diseasescorrelated with mouse BaP exposed forestomach expression profiles(Supplementary Table 4). Cancer was the 18th most correlated disease,supported by 641 studies. We then queried all of the significant genes(FDR P ≤ 0.05 and fold change ≥ 1.5) in the BaP-treated forestomachassociated with antigen processing and presentation, immune re-sponse, chemotaxis, and keratinocyte differentiation against curated
Fig. 2. (A) Graphical representation of upstream regulators and the significantly differentiaRegulator analysis. The IFNγ, P53, and NFκB regulators were selected for their relative signNetwork (IPA) analysis of activated and inhibited regulators significant in the BaP exposelines indicate indirect interaction.
studies in NextBio related to human esophageal (score 60), gastric(score 15), intestinal (score 64), and colon (score 38) cancers. Severalgenes associated with the aforementioned top GO processes werecommon between the BaP exposed forestomach responses and theaforementioned types of human cancers (Table 1), suggesting strongsimilarity between gene expression in the BaP-exposed rodentforestomach and various human cancers.
qRT-PCR validation of representative genes
A total of 84 genes were validated using RT-qPCR arrays (Supple-mentary Table 5). These genes included: (a) 54 involved in antigen pro-cessing and presentation, immune and inflammatory response, andkeratinocyte differentiation; (b) seven involved in xenobiotic metabo-lism; (c) eight involved in DNA damage response; (d) three growthfactors; and (e) 12 genes showing the highest fold changes.
Fifty six of the 84 genes were differentially expressed with P ≤ 0.05in at least one treatment group compared tomatched controls (Table 2)including: 16 related to antigen processing and presentation, 14 in theimmune and inflammatory processes, 14 involved in keratinocyte dif-ferentiation, six in DNA damage response pathways, and six involvedin xenobiotic metabolism. Three genes showed statistically significantdifferential expression by RT-qPCR, but not by microarrays. Theseincludedmurine double minute 2 (Mdm2), C-type lectin domain family2 member g (Clec2g), and amphiregulin (Areg).
Protein validation
In addition to the changes described above, our gene expressionanalysis also revealed distinct upregulation of immunoproteasomesubunits. Specifically, proteasome subunit beta type 9 (Psmb9: 2.8 and3.5-fold change in 50 and 75 mg/kg-bw/day dose groups, respectively)and 10 (Pmb10: 1.6 and 1.5-fold change in 50 and 75 mg/kg-bw/daydose groups, respectively) were perturbed by oral BaP exposure.
lly expressed genes associated with them. The graph was generated by IPA's Upstreamificance in the context of the dataset as determined by the Z-score. (B–D) Mechanisticd forestomach showing interconnections between regulators in the gene set. Dashed
Table 1Meta-analysis in NextBio of significant genes in the BaP-treated mouse forestomach and human profiles in which esophageal, gastric, intestinal, or colon cancer was a phenotype.
a The genes listedwere significantly differentially expressed (FDRP ≤ 0.05 and fold change ≥ 1.5) in our dataset and similarly differentially expressed in the curated studies inNextBio.b The number of studies refers to the total number of studies used in the meta-analysis that are positively correlatedwith our dataset. The individual studies used in themeta-analysis
can be found in Supplementary Table 1.
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These genes encode the immunoproteasome catalytic subunits β1i andβ2i, respectively, which are activated in place of the standardproteasome subunits β1 and β2 (Ferrington and Gregerson, 2012)under conditions of oxidative stress or IFNγ induction (Heink et al.,2005). Since immunoproteasomeactivation is specific to thepresentationofMHC class I antigens, and since they are the upstream regulators of theimmune response pathways altered in the present study,we investigatedthe biological relevance of the observed changes in Psmb9 and Psmb10gene expression at the protein synthesis level. Western blot analysisshowed a dose-dependent increase in immunoproteasome subunits β1i(from 1.7 to 3.6 fold) and β2i (from 1.9 to 4.6-fold) protein expressionrelative to matched controls, respectively (Fig. 3). However, we saw nochange in subunits β1 and β2, the normal proteasome subunits,suggesting specific BaP-induced activation of immunoproteasome inthe forestomach.
