Predictive Toxicogenomics Approaches RevealUnderlying Molecular Mechanisms ofNongenotoxic Carcinogenicity

Alex Y. Nie,1* Michael McMillian,1 J. Brandon Parker,1 Angelique Leone,1 Stewart Bryant,1 Lynn Yieh,2

Anton Bittner,2 Jay Nelson,2 Andrew Carmen,2 Jackson Wan,2 and Peter G. Lord1

1Johnson & Johnson Pharmaceutical Research & Development, LLC, Raritan, New Jersey2Johnson & Johnson Pharmaceutical Research & Development, LLC, La Jolla, California

Toxicogenomics technology defines toxicity gene expression signatures for early predictions and hypothesesgeneration for mechanistic studies, which are important approaches for evaluating toxicity of drug candidatecompounds. A large gene expression database built using cDNA microarrays and liver samples treated with over one

hundred paradigm compounds was mined to determine gene expression signatures for nongenotoxic carcinogens(NGTCs). Data were obtained from male rats treated for 24 h. Training/testing sets of 24 NGTCs and 28 non-carcinogens were used to select genes. A semiexhaustive, nonredundant gene selection algorithm yielded six genes

(nuclear transport factor 2, NUTF2; progesterone receptor membrane component 1, Pgrmc1; liver uridine diphosphateglucuronyltransferase, phenobarbital-inducible form, UDPGTr2; metallothionein 1A, MT1A; suppressor of lin-12homolog, Sel1h; and methionine adenosyltransferase 1, alpha, Mat1a), which identified NGTCs with 88.5% pre-

diction accuracy estimated by cross-validation. This six genes signature set also predicted NGTCs with 84% accuracywhen samples were hybridized to commercially available CodeLink oligo-based microarrays. To unveil molecularmechanisms of nongenotoxic carcinogenesis, 125 differentially expressed genes (P< 0.01) were selected by Student’st-test. These genes appear biologically relevant, of 71 well-annotated genes from these 125 genes, 62 were over-

represented in five biochemical pathway networks (most linked to cancer), and all of these networks were linked byone gene, c-myc. Gene expression profiling at early time points accurately predicts NGTC potential of compounds, andthe same data can be mined effectively for other toxicity signatures. Predictive genes confirm prior work and suggest

pathways critical for early stages of carcinogenesis. � 2006 Wiley-Liss, Inc.

INTRODUCTION

Drug-induced carcinogenesis is an issue withregulatory as well as scientific implications. Regula-tory carcinogenicity studies are performed late in thedrugdevelopmentprocess, andowing to a somewhatvariable rate of tumor formation inuntreatedcontrolrodents, results of these long-term studies are oftendifficult to interpret. For compounds or their meta-boliteswith directDNAdamaging properties,mostlyreactive electrophiles, a number of assays have beendeveloped to predict results in the long-term rodentstudies. These include bacterial mutagenicity assays[1], bioengineered rodents such as the Big BlueMouse and Rat [2,3], the micronucleus assay [4],and variations on the comet assay [5,6] for detectingdirect DNA damage. Generally, a robust positiveresponse in any of these genotoxicity assays isenough to stop development of a potential thera-peutic, unless its benefit exceeds its risk for a partic-ular prognosis. While these assays are predictive forDNA damage-induced carcinogenicity in rodents,over half of all chemical-induced carcinogenesis inrodents appears to be nongenotoxic in origin, andnongenotoxic carcinogens (NGTCs) have proven

more difficult to predict accurately in short-termassays.The term, nongenotoxic refers to a lack of direct

chemical effect on DNA, for carcinogens in theinitiation of tumors. There are many types of NGTC.Many have abrupt dose response effects (possiblyacting as anticancer agents at low doses) and conti-nued administration of a NGTC is often required fortumor development (these tumors are compounddependent). NGTCshave beenpostulated to act via anumber of mechanisms: increased mitogenesis,decreased apoptosis, interference with gap junctionintercellular communication, and interference withtubulin polymerization [7]. Some of these effects are

MOLECULAR CARCINOGENESIS 45:914–933 (2006)

� 2006 WILEY-LISS, INC.

Abbreviations: NGTC, nongenotoxic carcinogen; NC, noncarcino-gen; MA, macrophage activator; PP, peroxisome proliferator; SAM,S-adenosyl-L-methionine; TSH, thyroid stimulatory hormone; PCA,principal component analysis.

*Correspondence to: Johnson & Johnson PharmaceuticalResearch & Development, LLC, Raritan, NJ.

Received 9 August 2005; Revised 30 November 2005; Accepted17 January 2006

DOI 10.1002/mc.20205

Published online 18 August 2006 in Wiley InterScience(www.interscience.wiley.com)

not so different from those observedwithdirectDNAdamaging agents. For example, if a compound is amitogen, cytotoxic, or an irritant, there is prolifera-tion and/or compensatory regeneration of tissue,and this proliferating tissue accrues mutations at ahigher rate than the normal, quiescent tissue,especially if apoptosis is blocked, thus increasingrisk for tumor development [8]. Proliferation is anindicative risk factor for carcinogenesis, and mon-itoring early proliferative responses (within the 1stwk)byBrdUorPCNA labelingofnucleihasbeenusedto predict formation of tumors in response to drugsand chemicals [9–11]. Decreased apoptosis blocksthenormal removalofdamaged, sometimesmutatedcells and again the risk for cancerous growth isincreased [12]. The role of gap junction communica-tion in inhibiting carcinogenesis is less clear, butisolation of affected cells from control by surround-ing normal cells appears to be a common effect withmany NGTCs [13]. Inhibitors of tubulin polymeriza-tion disrupt chromosomal movements and separa-tions duringmitosis, and represent a specialized typeof carcinogen [7]. Rodents are typically used forregulatory carcinogenicity studies due to their shortlifetimes for compound administration. About halfof compounds tested in rodents are carcinogenic [8]and most of these are NGTCs [14,15]. Rat liverappears unusually sensitive to NGTCs, and hasbeen extensively studied to understand potentialmechanisms involved.Many recent publications have used microarrays

and expression profiling to study NGTCs in liver.Most investigators have characterized early hepaticresponses to a single NGTC or class, or a few NGTCswithin a very small set of compounds [16–19].Comparisons of peroxisome proliferators (PPs) withanother NGTC led to the conclusion that, despitepronounced effects on gene regulation, there are fewgene expression changes in common and that eachclass of nongenomic carcinogen exerts its effects bydifferent mechanisms [20–23]. NGTCs can becleanly distinguished from genotoxic carcinogensin vitro in HepG2 cells, a model which provides anearly screen but may involve different mechanismsfrom carcinogenesis in vivo [24].We have previously used cDNA microarray tech-

nology to characterize two classes of hepatotoxicNGTC: macrophage activators (MAs) and peroxi-some proliferators [25,26]. Compounds that activatemacrophages (Kupffer cells in the liver) mimicinflammation, and share characteristics with hepa-titis viruses. Generally these compounds produceacute liver damage and regeneration. Continuedadministration of MAs leads to fibrosis and even-tually to hepatic tumors [27,28]. MAs have a veryrobust, distinct gene expression signature responsein rat liver, and frequently cluster together evenwhen other types of hepatotoxicities are the foci ofexamination by microarray [25,26]. Similarly, PPs

have pronounced, distinct effects on genes, such asthose involved in b-hydroxylation of fatty acids,such that their gene expression signature responsesoverwhelm any similarities to other NGTCs. Peroxi-someproliferation is a direct result of PPARa receptoractivation in rat liver, and the resultant tumorsreflect, at least in part, the effects of increasedhydrogen peroxide levels derived from peroxisomes.In the present study, we have examined toxicoge-

nomic effects at 24hof a largenumberof compoundspreviously categorized as NGTCs or noncarcinogens(NCs) in the rat. Although we have focused onrat liver, we have also included NGTCs that onlyaffect other tissues, to see how well we can modelnongenotoxic carcinogenicity in general, at a single,early timepoint. Gene expression results from thesame set of liver RNA samples are associated with anumber of hepatotoxic endpoints, and these data arebeing extensivelymined for other gene signatures inaddition to that for NGTCs.

MATERIALS AND METHODS

Compounds

Compounds were obtained from Sigma-Aldrich(St. Louis, MO), except for troglitazone andWY14643, which were from Biomol (PlymouthMeeting, PA).

In Vivo Studies

A detailed description of in life studies was givenpreviously [26]. Single, high doses of chosen com-pounds were administered to male, 7-wk oldSprague–Dawley rats (Charles River Laboratories,Inc.), and rats were killed and livers removed 24 hlater. Each treatment was administered to three rats.High toxic doseswere selected typically as 30%–50%of the published LD50s for compounds, and anyadverse effects noted at necropsy were recorded(Appendix A in [26]). Compounds that producedminimal gene changes in liver and no obvious toxiceffects were given at a higher dose. In all instances,the animals were humanely handled in accordancewith IACUC guidelines.

