Systems-ADME/Tox: Resources and network approaches

29 Pages·2005·1.35 MB·English

JournalofPharmacologicalandToxicologicalMethods53(2006)38–66
www.elsevier.com/locate/jpharmtox
Appraisal of state-of-the art
Systems-ADME/Tox: Resources and network approaches
Sean Ekins*
GeneGo,500RenaissanceDrive,Suite106,St.Joseph,MI49085,USA
SchoolofPharmacyDepartmentofPharmaceuticalSciences,UniversityofMaryland,USA
Received23May2005;accepted23May2005
Abstract
The increasing cost of drug development is partially due to our failure to identify undesirable compounds at an early enough stage of
development. The application of higher throughput screening methods have resulted in the generation of very large datasets from cells in
vitroorfrominvivoexperimentsfollowingthetreatmentwithdrugsorknowntoxins.Inrecentyearsthedevelopmentofsystemsbiology,
databases and pathway software has enabled the analysis of the high-throughput data in the context of the whole cell. One of the latest
technology paradigms to be applied alongside the existing in vitro and computational models for absorption, distribution, metabolism,
excretionandtoxicology(ADME/Tox)involvestheintegrationofcomplexmultidimensionaldatasets,termedtoxicogenomics.Thegoalisto
provide a more complete understanding of the effects a molecule might have on the entire biological system. However, due to the sheer
complexityofthisdataitmaybenecessarytoapplyoneormoredifferenttypesofcomputationalapproachesthathaveasyetnotbeenfully
utilized inthis field.
ThepresentreviewdescribesthedatageneratedcurrentlyandintroducescomputationalapproachesasacomponentofADME/Tox.These
methods include network algorithms and manually curated databases of interactions that have been separately classified under systems
biologymethods.Theintegrationofthesedisparatetoolswillresultinsystems-ADME/Toxanditisimportanttounderstandexactlywhat
data resources and technologies are available and applicable. Examples of networks derived with important drug transporters and drug
metabolizing enzymesareprovidedto demonstrate the networktechnologies.
D 2005Elsevier Inc.All rights reserved.
Keywords:Algorithms;Human;Microarray;Mouse;Networks;Rat;Software;Toxicogenomics;Toxicoproteomics
1. Introduction in the industry as they have been evaluated primarily for
toxicology and metabolism assessment (Waring et al.,
Metabolism and safety assessment have witnessed some 2003, 2002, 2001) with some considerable focus on
growth in the number of new technologies and methods hepatotoxicity (Harris, Dial, & Casciano, 2004; Hartley
that have been introduced within the last decade. However, et al., 2004; Heijne et al., 2004; Huang et al., 2004;
according to a recent FDA white paper there is still Liguori et al., 2005; Ulrich, Rockett, Gibson, & Pettit,
considerable scope for additional new methods (FDA, 2004). For example, searching PubMed for publications in
2004). For example, recently various reports have the last 5 years with the keywords Fmicroarray and
described new software and methods for metabolism toxicology_ or Ftoxicogenomics_, indicates that the accu-
prediction (Balakin et al., 2004a, 2004b; Borodina et al., mulation of papers describing the latter is doubling every
2003; Borodina et al., 2004; Boyer & Zamora, 2002; year (Fig. 1) which perhaps is mirrored by the application
Korolev et al., 2003). Simultaneously the use of high in the pharmaceutical industry for predictive toxicology
throughput (HT) methods for genomics, proteomics and (Suter, Babiss, & Wheeldon, 2004).
metabonomics have taken off in terms of their acceptance To date toxicogenomics experiments have been carried
out under non-standardized conditions. Most of the studies
have been conducted with rats, less often mice, using
*Correspondingauthor.Tel.:[phone];fax:[phone].
E-mailaddresses: [email protected],[email protected]. multiple different microarray formats and statistical proce-
1056-8719/$-seefrontmatterD2005ElsevierInc.Allrightsreserved.
doi:10.1016/j.vascn.2005.05.005
S.Ekins/JournalofPharmacologicalandToxicologicalMethods53(2006)38–66 39
n 100 The perturbing effect of a molecule on the complete
s i biological system can be observed across all metabolic
n 80
o and signaling pathways or networks and can provide some
ati
cd 60 limited insight into the binding to multiple proteins or
bliMe effectsongeneexpressionsimultaneously.Thisrequiresthe
ub
of pPu 40 collection of high-throughput data, including global gene
r expression, protein content, metabolic profiles for the same
e 20
mb samples as well as individual genetic, clinical and pheno-
Nu 0 typic data. However there are difficulties with such an
2000 2001 2002 2003 2004 2005 approach as there are likely to be differences between the
Year Fstimulus to effect_ durations for all the gene–protein
relationships (Nicholson, Holmes, Lindon, & Wilson,
Fig.1.Annualfrequencyofarticlesappearingwiththewords‘‘toxicoge-
2004).
nomics’’(squares)or‘‘microarrayandtoxicology’’(diamonds).