Discussion
We analyzed global gene expression profiles in the forestomach ofmice exposed to the genotoxic carcinogen BaP in order to explore themolecular mechanisms associated with forestomach cancer. Micewere exposed via oral gavage for 28 consecutive days to 25, 50 and75 mg/kg-bw/day BaP. The top dose is known to induce forestomachtumors in the mouse strain examined (Hakura et al., 1998). Systematicanalysis of all differentially expressed genes revealed three majorfeatures: 1) activation of DNAdamage response pathways; 2) activationof immune response including antigen presentation pathways andimmunoproteasome; and 3) potential for oncogenic transformationreflected by induction of genes involved in keratinization in the high
dose. We present results that delineate the molecular dynamics ofdose-dependent transitions in the response to BaP, explore the relevanceof the BaP-induced gene expression profiles to humandisease, andfinally,discuss the applicability of rodent forestomach tumor data for humanhealth risk assessment.
BaP-induced DNA damage response in the forestomach
The most established mechanism underlying BaP-induced carcino-genesis involves the metabolic activation of BaP, and subsequent inter-action of BaP metabolites with DNA, leading to DNA adducts andmutations in tumor suppressor genes or oncogenes (IARC, 2012).However, rates of BaPmetabolism, levels of DNA adducts, andmutationfrequencies do not correlate with the extent of tumor formation in thetissues studied (Godschalk et al., 2003), suggesting that additionaltissue-specific factors may contribute to carcinogenic events. Althoughwe did not directly measure DNA adducts in the forestomach, a pre-vious study by our group did note a dose-dependent increase in DNAadducts and lacZ mutant frequency in the glandular stomach of miceexposed to BaP (Lemieux et al., 2011), and previous studies haveestablished that levels of DNA adducts in the glandular stomachand forestomach from mice orally exposed to BaP for 24 h and fivedays are comparable (Arlt et al., 2008). Although the finding of Arltet al. (2008) refers to acute exposure effects, it should be acknowledgedthat the differences between the rate of cellular proliferation and themetabolic capacity to form DNA-reactive BaP metabolites in theforestomach and glandular stomach, could all influence the extent ofDNA damage and the outcome following 28 days of repeated exposure.For instance, basal cellular proliferation rates, as determined by BrdU
Table 2Genes validated by real-time PCR in the forestomach of BaP treated mice divided byfunction. All genes shown were significantly differentially expressed with a FDRP ≤ 0.05 and fold change ≥ 1.5 in either direction. ↑ indicates upregulated; ↓ indicatesdownregulated, and – indicates no change.
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incorporation and/or Ki67 staining (marker of proliferation) in theforestomach are approximately 50–67% that of the glandular stomach(Shibata et al., 1990; Tanaka et al., 2011). While not the same endpoint,
other studies have usedhyperplasia asmarker of increased cellular prolif-eration, and have shown increased hyperplasia in the forestomach fol-lowing BaP oral exposure. Other studies comparing cellular proliferationrates after oral exposure to okadaic acid-like compounds (non-PAHs)have shown higher proliferation levels in the forestomach compared tothe glandular stomach (Yuasa et al., 1994). These results imply thatupon stimulation by BaP for 28 consecutive days, cellular proliferationcould be expected to significantly increase in the forestomach comparedto 24 h or five days of exposure. Another determinant of the potentialdifferences between the two tissues that plays an important role in theDNA damage induced by BaP could be differences in phase I metabolismby cytochrome P450 enzymes, which is necessary for the generation ofDNA reactive BaP metabolites, such as BPDE. Indeed, it has been shownthat, following five days of oral BaP exposure, the mRNA and proteinlevels of Cyp1a1 and Cyp1b1 are higher in the forestomach comparedto the glandular stomach (Uno et al., 2008). In toto, we can reasonablyassert that following 28 days of repeated exposure, due, at least in part,to plausible increases in the cellular proliferation and higher metaboliccapacity, the forestomach would be expected to show comparativelyhigher DNA adduct levels and increases in the lacZ mutant frequency.However, further studies are needed to conclusively define the mecha-nisms underlying the similarities and differences in the responses offorestomach and glandular stomach to oral PAH exposures.