RNA Isolation

A strip of liver from the medial lobe (approxi-mately 200 mg) was snap-frozen in liquid nitrogen.Liver samples were stored at �708C until RNAextraction. Total RNA was extracted using QiagenRNEasyMidi kits as per kit instructions. The amountof RNA in samples was determined spectrophotome-trically by absorbance at 260 nm. The quality of RNAsamples was assessed using rRNA peaks determinedby an Agilent 2100 Bioanalyzer.

cDNA Microarrays

RNA and probe preparation for microarray analy-sis—One round of T7 polymerase-based linear RNA

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amplificationwasperformedby reverse transcriptionof RNA with a T7 promoter oligo(dT) primer.Cy3-dCTP labeled fluorescent cDNA probe wassynthesized from the amplified RNA as described[29]. Probes were then purified with a PCR purifica-tion kit (Qiagen, Inc., Valencia, CA), vacuum-dried,and resuspended in 55 mL of hybridization buffer(Version 2 hybridization buffer (Amersham Pharma-cia Biotech, Piscataway, NJ) with 50% formamide)containing rat Cot1 DNA (Applied Genetics Labora-tories, Melbourne, FL).cDNA microarrays were prepared as described

previously [26] but with a different collection ofgenes. The 1471 genes on the current microarraywere selected from the last version based on theirperformance or directly picked from literature. Eachslide contained four identical panels of 1471 genesplus various types of control probes. The list of geneson the cDNA microarray used for these analyses isavailable at: http://mimicell.com/toxicogenomics/.Clones for genes of interest were obtained fromResearch Genetics (IMAGE consortium), sequenceverified, PCRamplified, purified, and spotted inquad-ruplicate on aminosilane-coated slides (Corning)using a contact pin microarrayer (Generation IIIArray Spotter, Molecular Dynamics).The sample cDNA probes were hybridized to

the microarrays and washed as described previously[26]. After drying, the microarrays were scannedwith a confocal laser scanner (Array Scanner,Molecular Dynamics). Intensity values for each spotof the array were obtained using Autogene software(BioDiscovery).

CodeLink Oligo Microarrays

One round of T7 polymerase-based linear RNAamplificationwasperformedby reverse transcriptionof RNA with a T7 promoter oligo(dT) primer,followed by in vitro transcription using the Ribo-Beast 1-Round Aminoallyl-aRNA Amplification Kit(Epicentre, Madison, WI). This protocol was per-formed as specified in the manufacturer’s protocolwith the following modifications: SuperscriptII(Invitrogen, Carslbad, CA) was used as the reversetranscriptase, and the Rneasy96 block was used forpurification (Qiagen, Valencia, CA). Following pur-ification, the cRNA was conjugated with biotin-ester(Biotium, Hayward, CA), purified again with theQiagen96 block, and concentrated by Speed-Vac(Thermosavant, Waltham, MA). Biotin-labeledcRNA was fragmented at 948C for 20 min and addedto Codelink hybridization buffer A and B (GEHealthcare, Chandler, AZ).EachcRNAsamplewas applied to twoCodelinkRat

Whole Genome microarrays (GE Healthcare) usingthemanufacturer’s protocol, and hybridized at 378Covernight. Slides were washed using the manufac-turer’s protocol and stained with an Alexa555-streptavidin conjugate (Invitrogen), and then

scanned at 532 nm with an Agilent G2565BAMicroarray Scanner (AgilentTechnologies, PaloAlto,CA). Fluorescence intensity for each feature of thearray was obtained by using Codelink EXPv4.1software (GE Healthcare).

Microarray Data Normalization

Data were normalized, day-to-day hybridizationdifferences corrected, and outlier data removed asdescribed in detail previously [26]. In short, geneexpression responses were measured with twoslides composed of two (CodeLink) or eight (cDNAmicroarrays) identical panels; data were Splinenormalized, the average was taken among allpanels, and the geometric mean of controls wasused to correct for day-to-day variability and togenerate ratios. The average was then takenamong three replicate rats to generate the averageratio for each individual treatment, which was thenused in the following cross validation and geneselection.

Gene Selection

Two different methods were used to select genes,one of which aimed at finding differentiallyexpressed genes between NGTC and NC treatments.Genes selected with this method were used tohelp understand the mechanisms of nongenotoxiccarcinogenicity. The other method was designed toselect the gene expression signature with the mini-mum number of genes and the highest predictingpower.

Selection of Differentially Expressed Genes

Student’s t-test was used to evaluate each gene forits expression difference between the NGTC- andNC-treated samples. Each of the genes, with itsP-value, average log ratio among all NGTC treat-ments, and average log ratio among all NC treat-ments was then loaded into Ingenuity’s networktools (Ingenuity1 Systems, www.ingenuity.com).Each gene identifier was mapped to its correspond-ing gene object in the Ingenuity Pathways Knowl-edge Base. A P<0.01 cutoff value was set to identifygenes whose expression was significantly differen-tially regulated. Significant genes that had directinteractionswith other genes in the database, the so-called Focus Genes, were overlaid onto a globalmolecular network contained in the Ingenuity Path-ways Knowledge Base. Networks of the Focus Geneswere then algorithmically generated based on theirconnectivity using the Ingenuity tools as describedin [30]. Briefly, the specificity of connections for eachfocus gene was calculated by the percentage of itsconnections to other significant genes, and thepathways grew from the genes with the highestspecificity until it reached 35 genes. Significantpathways were then integrated to form a compositenetwork useful for biological interpretation.

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Selection of NGTC Gene Signature

A two-step gene selectionmethodwas used for thispurpose (Figure 1). At the first step, a list of genes wasselected by each of the following gene selectionmethods: Student’s t-test, genetic algorithm, forwardselection, and backward elimination [31]. Addition-ally, a number of top-ranked gene pairs were pickedby exhaustively searching through all possiblecombinations of two genes. The five gene listswere then pooled together and the genes ranked bythe number of appearances. P-values from the t-testwere used to break the tie whenever needed. At thesecond step, the final gene expression signature wasdetermined by exhaustive search within the top50genes fromthepooled list.Due to the limitationofcomputing power, an exhaustive search could onlybe performed for gene expression signatures with upto six genes.The number of genes selected from each method

was determined experimentally. For the t-test, afterthe P-values from the t-test were calculated, thenumber of significant P-values was so chosen that5% of the genes might be falsely discovered [32].For the forward selection, backward elimination, andgenetic algorithms, 20 genes each were selected

because selection of more than 20 genes (up to 50genes tested) by forward selection did not show anyimprovement in discriminant analysis and a largernumber of genes could lead to overfitting [33]. Forgene-pair selection, the best 20 pairs were selected.For forward selection, backward elimination, and

genetic algorithm, linear discriminationanalysiswasused to evaluate the selected gene set. For gene-pairselection, quadratic discrimination analysis wasused. A posterior error score was given to representthe performance. Higher performance meant betterseparation between NGTCs and NCs. Gene selectionand evaluation were aided by PartekTM software(Partek, Inc., St. Charles, Missouri).

Cross-Validation of the Gene Selection Process andthe Final Gene Set

Gene selection was performed on the trainingsamples after the samples were partitioned intotraining and testing sets. The discriminant ruleassociated with the selected genes was also deter-mined with the training sample at the same time.The performance of the selected genes and thecorresponding discriminant rule were evaluatedwith the testing samples. This process was iterated

Figure 1. Gene selection procedure. See Materials and Methods for details. Numbers on the edges reflect thenumber of genes selected by the specific method (e.g., forward selection) or passing through the selection criteria(e.g., P< 0.01). These numbers are the actual gene numbers during the final gene selection after cross-validation.

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when the samples were repartitioned. Ten-fold crossvalidation was used in the present study, that is, ateach iteration, 10% of the samples were used fortesting and the testing samples did not repeat so thatevery sample had a chance to be tested. Theestimated error rate was calculated as the numberof misclassified testing samples divided by the totalnumber of samples.After cross-validation, the final gene set and

discriminant rule were decided with all the samples,and the estimated error rate was the one determinedin the cross-validation.The final gene set and discriminant function were

also used to predict the treatments in the databasethat were not included in the training/testingexercise. For each treatment, a posterior probabilityscore was calculated for each class: p(NGTC) andp(NC), where p(NGTC)þp(NC)¼1. If p(NGTC)>p(NC), then the compoundwaspredicted as aNGTC.For the purpose of simple representation (Table 1),we used aþ/� system:þþþ for p(NGTC)�0.99;þþ,0.99>p(NGTC) �0.95; þ, 0.95>p(NGTC) >0.5; �,0.95>p(NC)between�0.5;��, 0.99>p(NC)�0.95;and ���, p(NC) �0.99.

RESULTS

Selection of NGTC and NC Compounds

Compounds were labeled as NGTC or NC basedon published 2-yr carcinogenicity studies (Table 1,[14,34]). Our focus was on NGTC from rodentstudies; however, no distinction was made betweenthe compounds that act in liver or in extrahepatictissues. In some cases, such as cyproterone acetate,the compound is known to be a NGTC in mouse,however the data were not clear-cut for the rat; forour broad screeningpurposes the compoundwas stillclassified as a NGTC. Two well-studied classes ofNGTC, PP, and MA, are easily identified in rat liver[26,35], but their gene expression signatures over-whelm gene expression responses to other classes ofcompounds, so PPs andMAs were removed from thetraining/testing sets of compounds. Fifty-two com-poundswere so chosen, 24ofwhichwereNGTCs and28 NCs. Only the highest dose for a compound wasused if multiple doses of that compound were tested(Table 1).