We can now use either thegrowingnumber ofacademic
or commercially available pathway database and network
dures (Tables 1-4). There have been relatively few cross- building tools with expression data. These enable the
platform toxicogenomics studies under controlled condi- connection of interacting, differentially expressed genes as
tions (Thompson et al., 2004). Despite the suggested poor networks(Barabasi&Oltvai,2004;Hanisch,Zien,Zimmer,
compatibility between the different array types, this latter & Lengauer, 2002; Ideker, Ozier, Schwikowski, & Siegal,
study demonstrated a high (90%) consistency between the 2002;Idekeretal.,2001;Segaletal.,2003a;Segal,Wang,&
expression of the genes that were shared between the Koller, 2003b; Spirin & Mirny, 2003; Tornow & Mewes,
platforms. The development of methods to visualize such 2003) as well as allowing the reverse engineering of
complex expression data has also expanded beyond the functional connections (Somogyi, Fuhrman, &Wen, 2001).
widelyusedclusteringmethods(Eisen,Spellman,Brown,& The use of such network visualizations suggests an
Botstein, 1998). With the outcome of microarray analysis organized modularity in complex systems (Han et al.,
being dependent on the widely used statistical procedures 2004) which has also been applied to interpret the
applied to derive those genes that are significantly differ- connectivity of small molecules and their interaction with
entially expressed (Butte, 2002), newer approaches that do proteinsinthesubfieldofchemogenomics(Bredel&Jacoby,
notnecessarilyrequiredataclusteringmaybeanadvantage. 2004; Csermely, Agoston, & Pongor, 2005; Parsons et al.,
As rat and mouse are the most widely used in vivo toxicity 2004; Sharom, Bellows, & Tyers, 2004). The parallel
models it is assumed that acute and chronic toxicity shown developmentofHTmethods,databases,ADME/Toxmodel-
inanimalslargelycoincideswith human toxicity.Therefore ing and systems modeling is ongoing (Ekins, Nikolsky, &
differential expression patterns in animal models are also Nikolskaya, 2005e). The present review is therefore timely
assumed to be predictive of the end point toxic response in as it discusses some of the data resources, limitations and
human. This is not always the case due to differences technologies that are available for Systems-ADME/Tox
between human and rodent physiology, genetics, metabo- (Fig. 2) along with some illustration of their applications to
lism and signaling pathways. For example, the mechanism drug metabolism and drug transport which are key com-
of toxicity for pyrazinamide has been reconstructed ponents of the ADME/Tox process. The ultimate aim of
(Bugrim, Nikolskaya, & Nikolsky, 2004) to illustrate that this discussion is to provide awareness of an integrated
the accumulation of uric acid occurs in human, but not in approach rather than a technology silo mentality, represent-
mice,andthisresultsintoxicityintheformer.Therelatively ing thelatestproposed research model in thefield (Fig. 2).
poor understanding of such species differences may be
reflected in the relatively large number of late stage
molecules that have undergone in vivo toxicity assessment 2. Data available
yet have been later withdrawn due to adverse events in
humans. A recently published book provides an excellent over-
Inrecentyears theappearanceofsystems biologywhich view of toxicogenomics and the reader is referred to this to
uses the relationships of all elements of biology rather than gain more insight into the applications and limitations
approaching them separately has been evident and will (Hamadeh & Afshari, 2004). The growing number of
likely reunite biological fields (Harrison, 2004; Hood & toxicogenomic datasets derived from in vivo studies with
Galas, 2003). These systems approaches are the latest rat (Table 1) and mouse (Table 2) as well as in vitro cell
incarnation of the importance of the Fparts vs wholes_ derived data (Table 3) highlights the different strains,
debate(Ekins&McGowan,2001)andinterpretingADME/ microarraytypesandcompoundsthatareroutinelyassessed.
Tox in this context may improve our understanding and Alsotherearenumerousinstancesofmultiplegroupstesting
ultimatepredictions(Bugrimetal.,2004;Ekins,Boulanger, the same compound at similar or different doses, e.g. well
Swaan, & Hupcey, 2002a; Kitano, 2002a; Werner, 2003). knownhepatotoxicantsornephrotoxicantssuchasclofibrate
40 S.Ekins/JournalofPharmacologicalandToxicologicalMethods53(2006)38–66
Table1
Literaturetoxicogenomicsdataderivedfromratinvivostudies
Compounds Ratstrain Microarraytype Compounddose Microarraydata Reference
availability
Acetaminophen;furan; MaleSprague– Phase1Molecular Acetaminophen Genename,accession (Huangetal.,
methotrexate; DawleyVAF+ toxicologyarray (4500mg/kg/day); numberandfoldchanges 2004)
methapyrilene; albino methotrexate(1mg/ areprovidedina
phenytoin kg/day); manuscripttable
methapyrilene(100
mg/kg/day);furan(40
mg/kg/day)or
phenytoin(300mg/
kg/day)
A-277249;3MC; Sprague–Dawley Affymetrixrat 10or100mg/kg Genenameand (Waringetal.,
Aroclor toxicologyU34array AffymetrixIDandfold 2002)
changeinamanuscript
table
Clofibrate;paracetamol; MaleSprague– Customchip CLOat250mg/kg Genenameandfold (Cunningham,
benzoapyrene Dawley BPat10mg/kgbw changeinamanuscript Liang,Fuhrman,
given3timesper table Seilhamer,&
weekfor2weeks, Somogyi,2000)
APAPat1000mg/kg
L-742694 Female 25Kratmicroarray L-742694(50mg/kg/ Tablesofaccession (Hartleyetal.,
Dexamethasone Sprague–Dawley day);DEX(50mg/ numberandgenenames 2004)
kg/day) onlyinamanuscript
tableforliverand
intestine
Bromobenzene MaleWistar Custom3000ratgene 0.5,2,5mmol/kg Genename,accession (Heijneetal.,
array numberandlogfold 2004)
changefor3doselevels
inamanuscripttable.