In alignmentwith the observed levels of DNA adducts and inductionof mutations in the glandular stomach, analysis of all differentiallyexpressed genes and perturbed biological pathways in the forestomachrevealed activation of the DNA damage response. The cellular DNAdamage response to BaP exposure is regulated by p53 (Park et al.,2006), a transcription factor that suppresses tumor formation bymodulating the expression of genes involved in cell cycle control,DNA repair, inflammatory modulators, and growth factors. Our previ-ous work has shown that the p53-regulated DNA damage response isa primary pathway induced in the lungs and the liver of the samemice as those studied here (Labib et al., 2012; Malik et al., 2012).Genes within the p53-pathway exhibited a dose-dependent responsein the liver and lung, indicating the important role of p53 mediatedresponses in coping with increasing amounts of DNA damage in thesetissues. Analysis of the forestomach transcriptome revealed alterationof 21 p53-responsive genes (Fig. 2) in all of the doses combined.Seven of these genes are directly involved in the DNAdamage response,including the cell cycle regulatory genes cyclin dependent kinase inhib-itor 1a (Cdkn1a) and cyclin-G1 (Ccng1), the DNA repair enzyme O-6-methylguanine-DNA methyltransferase (Mgmt), and pro-apoptoticgenes Pmaip1, Trp53inp1, Trp3, and Trp73. The p53 transcription factorwas ranked 47th of 139 upstream regulators in the analyses conductedhere (see Supplementary Table 3). These results imply that the DNAdamage and activation of DNA repair mechanisms may be involved inthe onset and progression of cancer in the forestomach; however,additional perturbations of other biological processes such as activa-tion of immune response, and antigen processing and presentationpathways are likely contributing to the development and progressionof forestomach tumors.
BaP activates antigen processing and presentation pathway
The other predominantly affected pathways in the BaP-exposedforestomach involved the activation of genes involved in immun-oproteasome activation, immune response, inflammatory response,and antigen presentation. Specifically, significant enrichment was ob-served for MHC class genes involved in antigen processing and presen-tation (Table 2). MHC molecules (classes I and II) are cell surfaceglycoproteins that are necessary for the presentation of peptide antigensto T lymphocytes, including T helper cells, cytotoxic T lymphocytes, andNKT cells (Garcia-Lora et al., 2003). Antigenic peptides are generatedfromdegraded endogenous proteins during lysis or degradation of an in-vading pathogen's cellular components by specialized multi-catalytic
Fig. 3. Activation of the immunoproteasome in the BaP exposed forestomach. Western blot analysis confirming the expression of immunoproteasome subunits β1i and β2i andproteasome subunits β1 and β2 in the Muta™Mouse forestomach exposed to 25 (green), 50 (blue), and 75 (red) mg/kg-bw/day BaP. The band intensities were normalized toβ-actin. *P ≤ 0.1, **P ≤ 0.05.
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protease complexes called the immunoproteasomes (Ferrington andGregerson, 2012). Antigen presentation is the mechanism by whichMHC classmolecules bind antigens in the formof peptides on the surfaceof bacterial or virus-infected cells and present them for recognition andremoval by the immune cells. Activated MHC class I molecules in nucle-ated cells and MHC class II molecules on antigen presenting cells andlymphocytes initiate activation of cytotoxic T lymphocytes and NKTcells (class I), and T helper cells (class II). Thus, initiated Tcell-mediated signaling leads to a cascade of events ranging from cyto-kine production, cellular proliferation, and target cell lysis (Weltzienet al., 1996). Excessive T cell activation and cytokine productionwill ultimately lead to apoptosis of cells probably consisting of cellu-lar debris or chemical metabolites. Phagocytic engulfment of suchdamaged cells will ultimately lead to the efficient removal of the threat.
Various chemicals trigger an immune response via T cell activation(Weltzien et al., 1996). Several studies have demonstrated the activa-tion of T cells following exposure to BaP. For example, peritoneal mac-rophages from mice exposed to BaP are shown to have an increasedability to present MHC class II antigens (Myers et al., 1987). Andersonet al. (1995) showed that AhR binding and phase I metabolism ofdimethylbenz(a)anthracene (another immunogenic PAH) is essentialfor the induction of immune response involving CD8+T cells that is me-diated byMHC class Imolecules in C3G/HeNmice. Transcriptomic profil-ing of peripheral blood mononuclear cells from healthy 25–30 year-oldnon-smoking humans exposed for 20 h to three concentrations(10-fold dilution series) of eight genotoxic carcinogens (4-hydroxy-2-nonenal, malondialdehyde, acrylamide, 2-amino-3-methyl-3H-imidazo[4,5-F]quinolone, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine,aflatoxin B1, dimethylnitrosamine, and BaP) and three non-genotoxiccarcinogens (ethanol, polychlorinated biphenyl 153, and 2,3,7,8-tetrachlorodibenzo-p-dioxin), showed that genes related to immuneresponse activation were uniquely found in the transcriptomic finger-prints associated with eight genotoxic carcinogens, including BaP(Hochstenbach et al., 2012). These results suggest that this type ofMHC class-induced response is common to a diverse array of xenobiotics,and they raise important questions about the role of immune responsesin the recognition and removal of toxicants.