NGTC Gene Signature

The two-step gene selection method was firsttested by cross-validation as described in methods.After cross-validation, the final gene expressionsignature was then determined using all samples.Specifically, at thefirst step, 26geneswere selectedbyStudent’s t-test, 20 pairs of genes (21 individualgenes) were selected by exhaustive gene-pair selec-tion, and each of the other methods produced20 genes. The gene lists were pooled together andgenes ranked by the number of appearances. The top

50 genes from the pooled 61-gene list were used forthe next step of gene selection and the best six genes(Table 2 and highlighted in Table 3) were thenselected with the exhaustive selection method(Figure 1).Of the six genes selected, three geneswere induced

by NGTCs relative to NCs: nuclear transport factor 2(NUTF2), progesterone receptor membrane com-ponent 1 (Pgrmc1), and liver uridine diphosphateglucuronyltransferase, phenobarbital-inducibleform (UDPGTr2). Three genes were repressed byNGTCs relative to NCs: metallothionein 1A (MT1A),suppressor of lin-12 homolog (Sel1h), and methio-nine adenosyltransferase 1, alpha (Mat1a). Geneexpression changes were not dramatic (Table 2),but these six genes used in concert performedextremely well. With this gene selection procedure,the prediction accuracy estimated by cross-valida-tion was 88.5% (6 of 52 testing samples wereincorrectly predicted). As a comparison, the highestaccuracy of prediction with 1�20 genes selected byforward selection was 84.6%. Not surprisingly, allcompounds in the training/testing sets could becorrectly predicted with this final gene signature. Aprincipal component analysis (PCA) plot was gener-ated to illustrate the separability between NGTCsand NCs using the selected genes (Figure 2a).

Verification of the NGTC Gene Signature onCodeLink Microarrys

RNA samples from 50 of the 52 training/testingtreatments were rerun on the CodeLink oligo-basedmicroarray platform (trans anethole andmonocrota-line were not included). A PCA plot was generatedwith the six genes selected from the cDNA micro-array for comparison (Figure 2b). The predictionaccuracy of this gene signature using expressionvalues from the CodeLink platform was 84%,estimated by leave-one-out crossvalidation (withoutgene selection). There was a degree of correlationobserved between the cDNAmicroarray data and theCodeLink data for the six genes (Figure 3) andCodeLink generally showed higher dynamic rangesfor gene expression changes (e.g., log ratio of Sel1hranged from�0.26 to 0.25 from sample to sample oncDNAmicroarray, but ranged from�1.2 to1.1 on theCodeLink chips (Figure 3b, Table 2).)

Prediction of Other Compounds in the Database

The selected six-gene signature and the associateddiscriminant rule were used to screen all thecompounds in the database. Among noncharacter-ized compounds in our database, several weredetected as NGTCs (Table 1). Although not used inthe training or testing sets, many PP compoundswere also detected (Table 1) as NGTCs. Only a fewMAs were detected despite strong evidence for thesecompounds being NGTCs; fortunately they areeasy to detect with their own gene signature [26].

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Table 1. Compounds Used in This Study

Compound Dose (mpk) Literaturea OSb TTc Predictedd

1-naphthyl-isothiocyanate 200 NRS (5) ���2-acetamidofluorene 200 DNA damager OS þþþ3-acetamidophenol 350 NC? OS �3-methylcholanthrene 125 DNA damager þ4,40-methylenedianiline 100 DNA damager þ4-methylthiazole 120 NRS (5) OS �Amiodarone 1000 NGTC (1); NRS (5) ���Amsacrine 20 DNA damager ���Aniline 200 NGTC (2) OS TT þþþAntimycin A3 2 NRS (5) ���Aspirin 600 NC (2) TT ��Atenolol 1500 NGTC (1) TT þþþBenzafibrate 500 NGTC; PP þþþBenzbromarone 200 NGTC; PP þþþBromobenzene 900 NRS (5) OS �Bromocryptine 200 NGTC (1) TT þþþBuspirone 100 NC (1) TT �Busulfan 100 DNA damager ���Butylated hydroxytoluene 1000 NGTC (2; mouse) OS TT þþþCadmium chloride 75 DNA damager ���Captopril 5000 NC (1) TT ���Carbamazepine 20 NGTC (1) þþCarbamazepine 120 NGTC (1) þþþCarbamazepine 225 NGTC (1) TT þþþCarmustine 30 DNA damager þCerium Chloride 50 NRS (5) �Chlorambucil 25 DNA damager ���Chlorpromazine 150 NGTC (4) OS TT þþþCisplatin 15 DNA damager þClofibrate 600 NGTC; PP þþþClozapine 150 NC (1) TT ��Cyclophosphamide 40 DNA damager ��Cyproterone acetate 200 NGTC (2; mouse) TT þþþDacarbazine 200 DNA damager ���Dantrolene 500 NGTC (1) TT þþDapsone 50 NGTC (1) TT þþþDexamethasone 75 NC (2) TT �D-Galactosamine 3000 MA þþþDichloroacetate 1500 NGTC; PP þþþDiclofenac 75 NC (1); MA TT ���Dieldrin 45 NGTC (2; mouse) OS TT þþþDiethylphthalate 1000 NGTC; PP þþþDiflunisal 750 PP?; NC (1) þþþDigoxin 15 NRS (5) ��Dimethylmaleate 400 NC? þþDipyridamole 5000 NC (1) TT ���Disulfiram 2010 NC (2) OS TT ���Doxorubicin 20 DNA damager ���Enalapril 1800 NC (4) ���Erythromycin estolate 1500 NC (2) ���Ethynylestradiol 500 NGTC (2) OS þþþEtoposide 60 DNA damager ���Etoposide 120 DNA damager ���Etoposide 150 DNA damager; MA þþþFamotidine 500 NC (1) TT ���Felbamate 180 NGTC (1) ��Felbamate 1000 NGTC (1) OS þþþFelbamate 2100 NGTC (1) OS TT þþþFenbufen 250 PP?; NC? þþþ

(Continued)

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Flufenamic acid 250 NC?; MA ���Fluoxetine 100 NC (1) TT ���Flurbiprofen 40 NRS (5); MA ���Flutamide 500 NGTC (4) OS þþþFurosemide 1500 NGTC (1) TT þþþGabapentin 180 NGTC (1) þGabapentin 1000 NGTC (1) þþþGabapentin 2160 NGTC (1) TT þþþGentamicin 750 NC? ���Glucosamine 2000 NC? ��Glybenclamide (glyburide) 5010 NC (1) �Hexachlorocyclohexane g (lindane) 80 NC (1) OS ���Hydrazine hydrate 60 DNA damager þþþIbuprofen 500 NC (2) þþIndomethacin 30 NC (1) TT ���Isoniazid 125 NGTC (1; mouse) OS TT þþþIsoproterenol 3 NC? ���Ketoconazole 150 NC (1) OS þþþLansoprazole 200 NGTC (1) TT þþþMebendazole 40 NC (1) TT ���Menadione 250 DNA damager �Metformin 750 NC (1) TT ��Methapyrilene 200 NGTC (2) TT þþþMethotrexate 300 NC (2) TT ��Methyldopa 1000 NC (1) TT ���Metoprolol 2000 NC (1) TT ��Mifepristone 80 NC? ��Monocrotaline 30 NGTC (2) TT þþþMycophenolic acid 500 NC (1) TT ���Naltrexone 1000 NC (1) TT ���Niacin 5010 NC (1; mouse) TT ���Nifedipine 750 NC (1) þþNimesulide 500 NC? OS þNizatidine 1000 NC (1) TT ���Paraquat 100 DNA damager ��Perfluorodecanoic acid 50 NGTC; PP þþþPerfluorooctanoic acid 150 NGTC; PP þþþPerhexilene 2010 NC (2) TT ���Phalloidin 1 NRS (5) þPhenacetin 1200 DNA damager �Phenobarbital 20 NGTC (2) þþþPhenobarbital 225 NGTC (2) TT þþþPhenylephrine 5 NC (2) TT ��Phenytoin 20 NGTC �Phenytoin 120 NGTC �Phenytoin 225 NGTC þþþPhorone 500 NC? ���Piperonyl butoxide 4000 NGTC (2) OS TT þþþPrecocene I 500 DNA damager OS ���Pregnenolone carbonitrile 200 NGTC þþþProgesterone 100 NGTC (3; mouse) þþþPuromycin 350 NC? þþþQuercetin dihydrate 4005 NC (2) ��Raloxifene 700 NGTC (1) TT þþþRanitidine 1000 NC (1) OS TT ���Retinol (vitamin A) 200 NC (2) TT ���Rifampicin 600 NGTC (2; mouse) TT þþþRosiglitazone 30 NC (1; mouse) ���Rosiglitazone 100 NC (1; mouse) ��Rotenone 6 NC (2) ���

Table 1. (Continued)

Compound Dose (mpk) Literature OS TT Predicted

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Additionally, many compounds associated withoxidative stress/reactive metabolites were alsodetected at high dose as NGTCs (Table 1).We did not routinely run low dose levels of com-

pounds (and NGTCs are not detected at low dose).However, multiple dose levels were run for a smallgroup of antiepileptic compounds (gabapentin,felbamate, and phenytoin), a class which includesknown NGTCs. Only the higher dose levels of thesecompoundswere detected by theNGTC signature set(Table 1).