Supplementaldataalso
available
1,25-Dihydroxyvitamin MaleSprague– Affymetrixhigh-density 730ng/kg Genename,Genbank (Kutuzova&
D3 Dawley(small ratoligonucleotide accessionnumberand Deluca,2004)
intestine) arrays(GeneChips foldchangeina
RG-U34A) manuscripttable
Fenofibrate;clofibrate; MaleCD Agilentarrays Doserangingforup Genenameandgenbank (Cornwell,Souza,
bezafibrate; to14days accessionnumberina &Ulrich,2004)
gemfibrozi; manuscripttable
ciprofibrate;
beclofibrate;etofibrate
Furan MaleSprague– NIEHSratchip¨7000 Exposedto4or40 GeneIDandgene (Hamadehetal.,
Dawley clones mg/kgfuranforup name—binarydataina 2004)
to14days manuscripttableDataset
alsoavailableonNIEHS
website
Paracetamol MaleF344/N RatNIEHStoxchip 0,50,150, Unigeneaccession (Heinlothetal.,
(http://dir.niehs.nih.gov/ 1500mg/kg number,genename,fold 2004)
microarray/chips.htm) changeinamanuscript
table
Carbontetrachloride MaleSprague– ADMERatexpression Upto14day Accessionnumbersand (Youngetal.,
Dawley bioarray(MotorolaLife treatment binarydataina 2003)
Sciences)consistsof manuscripttable
1040single-stranded
oligonucleotideprobes
Carbontetrachlorideand MaleSprague– TheratCTarrays 6,24,72hhighand Gene names and fold (Kieretal.,2004)
chloroform Dawley containsequencesfrom lowdoses changesatmultipletime
almost700ratgenes points in a manuscript
withknownor table
discovered
responsivenesstotoxic
treatments
(continuedonnextpage)
S.Ekins/JournalofPharmacologicalandToxicologicalMethods53(2006)38–66 41
Table1(continued)
Compounds Ratstrain Microarraytype Compounddose Microarraydata Reference
availability
Dimethylarsinicacid FemaleF344 Rat10Kchip(MWG 100ppm Genbankaccession (Wei,Arnold,
(bladder BiotechInc.)containing number,genenameina Cano,&Cohen,
epithelium) 10,000genes manuscripttable 2005)
Dexamethasone; FemaleSprague– RatHepatoChip DEX(50mg/kg/day); GenenameandGenbank (Meneses-Lorente
troleandomycin; Dawley TAO(500mg/kg/ accessionnumber—data etal.,2003)
miconazole; day);MIC(100mg/ displayedasaheatmap
clotrimazole; kg/day);CLOT(100 notreadilyextracted
isoniazid; mg/kgperday);ISN frompublication
methylclofanapate (100mg/kg/day);
MCP(75mg/kg/day)
TCDDPeCDFPCB126 FemaleHarlan AffymetrixGeneChip Exposedfor13weeks Accessionnumber,gene (Vezina,Walker,
PCB153 Sprague–Dawley Test3arrays totoxicologically nameandfoldchangein &Olson,2004)
equivalentdoses amanuscripttable
Paclitaxel Maleandfemale Genocheck4.8KcDNA 4mg/kg/daymale; Accessionnumbersand (Leeetal.,2004)
Sprague–Dawley 7mg/kg/dayfemale genenamesina
manuscripttable
Clofibrate;gemfibrozil; MaleSprague– 4.8KcDNAmicroarray Treatedwitheach Genenameandfold (Jungetal.,2004)
phenytoin DawleyVAF(+) inhouse compoundfor24h changeinamanuscript
albino and2weeks table
Clofibrate; Sprague–Dawley MerckDrugSafetyChip 30mg/kg/day Genename,GenBank (Gerholdetal.,
dexamethasone; 1443genes(rat,human accessionnumberand 2001)
phenobarbital; andmouse) relativefluorescence
3-methylcholanthrene levelsinamanuscript
table
Bemitradine;clofibrate; MaleCD;IGS IncyteRatGEM1.0 Low,midandhigh Abarchartof2genes (Krameretal.,
doxylamine; ¨7800ratcDNAs doses thatareaffectedby 2004)
methapyrilene; compounds
phenobarbital;
tamoxifen;
2-acetylaminofluorene;
4-acetylaminofluorene;
isoniazid
PhIP FemaleSprague– MousecDNA 75mg/kg/day Genenamesina (Shan,Yu,Schut,
Dawley microarraycontaining manuscripttable &Snyderwine,
9984cDNAclones 2004)
(NationalCancer
Institute
Ethinylestradiol Maleandfemale Customchip3776genes 0,0.01,0.1,and Accessionnumbergene (Katoetal.,2004)
Sprague–Dawley 1.0ppm nameandfoldchangeat
differentexposurelevels
inamanuscripttable
Paraquat MaleWistar 1090genes 7mg/kg/day Genenameand (Satomietal.,
expressionratioina 2004)
manuscripttable
Hexachlorobenzene FemaleBrown Affymetrixrat 0,150,or450mg/kg Accessionnumber,gene (Ezendametal.,
Norway RGU-34AGeneChip nameandfoldchange 2004)
microarray dataformultipleorgans
inamanuscripttable
N-methyl-N-Vnitro-N- Rat pyloric muco- AFFYMETRIXRat 83mg/l AFFYID,genename, (Yamashitaetal.,
nitrosoguanidine sae;malecongenic GenomeU34A genesymbolandfold 2004)
(MNNG) ratstrainthathasa changeinamanuscript
homozygous_LIZ table
transgeneof
BigBlue\rat
N-methyl-N-Vnitro-N- MaleACI/NJcI AffymetrixGeneChip 83mg/lfromtheage Accessionnumber,gene (Abeetal.,2003)
nitrosoguanidine (ACI) RatgenomeU34A of8weeksthroughto name,symbolfoldchange