MHC class I-mediated detoxification of BaP
Cellular debris arising from the degradation of invading pathogensgenerates antigenic peptides that are bound by MHC molecules andpresented to the immune cells for recognition and removal by thephagocytes; however, it isn't clear why or how xenobiotics, or BaPin particular, stimulate this response. What is clear is that the metab-olism of PAHs is a prerequisite for activation of the immune response(Anderson et al., 1995). Therefore, one possibility is that the accumula-tion of DNA- or protein- reactive metabolites stimulates the generationof antigenic peptides. The initial metabolism of BaP involves its oxida-tion to epoxides bymembers of the cytochrome P450 family of enzymes(IARC, 2012) that are further metabolized by epoxide hydrolases intoreactive BaP diol epoxides that can accumulate if not removed immedi-ately. Downregulation of the phase II enzymes sulfotransferase family1E member 1 and UDP-glucuronosyltransferase 2 family polypeptideB34, which conjugate a sulfonyl group and glucuronic acid to electro-philic phase I BaP metabolites, respectively, that was observed in theforestomach of mice treated with high doses of BaP, could contributeto the accumulation of hydrophobic reactive metabolites. If the metab-olites are not cleared efficiently, they can form covalent adducts withDNA and protein, the latter of which can induce BaP-specific antibodyproduction and immune response (Schellenberger et al., 2012). BPDEcan also bind the tripeptide glutathione (GSH), the most abundantcellular antioxidant, leading to its depletion. Glutathione-s-transferases(GST) mediate the formation of GSH–BPDE adducts by catalyzing thebinding of the sulfhydryl group of GSH to electrophilic BaP metabolites(Dostal et al., 1988; Singh et al., 2004). Although the process of GSH con-jugation effectively decreases the cellular pool of reactive BaP metabo-lites, excessive binding of GSH to BPDE depletes the cellular pool offree glutathione by 20 to 30% (Romero et al., 1997), and this can signalincreased activation of MHC class I antigen presentation (Makhadiyevaet al., 2012). MHC molecules can bind these GSH–BPDE adducts orBPDE directly and present them to phagocytes for their ultimate removal.In toto, these arguments suggest that activation of MHC class I moleculesin the forestomach following oral BaP exposure serves primarily as adetoxification mechanism.
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Another interesting detoxification pathway in the forestomach ofBaP-treated mice appears to involve the immunoproteasome. Theprimary role of the normal proteasomal degradation machinery isthe removal of abnormally folded proteins, peptides, cellular debris,and adducted proteins (Hill et al., 2007; Sommerburg et al., 2009).The immunoproteasome is a specialized proteasome complex activatedduring an immune response that plays a specific role in the generationof peptides that can fit into MHC class I molecules, leading to theproteasome-mediated degradation of cellular debris (Ferrington andGregerson, 2012; Krüger and Kloetzel, 2012). Significant and specific in-creases in the expression of genes encoding the β1i and β2i subunits(Psmb9 and Psbm10, respectively) and the corresponding proteinproducts (in the higher dose groups), supports the idea that immuneresponse-mediated removal of BaP plays amajor role in the forestomach.In support of this, Schellenberger et al. (2012) showed that BaP-peptideadducts induce the production of BaP-specific antibodies, whereas BaPbound to bulky proteins results in an immune response directedprimarily against the protein rather than against BaP (Grova et al.,2009). Antibodies generated against the peptide adduct complexesare able to sequester BaP and its metabolites, resulting in reducedexcretion in the urine and feces (Schellenberger et al., 2012). These re-sults collectively suggest that the immune response is a predominantroute bywhich BaP is detoxified in the forestomach, and that this processinvolves activation of the immunoproteasome and immune response.Fig. 4 shows the systematic arrangement of differentially expressedgenes in the BaP treated forestomach depicting the sequential activationof the aforementioned processes that we propose lead to the removal ofBaP.