Differentially Expressed Genes Between NGTC andNC Treated Samples

One hundred and twenty-five genes were signifi-cantly up- or downregulated (P-value <0.01) inNGTC treatments as compared to NC treatments,determined by Student’s t-test (Table 3). The accu-racy of the prediction with all differentially ex-pressed genes selected by this method was 77%,considerably lower than the more exhaustive genesearch carried out above. However, this largernumber of significantly differentially regulatedgenes was useful in determining which pathways

might be differentially regulated in early nongeno-toxic carcinogenesis. Out of the 125 genes, annota-tion could be found for 71 genes from Ingenuity’sPathways Knowledge Base upon which pathwayanalysis could be conducted. With Ingenuity’sannotation, 16of the 71geneswere related to cancer,19 tocellular growthandproliferation, 7 to cell cycle,10 to cell death, and 11 to immune and lymphaticsystem development and function (Table 3).With Ingenuity’s network-building tools, 62 of the

71 genes were incorporated into five networks(Figure 4, Table 4). Interestingly, theproto-oncogenec-myc was involved in four of the five networks eventhough c-myc itself was not differentially expressed.Eight genes showed direct interaction with c-myc,four of which were upregulated (phosphoglyceratekinase 1 (PGK1), iron responsive element bindingprotein 2 (IREB2), 20,50-oligoadenylate synthetase 1,40/46 kDa (OAS1), and lymphoid enhancer bindingfactor 1 (LEF-1)), and four were downregulated(Metallothionein 1 (MT1A), benzodiazapine recep-tor—peripheral (BZRP), fibronectin 1 (FN1), andearly growth response 1 (EGR1)) by NGTCs. Thesefive networks could then be integrated into a single

Rotenone 100 NC (2) TT ���Simvastatin 150 NGTC (1) TT þþþSodium orthovanadate 20 NRS (5) ���Streptozocin 140 DNA damager ���Sulfamethoxazole 2000 NGTC (3) OS TT þþþSulindac 400 NRS (5) þþþTacrine 50 DNA damager ���Tamoxifen 750 NGTC (1) þþþTannic Acid 3000 NC (2) OS TT ��Tetracycline 500 NC (2) �Tetracycline 600 NC (2) TT ���Thioacetamide 150 MA; DNA damager þþTransAnethole 600 NC (2) OS TT ���Troglitazone 100 NC (1) ���Troglitazone 500 NC (1) ��Valproic Acid 350 NGTC (1) þþþValproic Acid 500 NGTC (1) TT þþþVerapamil 75 NC (1) OS þþþWY-14643 100 NGTC; PP þþþ

Classifications were based on the following references:1 Snyder and Green, 2001 [14].2 L. S. Gold, The carcinogenic potency project, http://potency.berkeley.edu/cpdb.html, 2005.3 Chemical Carcinogenesis Research Information System (CCRIS), TOXNET, http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?CCRIS, 2005.4 Physician’s Desk Reference, 2003 [34].5 US EPA/Scorecard safety assessment, http://www.scorecard.org/chemical-profiles, 2005.aCompounds were classified in rodents (mouse where indicated, otherwise rat) as: noncarcinogen (NC), probable noncarcinogens (NC?;where no evidence for being a carcinogen), not a recognized or suspect carcinogen (NRS), nongenotoxic carcinogen (NGTC; whererodent carcinogen but lacking mutagenic or DNA damaging properties), DNA damager, macrophage activator (MA), peroxisomeproliferator (PP), and probable peroxisome proliferator (PP?) based on previous studies of these same RNA samples.bOS, treatment induces oxidative stress in rat liver.cTT, treatment in training/testing sets.dPredicted nongenotoxic carcinogenicity, see Materials and Methods (Section ‘‘Microarray Data Normalization’’) for description.

Table 1. (Continued)

Compound Dose (mpk) Literature OS TT Predicted

NONGENOTOXIC CARCINOGEN GENE SIGNATURE 921

Molecular Carcinogenesis DOI 10.1002/mc

Table

2.

Log2(r

atio)

of

the

Six

Genes

inth

eFi

nalSig

natu

refo

rall

Tra

inin

g/T

est

ing

Com

pounds

Com

pound

Dose

(mpk)

Cla

ss

CD

NA

mic

roarr

ay

CodeLi

nk

olig

och

ips

Mat1

aM

t1a

NU

TF2

Pgrm

c1Sel1

hU

dpgtr

2M

at1

aM

t1a

Nutf

2Pgrm

c1Sel1

hU

dpgpr2

NM

_012860

NM

_138826

BC

061569

NM

_021766

NM

_177933

NM

_173295

GE1268854

GE21633

GE1243390

GE20803

GE12572

GE21748

Asp

irin

600

NC

�0.1

0.6

70.0

60.0

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0.0

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1

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irone

100

NC

0.0

5�

0.1

0.0

70.1

4�

0.0

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8�

1.6

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4

Capto

pril

5000

NC

0.5

40.5

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40.0

20.1

21.3

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0�

0.1

8�

0.0

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61.1

8

Clo

zapin

e150

NC

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2�

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meth

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NC

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ulfi

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2010

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NC

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NC

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yldopa

1000

NC

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1.0

7�

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30.0

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pro

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NC

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ophenolic

aci

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NC

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NC

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9�

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1�

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NC

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Perh

exi

lene

2010

NC

�0.0

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Phenyl

ephrine

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0.1

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ine

1000

NC

0.6

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Retinol(v

itam

inA

)200

NC

0.5

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20.2

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NC

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Aci

d3000

NC

0.2

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0.0

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1.7

40.0

00.0

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acy

clin

e600

NC

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nsA

neth

ole

600

NC

�0.0

70.8

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Anili

ne

200

NG

TC

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nolo

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TC

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mocr

yptine

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TC

0.4

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0.0

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1.1

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10.7

8

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late

dhyd

roxy

tolu

ene

1000

NG

TC

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10.1

50.5

51.0

1�

0.1

2.7

4�

0.6

50.0

20.7

51.0

5�

0.6

42.8

0

Carb

am

aze

pin

e225

NG

TC

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6�

0.7

60.1

10.4

70.0

12.2

6�

1.3

80.0

00.7

70.8

50.0

62.6

8

Chlo

rpro

mazi

ne

150

NG

TC

�0.3

�0.2

30.1

80.4

7�

0.0

91.4

5�

1.7

9�

0.1

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0.0

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0.5

60.6

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4

Cyp

rote

rone

Ace

tate

200

NG

TC

�0.5

6�

1.1

40.1

70.0

6�

0.0

91.1

7�

0.0

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0.1

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50.0

01.6

7

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ole

ne

500

NG

TC

0.6

3�

0.0

80.0

10.1

5�

0.1

70.2

2�

0.9

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0.0

50.2

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1�

0.3

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1

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ne

50

NG

TC

0.3

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20

0.0

7�

0.2

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00.0

90.4

90.7

8�

0.8

91.0

7

Die

ldrin

45

NG

TC

�0.0

1�

1.0

50.1

30.2

40.0

52.0

9�

0.2

7�

0.0

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0.1

40.6

70.3

22.8

3

Felb

am

ate

2100

NG

TC

�0.3

6�

0.4

70.0

10.6

2�

0.0

52.5

4�

0.3

9�

0.0

10.4

10.8

7�

0.2

62.8

5

Furo

sem

ide

1500

NG

TC

�0.2

1�

1.1

8�

0.0

90.0

7�

0.2

50.3

30.2

2�

0.1

70.1

60.1

2�

0.3

30.9

0

Gabapentin

2160

NG

TC

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4�

0.4

30.3

40.2

40.0

10.4

1�

2.3

6�

0.1

80.2

7�

0.1

9�

1.0

9�

0.1

4

922 NIE ET AL.

Molecular Carcinogenesis DOI 10.1002/mc

network via c-myc and FN1 and therefore presented acomplete picture of gene expression changes inresponse to NGTC treatments (Figure 4).

DISCUSSION

The gene expression signature determined forNGTCs was 88.5% accurate in classifying samples.This gene expression signature is less robust thanthose determined for PPs and MAs with the samesamples previously [25,26], but should be morevaluable in evaluating drug candidates, becauseNGTCs are notably difficult to detect early indevelopment, and 2-yr carcinogenicity studies arerequired for definitive results. The NGTC geneexpression signature performed well across a broadset of mostly hepatotoxic pharmaceuticals, andpredicted a number of extrahepatic tumor-inducingcompounds. In addition to being a target of highconcentrations of gavage dosed NGTC, rat liver isresponsive to a number of growth factors andhormones (such as sex hormones and prolactin) thatmediate effects of some NGTCs. Some of theobserved gene expression changes in response toNGTCs may protect rat liver from tumors, whereasother tissues may lack these protective mechanisms.In a few cases, induction of hepatic genes may becausal for observed extrahepatic tumors; UDPGT is awell-characterized example, and was a critical genein theNGTC signature [36]. A high percentage of theNGTC gene expression signature set was linked to c-myc- andFN1-regulatedpathways, suggesting impor-tant roles for that proto-oncogene and for integrinsignaling. Importantly, genes related to DNAdamage and repair were missing from the NGTCsignature, and almost all of the genotoxic com-pounds detected as positives (e.g., phenytoinand some steroids) are known to also have NGTCeffects.The final NGTC gene expression signature draws a