arrays 40weeks inamanuscripttable;also
ratvshumanstomach
cancercomparison
Cisplatin Sprague–Dawley Differentarrays,tox 0.3–5mg/kgovera4to Unigene, gene ID, gene (Thompson et al.,
chip,incyte,phase1,etc. 144h name NIEHS ID and 2004)
datafor5platformsina
manuscripttable
42 S.Ekins/JournalofPharmacologicalandToxicologicalMethods53(2006)38–66
Table1(continued)
Compounds Ratstrain Microarraytype Compounddose Microarraydata Reference
availability
Clofibrate MaleSprague– AtlasRatToxicologyII High(250mg/kg/day)or Genbankaccession (Bakeretal.,
Dawley arrays(Clontech,Palo low(25mg/kg/day) numberanddatafor3 2004)
Alto,CA,USA) platformsina
containing465genes manuscripttable
Di(2-ethylhexyl) MaleSprague– Anin-housecDNA 20or2000mg/kg Genenames,Genbank (Kijimaetal.,
phthalate Dawley—(testes) microarray IDandfoldchangeina 2004)
manuscripttable
Ciprofibrate FemaleFischer Anin-housecDNA 50mg/kgbodyweight Genenames,symbol, (Yadetieetal.,
microarray accessionnumber,mean 2003)
ratioandSD
Methapyrilene MaleSprague– RatToxChip1.0 10or100mg/kg/day GeneName,accession (Hamadehetal.,
Dawley numberandindicationof 2002b)
upordownregulation;
originaldataavailableon
NIEHSwebsite
Ecteinascidin-743 FemaleWistar CustomchipcDNA 40ug/kg Dataavailableon (Donaldetal.,
(ET-743) microarrayscontaining laboratorywebsite—not 2002)
approximately4700 availableatpresent
hybridizablemouse
expressedsequencetags
derivedfromIMAGE
clonesobtainedfrom
ResearchGenetics
(Huntsville,AL)orfrom
theMRCHumanGene
MappingProject
Clofibrate;Wyeth MaleSprague– NIEHSratchipv1.0 Clofibrate(250 Genenamesandfold (Hamadehetal.,
14,643;Gemfibrozil; DawleyVAF+ mg/kg/day;Wyeth changesinamanuscript 2002a)
phenobarbital; 14,643(250mg/kg/ table
day);Gemfibrozil
(100mg/kg/day);
Phenobarbital
(120mg/kg/day)
Vinclozin;procymidone MaleSprague– ClontechAtlasRat1.2 200mg/kg Genename,accession (Rosen,Wilson,
Dawley—prostate Toxicologyarray number,averagefold Schmid,&Gray,
change 2005)
Cisplatin MaleSprague– RatToxMicroarrays 0.5or1mg/kg/day Genename,accession (Huangetal.,
DawleyVAF1 werepurchasedfrom number,foldchangein 2001)
albino(CRL: Phase-1Molecular kidney
CD(SD)BR Toxicology
Allylalcohol; MaleSprague– AffymetrixGeneChip Allylalcohol(40mg/kg Heatmapfiguresanda (Waringetal.,
miodarone;Aroclor Dawley Test2Array day);miodarone(100mg/ tableofAffymetrix 2001)
1254;arsenic; kg/day);Aroclor1254 namesforgenes
carbamazepine; (400mg/kg/day);arsenic correlatedwithclinical
carbontetrachloride; (20mg/kg/day); chemistrychangesina
diethylnitrosamine; carbamazepine(250mg/ manuscripttable
dimethylformamide; kg/day);carbon
diquat;etoposide; tetrachloride(1000mg/kg/
indomethacin; day);diethylnitrosamine
methapyrilene; (100mg/kg/day);
methotrexate; dimethylformamide(1000
monocrotaline; mg/kg/day);diquat(17.2
3-methylcholanthrene mg/kg/day);etoposide(50
mg/kg/day);indomethacin
(20mg/kg/day);
methapyrilene(250mg/
kg/day);methotrexate
(250mg/kg/day);
monocrotaline(50mg/kg/
day);
3-methylcholanthrene
(100mg/kg/day)
(continuedonnextpage)
S.Ekins/JournalofPharmacologicalandToxicologicalMethods53(2006)38–66 43
Table1(continued)
Compounds Ratstrain Microarraytype Compounddose Microarraydata Reference
availability
Microcystin-LR(MLR); MaleWistar Purpose-maderatDNA Various Accessionnumbergene (Buleraetal.,
phenobarbital(PB); microarray(Affymetrix, name,foldchanges 2001)
lipopolysaccharide SantaClara,CA) shownasaheatmap
(LPS);carbon containing1,600rat tableinpublication—
tetrachloride(CT); DNAsequences dataextractionwouldbe
thioacetamide(THA); laborious
andcyproterone
acetate(CPA)
Acetamidofluorene; MaleSprague– CustomRatMegaA Acetamidofluorene(200 Genename,accession (McMillianetal.,
aniline;bromobenzene; Dawley cDNAchip3434-gene mg/kg);aniline(200mg/ numberinamanuscript 2004)
butylhydroxytol; kg);bromobenzene(900 table
dieldrin;disulfiram; mg/kg);butylhydroxytol
ethinylestradiol; (1000mg/kg);dieldrin
hexachlorocyclohexane (30and45mg/kg);
g;4-methylthiazole; disulfiram(2000mg/kg);
nimesulide;piperonyl ethinylestradiol(500
butoxide;precoceneI; mg/kg);
pulegone;tannicacid; hexachlorocyclohexane
trans-anethole gamma(40,65,80mg/
kg);4-methylthiazole
(120mg/kg);nimesulide
(500mg/kg);piperonyl
butoxide(4000mg/kg);
precoceneI(500mg/kg);
pulegone(400mg/kg);
tannicacid(3000mg/
kg);trans-anethole
(600mg/kg)
NIEHSwebsiteathttp://dir.niehs.nih.gov/microarray/datasets/home-pub.htm.