Human relevance
In order to investigate the relevance of gene expression changes in therodent forestomach to effects that relate to human health, we conductedmeta-analyses of the altered gene sets (Fig. 1, antigen processing andpresentation, immune response, chemotaxis, and keratinocyte differenti-ation) using the NextBio HumanDisease Atlas Tool. The analysis revealeda strong similarity between the dataset of the present study and genesaltered during human bacterial infectious diseases, parasitic disease,respiratory disease, and viral infectious disease (Supplementary Table4), supported by 85, 82, 27, and 117 studies, respectively. Melo andRuvkun (2012) used the nematode Caenorhabditis elegansmodel to dem-onstrate that exposure to chemicals/xenobiotics or pathogens leads todisruption of the same essential cellular activities such as mitochondrialfunction, ribosome activities, protein translation, mRNA processing, etc.These disruptions collectively serve as the physiological cues that leadto activation of defense mechanisms, including detoxification pathwaysand immune responses. The results presented here suggest that the rec-ognition and removal of BaP and/or its reactive metabolites from theforestomach involves the same pathogen recognition mechanisms thatdefend the host against microbial attack.
The other highlight of the present dataset is the significantupregulation of a large number of genes involved in keratinocyte differ-entiation (Table 2), especially at the 50 and 75 mg/kg-bw/day doses(Fig. 1). We observed dose-dependent increases in the expression ofnumerous keratins including Krt6a, Krt6b, Krt12, Krt17, and Krt18, aswell as factors that are involved in the promotion of keratinocyte differ-entiation, such as small proline-rich proteins Sprr1b, Sprr2a2, Sprr2d,Sprr2e, Sprr2g, Sprr2h, Sprr2i, and Sprr2k. Analysis of this gene set inthe NextBio Human Disease Atlas Tool showed strong similarities be-tween the BaP-exposed forestomachs and malignant tumors of the in-testine and gastric cancers in humans (Table 1).
In the forestomach, the transformation of cuboidal epithelial cellsinto stratified squamous cells, which produce large amounts of keratins,is the first indication of initiation of carcinogenic events (Kossoy et al.,2006). The process is referred to as keratinocyte differentiation and isroutinely observed during the transformation of stomach epithelial
cells in adenocarcinomas caused by Helicobacter pylori (H. pylori) infec-tion in humans (where chronic infection eventually leads to intestinalmetaplasia) in humans. Keratins in squamous epithelia serve to protectthe cells from injury and are regulated bymany cancer-promoting stimulito promote cell growth, survival, andmotility. Excessive levels of keratinsare strongly associated with the development of various human cancers(Cao et al., 2011;Mosca et al., 2010; Somji et al., 2008) and are correlatedwith poor clinical outcome (Ide et al., 2012). A thick layer of keratin isnoted on pre-malignant papillomas and on the epithelial surface of theforestomach in BaP fed mice (Rigdon and Neal, 1966, 1969). Enhancedkeratinocyte differentiation and dysplasia are also observed in miceexposed to dimethylbenz(a)anthracene (Kossoy et al., 2006) via feed.Dimethylbenz(a)anthracene, an alkyl-substituted PAH, has also beenshown to induce similar carcinogenic effects in the forestomach of ex-posedmice. In another study, Shi et al. (2010) found squamous cell carci-noma in the preputial gland duct of Cyp1a1 and Cyp1b1 double knockoutmice following 12 weeks of oral BaP exposure that showed increasedlevels of keratin deposition. In a follow-up study using microarrays,these authors further demonstrated a significant upregulation of genesassociated with keratinization in the preputial gland duct after 8 weeksof exposure to BaP (Gálvez-Peralta et al., 2013). Although the role ofkeratinocyte differentiation in carcinogenesis is not well understood,Krt17 is known to positively regulate cell size and growth of keratinocytesby binding to the adaptor protein 14-3-3 sigma and by stimulatingmTORsignaling (Kim et al., 2006), which is involved in sustaining proliferativesignaling (a hallmark of cancer) (Hanahan and Weinberg, 2011). Theseresults collectively suggest that the key events leading to cancer are dif-ferent in the forestomach and somewhat distinct from those observedin other tissues, and include a strong immune response, persistent in-flammation, and initiation of keratinocyte differentiation. The strong sim-ilarities observed between our dataset and those that are associatedwithhuman stomach diseases, including cancers, provides evidence for use offorestomach tumor data for assessments of human cancer risks associ-ated with PAHs such as BaP.