different picture for the mechanisms underlyingnongenotoxic carcinogenicity. One of the objectivesof the gene expression signature selection methodwas to eliminate redundancy. The six genes in thefinal signature set thus do not appear coordinatelyregulated, and distinct mechanisms are suggested bytheir differential regulation. Several of these genesare not ones that changed most in magnitude orsignificance, but to the extent that our databaserepresents NGTCs, should give a good coverage ofpossible pathways involved in nongenotoxic carci-nogenesis.Phenobarbital-inducible UDP glucuronyltransfer-

ase is a NGTC indicator gene that has a well-characterizedmechanismof action.Thyroxine levelsin the rat are largely determined by clearance afterglucuronidation by UDPGT. When UDPGT isinduced, plasma levels of thyroid hormone fall,removing feedback inhibition of thyroid stimulatoryhormone (TSH) synthesis and secretion in theIs

onia

zid

125

NG

TC

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0.6

0.0

60.0

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7

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pra

zole

200

NG

TC

0.0

3�

0.1

80.1

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0.0

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0

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apyr

ilene

200

NG

TC

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0.0

50.0

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60.6

6�

0.7

01.2

7

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ota

line

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NG

TC

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4�

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0.0

80.3

5

Phenobarb

ital

225

NG

TC

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7

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nyl

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xide

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TC

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2

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TC

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0.0

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pic

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NG

TC

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0.4

9�

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0.1

9�

0.0

80.4

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90.9

4

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vast

atin

150

NG

TC

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8�

0.5

20.1

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70.1

60.0

70.0

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1

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eth

oxa

zole

2000

NG

TC

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6�

0.6

2�

0.0

50.0

1�

0.0

81.9

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1.4

60.0

10.3

20.1

70.3

72.0

0

Valp

roic

aci

d500

NG

TC

0.0

2�

0.0

9�

0.0

60.0

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0.2

50.2

3�

0.5

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0.0

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80.3

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0.1

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4

NONGENOTOXIC CARCINOGEN GENE SIGNATURE 923

Molecular Carcinogenesis DOI 10.1002/mc

Table

3.

Sele

cted

Genes

for

NG

TC

Acc

ess

ion

P-v

alu

eN

GTC

NC

Sym

bol

Annota

tion

Anno

aG

Ob

Focu

sc

NM

_138826

1.4

E–

07

�0.5

20.3

4M

T1A

Meta

lloth

ionein

1A

(funct

ional)

Aa,b

,c,d

g,

mN

M_177933

6.8

E–

07

�0.1

00.0

4Sel1

h(S

el1

h)

Sel1

(suppre

ssor

of

lin-1

2)

1hom

olo

g(C

.ele

gans)

a,b

gA

I763615

2.8

E–

06

�0.4

90.3

1Sim

ilar

toM

T1E_hum

an

meta

lloth

ionein

-IE

XM

_238057

6.3

E–

06

�0.1

60.3

2U

DP-N

-ace

tyl-alp

ha- D

-gala

ctosa

min

e:p

oly

peptide

N-a

cety

lgala

ctosa

min

yltr

ansf

era

se2

(pre

dic

ted)

(Galn

t2_pre

dic

ted)

XM

_216045

2.9

E–

05

�0.1

40.0

4STO

MSto

matin

XM

_219525

4.0

E–

05

0.1

40.0

0Sim

ilar

toA

a2-2

77

BF5

47633

4.7

E–

05

0.0

7�

0.0

2EST

NM

_031657

1.1

E–

04

�0.1

20.0

8G

RK

6G

pro

tein

-couple

dre

cepto

rkin

ase

6A

BC

061569

1.8

E–

04

0.0

8�

0.1

1N

UTF2

Nucl

ear

transp

ort

fact

or

2A

gN

M_012532

2.1

E–

04

�0.3

10.1

8C

PC

eru

lopla

smin

(ferr

oxi

dase

)A

BU

760351

2.2

E–

04

�0.2

10.1

0Sim

ilar

toalc

oholdehyd

rogenase

PA

N1B-lik

epro

tein

BG

373195

2.4

E–

04

0.0

1�

0.1

1EST

NM

_022407

2.5

E–

04

0.7

40.0

1A

LDH

1A

1A

ldehyd

edehyd

rogenase

1fa

mily

,m

em

ber

A1

Aa,b

NM

_031644

2.7

E–

04

�0.0

9�

0.0

1Ptg

ds2

(Ptg

ds2

)pro

stagla

ndin

D2

synth

ase

2N

M_012555

3.1

E–

04

�0.2

10.0

1ETS1

v-ets

ery

thro

bla

stosi

svi

rus

E26

onco

gene

hom

olo

g1

(avi

an)

Aa,c

,d,e

XM

_216334

3.3

E–

04

0.4

1�

0.0

8Sim

ilar

toheat

shock

pro

tein

86

NM

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3.5

E–

04

0.3

20.0

0PSM

A1

Pro

teaso

me

(pro

som

e,

macr

opain

)su

bunit,

alp

ha

type,

1A

NM

_022245

3.6

E–

04

0.2

0�

0.1

5C

YB5

Cyt

och

rom

eb-5

AN

M_019143

3.7

E–

04

�0.2

50.0

4FN

1Fi

bro

nect

in1

Aa,b

,c,d

,em

NM

_013103

5.1

E–

04

�0.1

30.0

5TC

F2Tra

nsc

ription

fact

or

2,

hepatic;

LF-B

3;

variant

hepatic

nucl

ear

fact

or

AX

M_340849

5.2

E–

04

0.0

4�

0.0

5Sim

ilar

toRab

fam

ilysm

all

GTPase

Rah

XM

_213571

6.3

E–

04

0.2

9�

0.1

0Sim

ilar

topro

teaso

me

26S

non-A

TPase

subunit

2N

M_031000

6.6

E–

04

0.3

00.0

1A

KR1A

1A

ldo-k

eto

reduct

ase

fam

ily1,

mem

ber

A1

(ald

ehyd

ere

duct

ase

)N

M_012771

6.7

E–

04

�0.3

20.1

0LY

ZLy

sozy

me

(renalam

yloid

osi

s)A

XM

_224623

7.5

E–

04

0.0

5�

0.0

4Sim

ilar

tocD

NA

sequence

BC

019806

(LO

C306270),

mRN

AN

M_147205

8.1

E–

04

�0.2

40.0

5SIA

T1

Sia

lyltra

nsf

era

se1

(beta

-gala

ctosi

de

alp

ha-2

,6-s

ialy

ltra

nsf

era

se)

NM

_017282

9.7

E–

04

0.1

2�

0.0

2PSM

A5

Pro

teaso

me

(pro

som

e,

macr

opain

)su

bunit,

alp

ha

type,

5A

NM

_017170

9.8

E–

04

�0.3

10.0

4A

PC

SA

myl

oid

Pco

mponent,

seru

mA

NM

_021766

1.1

E–

03

0.2

50.0

0Pgrm

c1(P

grm

c1)

pro

gest

ero

ne

rece

pto

rm

em

bra

ne

com

ponent

1g

NM

_012551

1.5

E–

03

�0.0

80.0

7EG

R1

Early

gro

wth

resp

onse

1A

a,b

,c,d

,em

NM

_130429

1.5

E–

03

0.1

0�

0.0

4LE

F1Ly

mphoid

enhance

r-bin

din

gfa

ctor

1A

a,b

,c,d

,em

NM

_012637

1.5

E–

03

�0.1

80.0

1PTPN

1Pro

tein

tyro

sine

phosp

hata

se,

non-r

ece

pto

rty

pe

1A

a,b

,dN

M_017159

1.6

E–

03

�0.4

6�

0.0

3H

al

(Hal)

his

tidin

eam

monia

lyase

XM

_342854

1.6

E–

03

0.0

2�

0.0

5N

FIB

Nucl

ear

fact

or

I/B

NM

_145778

1.6

E–

03

0.0

2�

0.0

6TU

BG

1Tubulin

,gam

ma

1A

NM

_017143

1.6

E–

03

�0.3

9�

0.0

6F1

0C

oagula

tion

fact

or

XA

a,e

NM

_012966

1.6

E–

03

0.1

9�

0.0

7H

SPE1

Heat

shock

10

kD

apro

tein

1(c

hapero

nin

10)

Ad,e

NM

_024382

1.7

E–

03

�0.2

40.0

1SERPIN

D1

Serine

(or

cyst

ein

e)pro

tein

ase

inhib

itor,

clade

D(h

eparin

cofa

ctor)

,m

em

ber

1A

NM

_031504

1.7

E–

03

�0.3

90.0

4C

4A

Com

ple

ment

com

ponent

4A

AN

M_031149

1.7

E–

03

0.3

50.0

6PSM

C5

Pro

teaso

me

(pro

som

e,

macr

opain

)26S

subunit,

ATPase

,5

AB

924 NIE ET AL.

Molecular Carcinogenesis DOI 10.1002/mc

NM

_013037

1.8

E–

03

0.0

7�

0.0

3IL

1RL1

Inte

rleukin

1re

cepto

r-lik

e1

Ab,e

NM

_012516

1.8

E–

03

�0.1

70.1

9C

4BPA

Com

ple

ment

com

ponent

4bin

din

gpro

tein

,alp

ha

AA

A858661

1.9

E–

03

�0.1

3�

0.0

1Sim

ilar

toN

otc

h4,

PBX

2,

RA

GE,

lyso

phatidic

aci

dacy

ltr

ansf

era

se-a

lpha,

palm

itoyl

-pro

tein

thio

est

era

se2

(PPT2),

CREB-R

PX

M_220754

2.0

E–

03

0.1

3�

0.0

6Sim

ilar

to26S

pro

teaso

me

non-A

TPase

regula

tory

subunit

11

D43623

2.0

E–

03

�0.2

1�

0.0

4C

PT1B

Carn

itin

epalm

itoyl

transf

era

se1B

(musc

le)