EDGEwebsiteathttp://edge.oncology.wisc.edu/.
andcisplatin,(Table1).Inthemajorityofcasestheresulting byprovidingfreelyaccessiblemicroarrayandothertoxicity
number of differentially expressed genes is a very small related data. Two of these databases being developed in the
subset of the starting number on the microarray following public domain are Chemical Effects in Biological Systems
clustering or other types of analysis. Upon closer examina- (CEBS) (http://www.niehs.nih.gov/nct/cebs.htm) (Mattes,
tion of these publications, the majority of them either Pettit, Sansone, Bushel, & Waters, 2004; Waters et al.,
provide images of a heat map and/or a table listing a gene 2003) which will accommodate gene expression profiles,
nameaccessionnumberandexpressionchange.Veryfewof proteomics and metabolomics data and allow complex
the published studies (Tables 1–3) provide the original raw queries (Hood, 2003a; Mattes et al., 2004). Similar goals
microarray data file at a freely accessible website, hence are being pursued in the development of the ArrayTrack
restrictingfurtheranalysisbyscientistsusingothersoftware. database at the FDA (Tong et al., 2003). The EDGE
Although in some cases it is possible to cut and paste the database (http://edge.oncology.wisc.edu/edge.php), an
gene expression data tables from the publication pdf files, expanding public effort at The University of Wisconsin,
this is not always the case. In the worst case scenario one contains mouse gene expression profiles following treat-
would have to manually retype gene lists or extract them ment with different toxic molecules (Hayes et al., 2005;
fromheatmapsasbinarytypedata.Asnotallcomputational Thomas et al., 2001). These separate efforts if widely
researchers will have a laboratory available to them to adopted should make published studies describing HT data
generate such quantities of toxicogenomics data, the latter more readily accessible, although it might have been more
points are important if we are going to continue to see efficienttoevolvetheseintoasingleglobaldatabaseinstead
innovation in software development for this data. This will of fragmented repositories.
require free unrestricted access to data published. Similarly Proteomics data has also been generated in a limited
ifwearetodiscern‘‘fingerprints’’formoleculesactingwith number of toxicology studies (Table 4), once again this has
a similar or identical mechanism we will need databases of been producedwith different strains ofrats andmice,using
many diverse chemical structures that have been tested in a different protein chips, 2-D gel electrophoresis (2-DIGE)
similar manner. There is therefore considerable interest in and mass spectroscopy methods (e.g. MALDI-MS). The
the current databases being developed by the NIEHS, FDA proteomic data is very rarely accessible to the reader for
andothergroupswhichshouldhelptoimprovethesituation their own computational analysis. Subsequently there have
44 S.Ekins/JournalofPharmacologicalandToxicologicalMethods53(2006)38–66
Table2
Literaturetoxicogenomicsdataderivedfrommouseinvivostudies
Compounds Mousestrain Microarraytype Compounddose Microarraydata Reference
availability
Benzene MaleandfemaleP53 AffymetrixandIncyte 300ppm,6hperday,5 Genename,accession (Yoonetal.,2003)
KOmiceand GEMsystem daysaweekfor2weeks numberandfoldchange
C57BL/6 datainamanuscript
table
Benzene Male129/SvJ AffymetrixMG-U74Av2 100ppm,6hperday,5 Genename,accession (Faiola,Fuller,
daysaweekfor2weeks numberandfoldchange Wong,&Recio,
datainamanuscript 2004)
table
Phenobarbital CARandwildtype NIEHSMousetoxchip 100mg/kgfor12h Accessionnumber,gene (Uedaetal.,2002)
8736genes nameandfoldchange
forwildtypeandknock
outmiceinamanuscript
table
Aroclor;BNF; C57BL/6J Customarraywith1200 Aroclor(200mg/kg); Genenamesandfold (Thomasetal.,
ciprofloxacin;cobalt cDNAs BNF(5mg/kg); changeasaheatmap 2001)