An acknowledged shortcoming of the present study is that we didnot compare the transcriptomic responses between the forestomachand glandular stomach, target and non-target organs for BaP-inducedcancers in rodents. Although the rodent glandular stomach is analogousto the human gastric stomach, it does not develop tumors after expo-sure to high doses of BaP (oral, intraperitoneal, or intramammaryroute) that induce significant DNA damage. Out of the 570 two-yearrodent cancer bio-assays conducted by the National Toxicology Program(NTP), very few resulted in glandular stomach cancers (Chandraet al., 2010). Therefore, it is quite possible that we will see differenttranscriptomic profiles in the two tissues; however, we do expect tosee similar changes in genes that respond to DNA damage. Our previouswork comparing transcriptomic responses in lung and liver tissues ex-posed to BaP have rightly pointed out the distinctmodes of actionwith-in each tissue that relate to their inherent function. For example, despitecomparable levels of DNA damage between the lung and liver tissues,our findings showed a rather subtle transcriptomic response in theliver (target organ for carcinogenesis by BaP) that was not directlysuggestive of cancer even at the higher doses. On the other hand,transcriptomics did reveal changes in the expression of importantDNA damage responsive genes consistent with the observed DNA ad-ducts in the liver. In the lung tissue, a dramatic response was observedat all doses tested, with incremental dose-related changes related tocarcinogenesis. These results suggest that the inherent functions oftissues, and the concomitant balance between biological pathwaysleading to enhanced cellular proliferation and pathways leading to celldeath, play important roles in chemically-induced carcinogenesis.Based on these arguments we should expect to see 1) changes in theexpression of DNA damage response genes in the glandular stomachsimilar to those observed in the forestomach, lungs (Labib et al., 2012),and liver (Malik et al., 2012) of mice exposed to BaP, and 2) parallel acti-vation of cellular proliferation and cell death genes. In addition, in
Fig. 4. Molecular relational network of genes modulated by BaP in the forestomach using MetaCore for all three doses. A network is a graphical representation of the molecularrelationships between molecules. Biological relationships between two molecules are represented by a line; green lines denote positive effect, red lines denote negative effect,and gray lines represent unspecified effect. Biological processes are presented in causal order of BaP-induced damage removal and eventually carcinogenic transformation. Hexagonsrepresent physical and functional interactions: B— binding,+P— phosphorylation, Z— catalysis, TR— transcription regulation, IE— influence on expression, and CS— complex subunit.The processes include genes that were differentially regulated in the 25 mg/kg-bw/day (green rectangle), 50 mg/kg-bw/day (blue rectangle), and 75 mg/kg-bw/day (red rectangle) dosegroups.
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accordance with the reported mechanisms underlying the formation ofglandular stomach tumors (i.e. the inhibition of gastric acid secretionleading to hypergastrinemia and hyperplasia (Chandra et al., 2010)),we may expect to observe induction of pathways counteracting theseprocesses. Since due to lack of available tissue, the present study couldnot provide evidence to substantiate the aforementioned assertions,additional experiments are warranted.
Conclusion
We present a model for the rodent forestomach responses to BaPand propose key pathways that we believe to underlie BaP-inducedforestomach carcinogenesis. We find that the gene expression signa-tures in the BaP-exposed forestomach are broadly similar to humancancers of the gastrointestinal tract, and that general detoxificationpathways including sulfation and glucuronidation are negatively regu-lated in the BaP-exposed forestomachs, potentially resulting in theaccumulation of BaP and BaP metabolites. Excessive accumulation ofDNA- or protein-reactive metabolites then serve as cues leading to theactivation of other detoxification/defense mechanisms such as theimmunoproteasome, antigen processing and presentation pathways, Tcell activation, cytokine-mediated inflammation, and interferon γ sig-naling. Exposure to very high levels of BaP (75 mg/kg-bw/day) leadsto unremitting activation of immune responses and inflammation, andultimately activation of keratinocyte differentiation, an importantbiological alteration that is considered a hallmark of forestomach carci-nogenesis. Despite functional and histological differences between therodent forestomach and the human stomach, there appear to be com-mon tissue-specific alterations in core cellular responses to invadingpathogens/toxicants and the identity and functions of individual genesthat are disrupted in the forestomach of mice exposed to BaP for28 days are consistent with the development of human cancers of theesophagus and gut. Thus, our results provide support for the relevanceand use of rodent forestomach tumor data in human cancer risk evalu-ation. More studies involving testing of other known forestomachcarcinogenswith awide range of doses and time pointswill be necessaryto further confirm these findings.
Conflict of interest
The authors declare that there are no conflicts of interest.
Funding
Health Canada's Genomics Research and Development Initiative;the Health Canada Chemicals Management Plan.
Acknowledgments
We thank Byron Kuo for duplicate removal. We would also like tothank Julie Buick and Dr. Nikolai Chepelev for their help reviewing themanuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.taap.2013.05.027.
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