AN

M_012618

2.0

E–

03

�0.0

70.0

3S100A

4S100

calc

ium

bin

din

gpro

tein

A4

(calc

ium

pro

tein

,ca

lvasc

ulin

,m

eta

stasi

n,

murine

pla

centa

lhom

olo

g)

AA

NM

_023983

2.1

E–

03

0.0

2�

0.0

7M

CA

MM

ela

nom

ace

lladhesi

on

mole

cule

a,b

NM

_012515

2.1

E–

03

�0.1

60.0

2BZRP

Benzo

dia

zapin

ere

cepto

r(p

eriphera

l)A

mN

M_022860

2.3

E–

03

�0.2

0�

0.0

2G

alg

t1(G

alg

t1)

beta

-4N

-ace

tylg

ala

ctosa

min

yltr

ansf

era

sea,b

,d,e

BE106243

2.7

E–

03

�0.0

90.0

2EST

XM

_215733

2.7

E–

03

�0.1

8�

0.0

3PK

P4

Pla

kophili

n4

AN

M_022592

2.8

E–

03

0.5

80.1

3TK

TTra

nsk

eto

lase

(Wern

icke-K

ors

akoff

syndro

me)

AX

M_213534

2.8

E–

03

�0.2

3�

0.0

3Sim

ilar

toBeta

heavy

chain

of

oute

r-arm

axo

nem

aldyn

ein

ATPase

XM

_233556

3.0

E–

03

�0.1

10.0

3Sim

ilar

toSer/

Arg

-rela

ted

nucl

ear

matr

ixpro

tein

NM

_053291

3.1

E–

03

0.2

40.0

0PG

K1

Phosp

hogly

cera

tekin

ase

1A

bm

NM

_017073

3.1

E–

03

�0.4

60.1

2G

LUL

Glu

tam

ate

-am

monia

ligase

(glu

tam

ine

synth

ase

)A

NM

_080394

3.2

E–

03

�0.1

1�

0.0

1RELN

reelin

AN

M_020071

3.2

E–

03

�0.1

70.1

1FG

BFi

brinogen,

Bbeta

poly

peptide

AN

M_138913

3.2

E–

03

0.0

0�

0.0

8O

AS1

20 ,50 -

olig

oadenyl

ate

synth

eta

se1,

40/4

6kD

aA

mBF4

17097

3.2

E–

03

0.0

1�

0.1

0Sim

ilar

toce

lldiv

isio

ncy

cle

34

BI2

76078

3.3

E–

03

�0.1

9�

0.0

1Sim

ilar

toD

a1-6

XM

_213372

3.4

E–

03

�0.1

0�

0.0

2Sim

ilar

tom

acr

osi

alin

BG

373142

3.5

E–

03

0.4

50.0

3Sim

ilar

toG

luta

thio

neS-t

ransf

era

se,

theta

3N

M_177426

3.6

E–

03

0.5

2�

0.0

3G

STM

2G

luta

thio

neS-t

ransf

era

seM

2(m

usc

le)

AN

M_011740

3.6

E–

03

0.1

0�

0.0

1Y

WH

AZ

Tyr

osi

ne

3-m

onooxy

genase

/try

pto

phan

5-m

onooxy

genase

act

ivation

pro

tein

,ze

tapoly

peptide

A

NM

_031723

3.7

E–

03

�0.0

70.0

8Spc1

8(S

pc1

8)

signalpeptidase

com

ple

x18kD

NM

_013048

3.7

E–

03

�0.1

80.0

1Ttp

a(T

tpa)

toco

phero

l(a

lpha)

transf

er

pro

tein

NM

_057146

3.7

E–

03

�0.2

50.1

2C

9C

om

ple

ment

com

ponent

9A

a,b

,c,d

,eN

M_017272

3.8

E–

03

1.1

70.3

2A

ldh1a7

Ald

ehyd

edehyd

rogenase

fam

ily1,

subfa

mily

A7

NM

_057122

3.9

E–

03

0.1

8�

0.0

5PSM

C4

Pro

teaso

me

(pro

som

e,

macr

opain

)26S

subunit,

ATPase

,4

Ab

NM

_031057

4.0

E–

03

�0.0

90.0

6M

msd

h(M

msd

h)

meth

ylm

alo

nate

sem

iald

ehyd

edehyd

rogenase

gene

BM

388213

4.1

E–

03

�0.0

40.0

4EST

NM

_130749

4.1

E–

03

�0.0

30.0

7M

ARK

3M

AP/m

icro

tubule

affi

nity-

regula

ting

kin

ase

3A

NM

_013073

4.4

E–

03

�0.1

20.0

4PC

MT1

Pro

tein

- L-iso

asp

art

ate

(D-a

spart

ate

)O

-meth

yltr

ansf

era

seA

NM

_012844

4.5

E–

03

1.3

00.4

0EPH

X1

Epoxi

de

hyd

rola

se1,

mic

roso

mal(x

enobio

tic)

BC

062034

4.5

E–

03

�0.0

80.0

3A

KR1B1

Ald

o-k

eto

reduct

ase

fam

ily1,

mem

ber

B1

(ald

ose

reduct

ase

)A

CA

510059

4.5

E–

03

�0.1

4�

0.0

1EST

CA

510162

4.7

E–

03

�0.3

5�

0.0

9Sim

ilar

toK

DEL

(Lys

-Asp

-Glu

-Leu)

endopla

smic

reticu

lum

pro

tein

rete

ntion

rece

pto

r3

NM

_017321

4.7

E–

03

0.0

2�

0.1

1A

CO

1A

conitase

1,

solu

ble

AX

M_343770

4.8

E–

03

�0.1

50.0

1Sim

ilar

tohyp

oth

etica

lpro

tein

MG

C31104

NM

_022184

4.8

E–

03

0.0

4�

0.0

3C

ASK

Calc

ium

/calm

odulin

-dependent

serine

pro

tein

kin

ase

(MA

GU

Kfa

mily

)A

AA

858640

5.2

E–

03

�0.1

70.1

9EST

(Continued

)

NONGENOTOXIC CARCINOGEN GENE SIGNATURE 925

Molecular Carcinogenesis DOI 10.1002/mc

NM

_031360

5.2

E–

03

�0.0

90.0

2SM

PD

2Sphin

gom

yelin

phosp

hodie

stera

se2,

neutr

alm

em

bra

ne

(neutr

al

sphin

gom

yelin

ase

)N

M_019226

5.2

E–

03

0.0

2�

0.1

0D

NC

H1

Dyn

ein

,cy

topla

smic

,heavy

poly

peptide

1A

a,b

XM

_216805

5.5

E–

03

0.0

9�

0.0

1Rat

igdelta

heavy

chain

const

ant

regio

nand

30ut,

mrn

aN

M_031714

5.6

E–

03

0.0

00.3

2H

rsp12

(Hrs

p12)

heat-

resp

onsi

vepro

tein

12

AA

900536

5.6

E–

03

�0.1

1�

0.0

2EST

NM

_012803

5.8

E–

03

�0.2

8�

0.0

5PRO

CPro

tein

C(inact

ivato

rof

coagula

tion

fact

ors

Va

and

VIII

a)

Ad,e

M75148

5.9

E–

03

0.0

6�

0.0

3K

NS2

Kin

esi

n2

60/7

0kD

aA

NM

_053626

5.9

E–

03

0.0

00.0

6D

AO

D-a

min

o-a

cid

oxi

dase

AA

Y310161

6.0

E–

03

�0.2

30.0

6FG

AFi

brinogen,

Aalp

ha

poly

peptide

Aa,e

XM

_216371

6.3

E–

03

�0.0

20.0

4EST

NM

_052809

6.4

E–

03

�0.6

2�

0.0

8C

DO

1C

yste

ine

dio

xygenase

,ty

pe

IA

BM

385722

6.4

E–

03

0.0

6�

0.1

0Sim

ilar

toriboso

malpro

tein

S18,

cyto

solic

BQ

195702

6.7

E–

03

�0.0

70.0

6TTC

11

Tetr

atr

icopeptide

repeat

dom

ain

11

NM

_022518

6.8

E–

03

0.0

9�

0.0

6A

RF1

AD

P-r

ibosy

lation

fact

or

1A

bBF4

16253

6.9

E–

03

0.1

00.0

2RPL1

8A

Rib

oso

malpro

tein

L18a

AA

W522260

7.0

E–

03

0.0

5�

0.0

3EST

CK

603120

7.2

E–

03

�0.0

60.0

2EST

XM

_341663

7.2

E–

03

0.0

3�

0.0

2Sim

ilar

toheat

shock

pro

tein

40

NM

_006082

7.3

E–

03

0.3

60.0

6K

-ALP

HA

-1Tubulin

,alp

ha,

ubiq

uitous

AX

M_227084

7.3

E–

03

�0.0

60.1

3N

nt

(Nnt)