chloride;TCDD;IL-6; ciprofloxacin(250mg/ Dataisalsoavailablein
LPS;PCB-153; kg);cobaltchloride(60 theEDGEdatabase
phenobarbital; mg/kg);TCDD(10ug/
phenylhyrzn;TNFa; kg);IL-6(25ug/kg);
WY-16,463 LPS(1mg/kg);
PCB-153(80mg/kg);
phenobarbital(100mg/
kg/day),3days;
phenylhyrzn(100mg/
kg);TNFa(50ug/kg);
WY-16,463(0.125%w/v)
TCPOBOP CD-1female Custom9000cDNA 1–3htreatment,3mg/ Accessionnumbersand (Lockeretal.,
mousearray kgbodywt foldchangedataina 2003)
manuscripttable
3H-1,2-dithiole-3-thione Malewild-typeand Affymetrixmurine 0.5mmol/kg Accessionnumber,gene (Kwaketal.,
(D3T) nrf2-disrupted genomeU74Av2 name,foldina 2003)
GeneChip manuscripttable
MDMA MalealbinoSwiss– 15KmousecDNAclone 47mg/kg, Genenameandfold (Xieetal.,2004)
Webster,(neurons) set changeinamanuscript
table
Phenytoin FemaleC57BL/6and Murinegenome-U74Av2 300mg/l Genename,accession (Trochoetal.,
LDLR(cid:1)/(cid:1) numberandfoldchange 2004)
inamanuscripttable
Genistein(1000Ag/ ICR(testes) CustomcDNA Genistein(1000Ag/ Accessionnumbergene (Adachietal.,
mouse/day)or microarray,containing mouse/day); nameandfoldchangein 2004)
diethylstilbestrol 1754cDNAprobes diethylstilbestrol(50Ag/ amanuscripttable—note
(DES)(50Ag/mouse/ mouse/day) veryfewgenes
day)
Cocaineand MaleICR MouseDiscoveryArrayi 40mgkg—1cocaine Dataapparentlynot (Hayase,
buprenorphine typeIarraycontaining onceadayfor4days;40 available Yamamoto,
2688brain-derivedprobes mgkg—1cocaineplus Yamamoto,Muso,
(DisplaySystemsBiotech 0.25mgkg—1BUPfor &Shiota,2004)
Inc.,Copenhagen, 4days
Denmark)
Methamphetamine MaleC57BL/J6 Affymetrixmouse 40mg/kg Geneaccessionnumber, (Thomas,
(striatum) genechip,mg-U74A.v2. geneIDandnameand Francescutti-
oligonucleotidesarrays, signallogratioina Verbeem,Liu,&
12488genes manuscripttable Kuhn,2004)
Di(2-ethylhexyl) MaleC57BL/6 MurinegenomeU74Av2 1.0%dietaryDEHPfor Genbankaccession (Wong&Gill
phthalate Arrays(MGU74Av2) 13weeks numbers,genename;log 2002)
ratiodatapresentasabar
chartinthepublication;
dataextractionwould
takesomeeffort
Diethylhexylphthalate MalePPARanulland Custommadecontaining 1150mg/kg/day Genenamesavailableon (Hasmalletal.,
wildtype 600toxgenes abarchart—quantitative 2002)
datanoteasilyaccessible
S.Ekins/JournalofPharmacologicalandToxicologicalMethods53(2006)38–66 45
Table2(continued)
Compounds Mousestrain Microarraytype Compounddose Microarraydata Reference
availability
Acetaminophen C57B1/63129/Ola Test-2Chips(Affymetrix) 300mg/kg GenbankorSwissProt (Reillyetal.,
hybrid thenindividual IDandfoldchangedata 2001)
oligonucleotide inamanuscripttable
microarrays(Mul1Ksub
AandsubB;Affymetrix)
thatcandetectthe
expressionof11,000
knowngenesand
expressedsequencetags
(ESTs)
Cadmiumchloride; MaleSwissWebster CustomChips Various Dataintableform,few (Bartosiewicz,
benzo(a)pyrene(BaP); containing148unique genesaffectedforBAP Penn,&Buckpitt,
andtrichloroethylene genes andTCE 2001)
(TCE)
been only a very small number of studies that have statistically evaluated, as demonstrated in a very large
combined both transcriptomic and proteomic methods with number of published examples (Dobrin, Beg, Karabasi, &
asingleanimalstrainaftertreatmentwithadrug.Hopefully Oltvai, 2004; Fiehn, 2001; Han et al., 2004; Hanisch et al.,
we will see this change in the future, but this will in turn 2002;Idekeretal.,2002;Jeong,Mason,Barabasi,&Oltvai,
present considerable challenges as huge amounts of 2001; Jeong, Tombor, Albert, Oltvai, & Barabasi, 2000; Li
proteomic data are combined with the equally large etal.,2004;Miloetal.,2002;Nikitin,Egorov,Daraselia,&
transcript data files. Mazo, 2003; Pereira-Leal, Enright, & Ouzounis, 2004;
Rives &Galitski, 2003;Segaletal.,2003a; Somogyietal.,
2001; Spirin & Mirny, 2003; Tornow & Mewes, 2003;
3. Network analysis and databases Vasquez, Flammini, Maritan, & Vespignani, 2003; Yeger-
Lotem & Margalit, 2003; Yu, Zhu, Greenbaum, Karro, &
Fromsomeoftheearlyreviewsofsystemsbiologythere Gerstein, 2004).