nic

otinam

ide

nucl

eotide

transh

ydro

genase

BI2

92938

7.4

E–

03

�0.1

90.0

3EST

XM

_218976

7.4

E–

03

0.0

1�

0.0

7Sim

ilar

toN

up98-N

up96

pre

curs

or

AA

957282

7.7

E–

03

�0.1

7�

0.0

4EST

NM

_145779

7.9

E–

03

�0.3

10.0

1PZP

Pre

gnancy

-zone

pro

tein

AN

M_021593

8.0

E–

03

�0.1

10.1

4K

mo

(Km

o)

kyn

ure

nin

e3-h

ydro

xyla

seBM

386092

8.1

E–

03

�0.0

60.1

1EST

NM

_012860

8.3

E–

03

�0.3

00.0

5M

at1

a(M

at1

a)

meth

ionin

eadenosy

ltra

nsf

era

seI,

alp

ha

gN

M_006082

8.4

E–

03

0.2

50.0

1K

-ALP

HA

-1Tubulin

,alp

ha,

ubiq

uitous

AN

M_017099

8.4

E–

03

�0.1

4�

0.0

6K

CN

J8Pota

ssiu

min

ward

ly-r

ect

ifyi

ng

channel,

subfa

mily

J,m

em

ber

8N

M_022863

8.4

E–

03

0.1

30.0

3IR

EB2

Iron-r

esp

onsi

veele

ment

bin

din

gpro

tein

2A

mBU

759412

8.5

E–

03

0.0

4�

0.0

2Sim

ilar

tohyp

oth

etica

lpro

tein

MG

C10120

NM

_017284

8.7

E–

03

0.0

8�

0.0

5PSM

B2

Pro

teaso

me

(pro

som

e,

macr

opain

)su

bunit,

beta

type,

2A

bBQ

211102

8.8

E–

03

0.0

4�

0.0

3Sim

ilar

toH

SPC

263

NM

_019299

8.9

E–

03

0.0

8�

0.0

4C

LTC

Cla

thrin,

heavy

poly

peptide

(Hc)

AN

M_031595

8.9

E–

03

0.0

6�

0.0

5PSM

C3

Pro

teaso

me

(pro

som

e,

macr

opain

)26S

subunit,

ATPase

,3

Ab

NM

_017070

8.9

E–

03

�0.0

30.2

4Srd

5a1

(Srd

5a1)

stero

id5

alp

ha-r

educt

ase

1N

M_017280

9.0

E–

03

0.1

30.0

2PSM

A3

Pro

teaso

me

(pro

som

e,

macr

opain

)su

bunit,

alp

ha

type,

3A

BI2

80199

9.0

E–

03

�0.1

9�

0.0

3A

P1S1

Adapto

r-re

late

dpro

tein

com

ple

x1,

sigm

a1

subunit

ABM

391072

9.2

E–

03

0.0

4�

0.0

2Sim

ilar

toN

AD

Hdehyd

rogenase

NM

_022615

9.2

E–

03

0.0

70.0

1TO

P1

Topois

om

era

se(D

NA

)I

Aa,b

,cBQ

202155

9.5

E–

03

�0.0

70.0

4Sim

ilar

toN

AD

H-u

biq

uin

one

oxi

dore

duct

ase

B9

subunit

Table

3.

(Continued)

Acc

ess

ion

P-v

alu

eN

GTC

NC

Sym

bol

Annota

tion

Anno

aG

Ob

Focu

sc

926 NIE ET AL.

Molecular Carcinogenesis DOI 10.1002/mc

pituitary gland. Increased TSH levels lead toincreased production of thyroid hormones, whichare removed by glucuronidation. With chronic TSHsecretion, overstimulation and hyperplasia of thethyroid gland occurs and tumors can eventuallydevelop [36]. InductionofUDPGT is a fairly commonmechanism of nongenotoxic carcinogenesis in therat, and it is not surprising that this gene made ourfinal gene set.A second NGTC signature gene with strong

literature support for a role in carcinogenesis ismethionine adenosyltransferase 1 alpha (Mat1a).Mat1a is the hepatic-specific isozyme; extra-hepatictissues, fetal and regenerating livers and hepatomasexpress Mat2a, and these isozymes tend to be op-positely regulated [37–40]. The product of methio-nine adenosyltransferase activity, S-adenosyl-L-methionine (SAM), is atmuchhigher concentrationsin liver,which expressesMat1a, than inother tissues,which express Mat2a. High levels of SAM are asso-ciated with methylation of the promoter region ofthe Mat2A gene and decreased expression of thisisozyme [41].High levels of SAM or its metabolites are believed

to be critical for DNA methylation and silencing ofhepatic genes, the proposed basis for anticancereffects in the liver.Mat1a-knockoutmice havemuchreduced SAM levels, their livers are much bigger andmicroarray analysis indicates many acute phase,proliferative and MA-responsive genes are inducedin the livers of these animals [42]. Mat1a-knockoutmice spontaneously develop steatosis, and in com-mon with rodents treated with MA, also eventuallydevelop hepatocellular carcinomas [43]. Thioaceta-mide, aMA in rat liver, produced a decrease inMat1aand SAM levels, but an increase in Mat2a andhypomethylation of DNA [44]. SAM apparentlyenhances IkB activity, which in turn inactivatesNFkB. Induction of NFkB-regulated genes protectsdamaged or preneoplastic cells from apoptosis andmay also stimulate proliferation, eventually leadingto lesions and tumors [45]. Decreased levels of SAMalso prime hepatocytes to respond to growth factors[46]. Mat1a repression in the NGTC signaturesupports thehypothesis suggesting that hypomethy-lation is an important mechanism for nongenotoxiccarcinogenesis [47,48]. Hypomethylation alsoreportedly is responsible for increased expression ofc-myc [49].A third gene in theNGTC signature ismetallothio-

nein1 a (MT1a).Metallothioneins are small cysteine-rich proteins capable of complexing metals. MT1a isone of several isozymes important in protecting cellsfromheavymetal toxicity, is greatly induced by tracemetals such as zinc and cadmium, but also is inducedby other nonmetallic stressors, and the cysteineresidues may be important in protection fromoxidative stressors. Decreased expression or induc-tion of MT1a promotes carcinogenesis [50], and theN

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NONGENOTOXIC CARCINOGEN GENE SIGNATURE 927

Molecular Carcinogenesis DOI 10.1002/mc

Figure 2. Principal component analysis plots of the training/testing compounds using all six genes in the finalgene expression signature. Black circles represent NGTCs and gray triangles represent NCs. a: With gene expressiondata from cDNA microarrays; b: with gene expression data from CodeLink microarrays. The percentages in the axesreflect the contribution to the total variances from each principal component. The percentage in the title is the totalcontribution of variances from all three principal components.

Figure 3. Scatter plots of gene expression data (log ratio) between the cDNA and CodeLink platforms on the sixgenes in the final gene signature. x-axis, cDNA microarray; y-axis, CodeLink microarray. The correspondingCodeLink probe ID’s are shown on the axis axels. Gray circles represent NCs and black ones represent NGTCs.

928 NIE ET AL.

Molecular Carcinogenesis DOI 10.1002/mc

expression of MT1a can be inhibited by myc protein[51].Progesterone receptor membrane component 1

(Pgrmc-1, similar to Hpr6.6 and DAP1p) is expressedin the endoplasmic reticulum in liver. This gene isstrongly induced by dioxin and is hypothesized tomediate interactions between endogenous steroids,such as progesterone and estrogen, and arylhydro-carbon receptor agonists. Most of these compoundsare carcinogenic at high doses. The human homologHpr6.6 is induced at high levels inmany tumors, andhas beenpostulated tomediate cell death in responseto oxidative stress in breast cancer cells [52]. In yeast,deletion of DAP1p (damage resistance protein 1related to the membrane progesterone receptor)leads to loss of mitochondrial function, elongatedtelomeres, and inhibition of ergosterol synthesiswith accumulation of precursors squalene and

lanosterol. DAP1p binds heme and stabilizes a yeastcytochromeP450, Erg11p/Cyp51p. A role of Pgrmc-1in nongenomic carcinogenesis is far from clear, butincreased CYP activities and changes in sterolmetabolism are often associated with hepatomegalyand NGTC.Nuclear factor 2 (NUTF2; p10) is essential for ran-

mediated shuttling of molecules from cytosol intothe nucleus. Knockouts of this gene are lethal, andlowered expression in Drosophila is associated withfailure of certain transcription factors, such as NFkBhomologs, to transport into the nucleus [53]. Whilethe observed induction in theNGTCgene expressionsignaturemay reflect increased activity and turnoverof NUTF2, regulation of this gene is largely unchar-acterized.Another gene in the NGTC signature, suppressor

of lin-12 homolog (SEL1h) inhibits the function of

Figure 4. Gene regulation networks constructed with 62 genessignificantly up- or downregulated by NGTC treatments compared toNC treatments. Each edge indicates an interaction and each shape(e.g., circle) represents a gene. Warm color (red or pink) indicatesupregulation and cool color (green) indicates downregulation. Emptyshapes represents genes not on the microarray and gray onesrepresent genes on the microarray but were not significantly up- or

downregulated. In panel a–e, dark lines represent genes andinteractions in the specific network, and genes and interactions fromall other networks are represented by gray lines for reference. c-mycand eight genes from the gene list that directly interact with c-mycare circled. See Table 4 for lists of genes and related functions. a–e,networks a–e; f: all networks merged via c-myc and FN1.

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Molecular Carcinogenesis DOI 10.1002/mc

lin-12 and its homolog Notch, receptor pathwaysimportant in cell fate determination. SEL1h isexpressed basally at high levels in pancreas andstomach, and disappears in tumors from thesetissues. Notch pathways appear important in bileduct development and differentiation. Notch-1 alsohas been implicated in development of cholangio-carcinoma; blockade of Notch-1 activity leads toapoptosis of bile duct cells during inflammation [54].A role for SEL1horNotch inparenchymal liver cells isless clear. Repression of Sel1h sometimes leads togrowth factor-like and dedifferentiation effects inother tissues [55], but remains uncharacterized in ratliver.For pathway determination using the annotated

significant genes, genes that were either up- ordownregulated by NGTC treatments overrepre-sented a number of gene categories: cancer, cellulargrowth and proliferation, cell cycle, cell death, andimmune and lymphatic system development andfunction (Table 2). c-myc provides a nexus for allthese pathways. c-mychas longbeen recognized as anproto-oncogene whose activation by virus infection,mutation, DNA hypomethylation, or other meansinitiates or promotes tumor progression. It is notsurprising to see that all of the five networks built up

with genes selected from this study could be inter-connected via c-myc. Activation of PGK1 by c-mycelevates the glucose uptake in cancer cells that relyon glycosis for energy [56]. Upregulation of IREB2 byc-myc results in an increase of intracellular ironconcentration essential for c-myc-induced cell trans-formation andproliferation [57]. LEF-1 is a transcrip-tion factor initially identified inpre-B andT-cells andimplicated in Wnt signaling and tumorigenesis.Overexpression of LEF-1 occurred frequently incolon cancer, and knockout of LEF-1 resulted inincreased sensitivity to apoptosis [58,59]. Theexpression of MT1a can be inhibited by myc protein[51] and the lack of MT1a in the cell promotescarcinogenesis [50]. FN1 encodes an extracellularmatrix protein fibronectin, which plays an impor-tant role in cell adhesion. Activation of c-myc leads torepressionof FN1 in cancer cells [60].Overexpressionof EGR1 in cancer cell lines downregulates c-mycexpression and initiates cell differentiation [61].Both OAS1 and BZRP have been shown to beregulated by c-myc but their functions are not veryclear [62]. However, research has shown that OAS1influences host susceptibility to viral infection [63]and BZRP can be a potential prognostic factor forcolorectal cancer [64].

Table 4. Genes Organized in Ingenuity’s Networks

Network ID Genes in network Top functions

A ADRB2, CLIC4, CLTC, CTPS, CYB5, CYCS, DNCH1, GRK6,GSTM2, GSTP1, JRK, K-ALPHA-1a, KPNA2, KRT8, MARK3,M-RIP, MT1A, myc, NUTF2, PCTK2, PGK1, PHLDB2, PPFIBP1,RAN, S100A4, TGM2, TOP1, TP53, TUBA2, TUBB, TUBB2,TUBG1, YWHAD, YWHAG, YWHAZ

CancerCell cycleReproductive system disease

B AP1B1, AP1S1, ARF1, C4A, CP, CPT1B, EP300, ETS1, ETS2,ETV4, F2, F5, FGA, FGB, FGG, FOS, HNF4A, IL1RL1, KRT8,MMP1, MTPN, myc, NCOA3, PCMT1, POU1F1, PPARA, PROC,RARG, RUNX2, RXRA, SERPIND1, STAT6, TCF2, THBD, TNF

Hematological diseaseGene expressionViral function

C APBA1, APCS, APP, C4BPA, C4BPB, CASK, CDO1, CTSG,DAO, DCN, DLG1, F10a, FN1a, GLUL, HTR2C, IGFBP5, ITGAV,KIF5A, KIF5B, KIF5C, KNS2, LIN7B, LIN7C, MMP2, MYOD1,PKP4, PLG, PROS1, PSEN1, PZP, RELN, STXBP1, TGFB1,THBS1, TKT

Cell-to-cell signaling and interactionCellular movementCellular assembly and organization

D ACO1, AKR1B1, AKT1, ALDH1A1, BZRP, C9, CEBPA, CLU,DBI, EEF2, ETS2, FASN, HSPD1, HSPE1, IREB2, ITGA3, JUP,LEF1, MTPN, MYB, myc, MYCN, NFYA, OAS1, PTK2, PTPN1,RPL18A, RPL19, RPL3, RPL5, RPS7, RUVBL1, RUVBL2, SOD2,TNFSF6

Protein synthesisCancerCell death

E ATXN3, CD44, EGR1, FN1a, ITGAM, LYZ, MMP14, myc, p44S10,PLK1, PSMA1, PSMA2, PSMA3, PSMA4, PSMA5, PSMA6,PSMA7, PSMB1, PSMB10, PSMB2, PSMB3, PSMB4, PSMB5,PSMB6, PSMB7, PSMB8, PSMB9, PSMC2a, PSMC3, PSMC4,PSMC5, PSMD1a, PSMD4, TGFB1, TNF

Cell-to-cell signaling and interactionHematological system development

and functionImmune and lymphatic system

development and function

For each network, all genes are shown and those from the selected gene list are bolded. The interactions between genes are visualized inFigure 3. The top implicated functions from the genes in the network are listed in the third column.Bold—focus genes. Gene identifiers that made the user-defined cutoff and map to the Global Molecular Network are displayed withbold text.aDuplicates. Gene IDs marked with an asterisk indicate that multiple identifiers from the input list mapped to a single gene in the GlobalMolecular Network.

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The NGTC signature gene set defined in this studyperforms well with the pharmaceuticals studied, butmay need revision if very different types of com-pounds (e.g., herbicides and insecticides) are exam-ined. Only relatively few of these compounds weretested (rotenone, dieldrin, and lindane). This wouldbe an interesting area to explore further and possiblyexpand or modify the NGTC gene expression signa-ture. Similarly incomplete is our gene list on themicroarray. Mat2a was missing, and the expectedreciprocal regulation with Mat1A could not beexamined. At least one well-characterized mechan-ism of NGTC action was missed because of a geneabsent from our microarray: the alpha 2u globulingene is induced in male rat liver by a number oftoxicants (such as limonene and tertiary butylalcohol) and causes kidney tumors [65]. It was alsosurprising that PPs were generally detected by ourNGTC gene expression signature set, despite pro-nounced gene expression differences from mostcompounds, whereas MAs were largely missed(�30% of MAs were detected as NGTCs) by theNGTC gene expression signature.Compounds incorrectly classified by the NGTC

included strong false-positives ketoconazole, diflu-nisal, and verapamil, all classified as NC from rodentcarcinogen studies [14]. At the high dose (150mg/kgvs. 80 mg/kg/day in the rat carcinogenicity study)administered in the present study, ketoconazole isa strong suppressor of androgen biosynthesis, andapparently produces the same hepatic gene res-ponses as two other antagonists of androgen actions,flutamide and lansoprazole, both of which inducetesticular tumors in rats ([34], P3033, p3205).Diflunisal at high dose (750 mg/kg vs. 40 mg/kg/day in the rat carcinogenicity study) triggers thesame hepatic gene expression changes as PP [25],which are generally detected by theNGTC signature.Verapamil (75 mg/kg vs. 120 mg/kg/day in the ratcarcinogenicity study) appears to be a true falsepositive (cannot be explained by a lower dose inthe carcinogenicity study). A second NC calciumchannel blocker nifedipine also was detected as aweak false positive by the NGTC gene signature,suggesting commoneffects of this class of compoundon liver at high dose. Generally the doses used in thepresent short-term study were higher and bettertolerated than doses for carcinogenicity studies. Forseveral antiepileptics (including felbamate, pheny-toin, gabapentin, and carbamazepine), known carci-nogens in the rat, low doses were detected asnegatives or weak positives by the NGTC gene ex-pression signature, although high doses were alldetected as strong positives.The cDNAmicroarray used in this manuscript had

a small number of genes (1471). It was expected thata whole genome microarray would enable us to findmore and better genes to predict NGTC. In an effortto regenerate the toxicogenomic database on the

commercially available CodeLink Rat Whole Gen-omemicroarray, the validity of the final NGTC genesignature was verified. These genes showed higherdynamic changes in expression on the CodeLinkmicroarray and the prediction accuracy was 84%(Table 2).The microarray approach probably overestimates

the NGTC potential for the compounds examined,because higher doses are used in the present studythan in carcinogenicity studies. NGTCs often pro-duce tumors only at high doses, and some NGTCsmay even be anticancer agents at low doses [66]. Inaddition to providing a highly accurate predictivescreen for early detection of NGTCs among drugcandidates, the gene expression signatures in thisstudy provide starting points for further investiga-tion of pathways and mechanisms involved in earlystages of carcinogenesis.

ACKNOWLEDGMENTS

The support of Drs. Mark Johnson, Alfred Tonelli,WilliamPowers, andMichael Jackson for this projectis gratefully acknowledged.

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Molecular Carcinogenesis DOI 10.1002/mc