has been discussion of its application to drug discovery
(Kitano, 2002a,b) as well as the utility for ADME/Tox 3.1. Network applications
(Ekins et al., 2002a, 2000b). More recently several other
journalshavededicatedwholeissuestothefieldofsystems For example, one group has used as an inference the
biology.Howeveronecouldconsiderthisquiteabroadfield Bayesiannetworkmethodforanalysisoftissuetoxicityfrom
fromnetworkorpathwayanalysistoquantitativesimulation microarray data as well as a mechanistic simulation for a
oforganelles(Vo,Greenberg,&Palsson,2004),wholecells different pharmaceutically relevant molecule (Aksenov et
and organs. It is apparent that we are now understanding al., 2005). Pathway tools and various resources have also
organisms from the perspective of computationally gener- been applied to modeling the networks of nuclear hormone
atednetworksofproteinandligandinteractions(Barabasi& receptors and their connections with other genes and small
Oltvai, 2004). Network and pathway tools enable the molecules using a manually curated database, MetaDrug
analysisofHTdata inthecontextofallknowninteractions (Ekins, Kirillov, Rakmatulin, & Nikolskaya, 2005d) or
when using a database as the source. Individual reviews MetaCore (Ekins, Bugrim, Nikolsky, & Nikolskaya, 2005).
haveinsomecasesindicatedthatnetworkswillbevaluable Transcriptional regulation of many transporters, CYPs and
for understanding adverse events (Hood & Perlmutter, phaseIIenzymesareregulatedbythesereceptors,affecting
2004), drug target identification or validation (Butcher, endogenous molecule transport, metabolism, cell growth,
Berg, & Kunkel, 2004) and complex metabolic interactions proliferationandoxidativestress(Ulrich,2003;Ulrichetal.,
(Nicholson et al., 2004). A general schematic has been 2004).Whenthesignalingnetworksandinteractingligands
generatedinordertoprovideadescriptionoftheutilization for the transcriptional factors PPAR, FXR/RXRA, ESR1,
ofsuchpathwaydatabasesandnetworkbuildingalgorithms AHR,HNF4A,GCR-h, MCR,CAR-beta, GCR-a,LXR-a,
from the initial parsing of high throughput data to network CAR/RXR, HNF4, FXR, PXR/RXR heterodimer, PXR,
comparisons and visualization (Fig. 3). High throughput AHR/ARNT heterodimer, PPARa/LXRa, VDR, PPAR-a
data can be superimposed and visualized on the various arevisualizedaverycomplex pictureofinteractions canbe
proteininteractiondatabasesavailable.Thisisaccomplished created (Ekins et al., 2005d). This suggests that when we
by using either preset maps that capture current biological considera molecule bindingwith only one nuclearreceptor
knowledge or by building custom interaction networks we are observing only a fraction of the likely possible
usingmanydifferentalgorithmswhichcanbecomparedand feasibleinteractions, based on the data gatheredto date.
46 S.Ekins/JournalofPharmacologicalandToxicologicalMethods53(2006)38–66
Table3
Literaturetoxicogenomicsdataderivedfrominvitrocellstudies
Compounds Celltype Microarraytype Compounddose Microarraydata Reference
availability
4-Hydroxytamoxifen MCF-7breastcancer NIEHSToxChip 1uMhydroxytamoxifen DataavailableatNIEHS (Hodgesetal.,
estrogen microarrayconsistingof forayear,10nM website 2003)
1901genes 17b-estradiol
Trovafloxacin Humanhepatocytes AffymetrixU133Aarray 30–800uM 142genesavailablein (Liguorietal.,
supplementaltable—not 2005)
easilyextracted
Estrogen MCF-7breastcancer NIEHSToxChip 10–10M17b-estradiol DataavailableatNIEHS (Lobenhoferetal.,
microarrayconsistingof website 2002)
1901genes
Valproicacid NMRImiceembryo Customchipincluding 600mg/kgbodyweight Genesymbol,gene (Kultimaetal.,
andP19mouse 15KmousecDNAclone name,NIAESTlogfold 2004)
embryocarcinoma set changeinamanuscript
table
Sulindacsulfide Humancolorectal NIEHShuman12Kchip 10uM Genbankaccession (Bottone,
carcinoma SW-480 number,genenameand Martinez,Collins,
andHCT-116 foldchangeatvarious Afshari,&Eling,
timepointsina 2003)
manuscript table; data
alsoavailableonNIEHS
website
17Beta-estradiol;estriol; MCF-7 U95Aoligonucleotide 10nM(E2,estriol; Unigenename,gene (Terasakaetal.,
estrone;genistein; probearrays(Affymetrix estrone;DES)10AM nameandfoldchange 2004)
diethylstilbestrol; (genistein,bisphenolA, forestrogenresponsive
bisphenolA; nonylphenol,and andnonresponsiveina
nonylphenol; methoxychlor) manuscripttable
methoxychlor
Ouabain;laurylsulfate; HepG2 ClontechAtlasHuman Ouabain(43uM);lauryl Gene name, ratio, p- (Morgan et al.,
dimethylsulfoxide; StressToxicologycDNA sulfate(260uM); value in downloadable 2002)
cycloheximide; arrays(234genes) dimethylsulfoxide(1.28 tablesatjournalwebsite
tolbutamide;sodium M);cycloheximide(62.5
fluoride;diethyl uM);tolbutamide(12.8
maleate;buthionine; mM);sodiumfluoride(3
sulfoxamine; mM);diethylmaleate
potassiumbromate; (1.25mM);buthionine;
sodiumselenite; sulfoxamine(30mM);
alloxan;adriamycin; potassiumbromate(2.5
hydrogenperoxide mM);sodiumselenite(30
uM);alloxan(130mM);
adriamycin(40uM);
hydrogenperoxide
(4mM)
AflatoxinB(1)(AFB(1)), HepG2andprimary Genefilterarrays 1.0uMaflatoxinB1, None (Harrisetal.,
2-acetylaminofluorene hepatocytes containing31,000genes 4.0 mM acetaminophen, 2004)
(2AAF), 100uM
dimethylnitrosamine dimethylnitrosamine,
(DMN), 1.0uM
acetaminophen(APAP) 2-acetylaminofluorene
MitomycinC(MMC) L5178YTK(+/-) Affymetrixmouse Low,midandhighdoses Accessionnumber,gene (Huetal.,2004)
andcisplatin(CIS), mouselymphoma MG-U74AforMMC name,geneID,statistical
andanalkylating andMG-U745Av2 significanceateachtime
agent,methyl (AffymetrixInc.,Santa pointinamanuscript
methanesulfonate Clara,CA)forallthe table
(MMS); otherchemicals;atotal
indirect-acting of9977probesets
genotoxinsincluded (genesorESTs)common
hydroxyurea(HU),a tothesetwoarray
ribonucleotide models
reductaseinhibitor,
taxol(TXL),a
microtubuleinhibitor,
andetoposide
(ETOP),
S.Ekins/JournalofPharmacologicalandToxicologicalMethods53(2006)38–66 47
Table3(continued)
Compounds Celltype Microarraytype Compounddose Microarraydata Reference
availability
Hydroxyurea L5178YTk_/-mouse TheTwin-ChipMouse- 10ng/mlpaclitaxel,31.3 Genesymbolandfold (Leeetal.,2003)
(acarcinogen), lymphoma 7.4KDigitalGenomics ug/mlhydroxyurea,32 changeinamanuscript
p-anisidine cDNAmicroarray ug/mlp-anisidine table
(anoncarcinogen),
andpaclitaxel
Acetaminophen; WistarRat DualChiprathepato Asingleconcentration Accessionnumbersand (deLongueville
amiodarone;clofibrate; hepatocytes (Eppendorf,Hamburg, whichvariedforeach genename—fold etal.,2003)
erythromycinestolate; Germany) compound changesshownas
isoniazid;alpha- coloredheatmap—note
naphtylylisothiocyanate; easilyextractedfrom
beta-naphtoflavone; publication
4-pentenoicacid;
phenobarbital;
tetracycline;and
zileuton
Bleomycinandhydrogen Mouselymphoma ClontechMouse1.2K Bleomycin(2.5and20 Genenamesandfold (Seidel,Kan,
peroxide L5178Y/TK(+/-) cDNAmicroarray(1185 ug/ml);hydrogenperoxide changepresentedasbar Stott,Schisler,&
genes) (5and10ug/ml) chartsinpublication Gollapudi,2003)
Bupivicaine; HL-60 AgilenthumancDNA 1mM Genename,GenBank (Unami,
camptothecin microarray accessionnumber, Shinohara,
unigeneandratioina Ichikawa,&Baba,
manuscripttable 2003)
BisphenolA MouseSertoliTTE3 IntelliGenemouse 0–400uM Genename,GenBank (Tabuchi&
expressionglass accessionnumberand Kondo,2003)
microarrays(Version foldchangeattime
1.0,TakaraShuzo), pointsinamanuscript
whichwerespottedwith table
564cDNAfragmentsof
mouseknowngenesand
approximately301
expressedsequencetags
(ESTs)
MitomycinCor HepG2 85humangenecustom 10ummitomycinC, Barchartswithfold (Hong,Muller,&
doxorubicin array 2umdoxorubicinor changesinpublication; Lai,2003)
2%ethanol veryfewgenes
AmphotericinB Humanperipheral GF211FKnownGenes_ 5ug/ml Accessionnumbersand (Cleary,Rogers,&
bloodmononuclear GenefiltercDNAarray foldexpressionina Chapman,2001;
andTHP-1 (ResGen);thisarray manuscripttable Rogers,Pearson,
consistsof>4000 Cleary,Sullivan,
individualelements, &Chapman,
eachrepresentinga 2002)
knownhumangene
Benzo(a)pyrene;diol TK6human Human-350microarray, 0,0.01,0.10or Genenamesandfold (Akermanetal.,
epoxide lymphoblastoid aglassslidewith350 1.0ug/ml) changeatdosesina 2004)
spottedhumancDNA manuscripttable
probes(Phase-1
MolecularToxicology
Etomoxir HepG2 ClontechAtlasi 1mMetomoxir Genenamesandfold (Merrilletal.,
HumanStress changeinamanuscript 2002)
ToxicologycDNAarrays table
(234genes)
Tetrodotoxin Humangliomacell UsingAffymetrix 10and20mM AffymetrixID,Genbank (Raghavendra
lineHTB-138 GeneChip(HG-U133A ID,genename,gene Prasad,Qi,
symbolandfoldchange Srinivasan,&
inamanuscripttable Gopalakrishnakone,
2004)
Methotrexate; Humanacute AffymetrixU133Achip Low and high dose and Dataavailableas (Cheoketal.,2003)
mercaptopurine lymphoblastic combination supplementaldataonline
leukemia
Prednisolone; Humanacute AffymetrixU133Achip Various Dataavailableas (Hollemanetal.,
vincristine; lymphoblastic supplementaldataonline 2004)
asparaginase; leukemia
daunorubicin