Systems toxicology approach for the assessment of zebra sh neurotoxicity Roman Li1,2, Colette vom Berg1, Marja Talikka2, Sumit Madan3, Jens Dörpinghaus3, Justyna Szostak2, Florian Martin2, Manuel C. Peitsch2, Julia Hoeng2, Anze Zupanic1

1Swiss Federal Institute of Aquatic Science and Technology (Eawag), Überlandstrasse 133, CH-8600 Dübendorf, Switzerland. 2PMI R&D, Swiss Federal Institute of Aquatic Science Philip Morris Products S.A., Quai Jeanrenaud 5, CH-2000 Neuchâtel, Switzerland. 3Fraunhofer Institute for Algorithms and Scientific Com- and Technology puting, Schloss Birlinghoven, 53754 Sankt Augustin, Germany Corresponding author: [email protected]

Introduction and Objectives Biological Expression Language (BEL)

Recent years have seen an unprecedented rise in the use of zebrafish as a model to study chemical toxicity. This is Our systems toxicology approach involves the construction of a computable network. The network consists of molecular due in part to the following advantages: relationships curated from literature. Such molecular relationships are scripted in BEL, a language for representing sci- entific findings in a computable form. - External development - Amenability to high-throughput assays Evidence found in a publication (PMID 25568117): "Expression of tlx2 was downregulated in both sox9a and atoh1a - Rich and defined behavioral repertoire morphants at 24 hpf (Fig. 5A-C)." - Compliance with the 3Rs (replacement, reduction, refinement) principles - High-quality genome sequence (GRCz11) - 70% orthology with humans BEL statement: act(p(ZFIN:atoh1a)) increases r(ZFIN:tlx2) - Remarkably similar responses to chemicals in zebrafish and humans

Taking advantage of these benefits, we have selected zebrafish larvae for a comprehensive systems toxicology as- BEL function: Namespace: Relationship Entity: sessment of the insecticide Imidacloprid. -Abundance -HGNC -Gene -Processes -ZFIN - openbel.org Methods -Activity -CHEBI -Biological process

Standard toxicology Systems toxicology Zebra sh Neurotoxicity Network Exposure of newly fertilized Cardiac toxicity Scientific litera- Quantitative as- eggs to chemicals for 120 hours ture curation into sessment of net- BEL statements can be assembled into a computable biological network. The visualization of the zebrafish neurotoxicity (OECD 236) a computable work perturbation model model was created in Cytoscape and contains 660 BEL statements curated from 90 scientific publications.

p(ZFIN:hcrtr2)

actsIn

p(ZFIN:psen1) p(ZFIN:appb) act(p(ZFIN:hcrtr2))p(ZFIN:mib1)p(ZFIN:angpt1) p(ZFIN:)

actsIn actsIn increases actsIn actsIn actsIn

act(p(ZFIN:psen1)) act(p(ZFIN:appb)) r(ZFIN:aanat2)act(p(ZFIN:mib1))act(p(ZFIN:angpt1)) p(ZFIN:manf) p(ZFIN:zic2a) p(ZFIN:chd8) act(p(ZFIN:gbx2)) p(ZFIN:dixdc1a) p(ZFIN:pou5f3) p(ZFIN:fgf3) p(ZFIN:tcf3a)

increases decreases * decreases decreases increases decreases increases decreases decreases increases decreases Morphology translatedTo increases increases decreases decreases actsIn actsIn actsIn actsIn actsIn actsIn actsIn * decreases decreases increases increases decreases decreases decreases increases

increases decreases decreases

decreases r(ZFIN:dla) r(ZFIN:her6) a(CHEMBLID:CHEMBL255682)r(ZFIN:notch1a) p(ZFIN:aanat2) a(CHEBI:"1−methyl−4−phenyl−1,2,3,6−tetrahydropyridine")a(CHEBI:"triatomic arsenic") r(ZFIN:slc6a3) r(ZFIN:dld)act(p(ZFIN:manf))r(ZFIN:elavl3) act(p(ZFIN:zic2a)) r(ZFIN:msi2b)r(ZFIN:pou5f3)act(p(ZFIN:chd8)) r(ZFIN:dlb)r(ZFIN:otx1b)r(ZFIN:gbx1)r(ZFIN:hoxb5b)r(ZFIN:emx3)r(ZFIN:pou3f2b) act(p(ZFIN:dixdc1a))r(ZFIN:lhx8a)r(ZFIN:six3b)act(p(ZFIN:pou5f3))composite(p(ZFIN:eng2a),p(ZFIN:eng2b))r(ZFIN:) r(ZFIN:bmp2b) act(p(ZFIN:fgf3)) act(p(ZFIN:tcf3a)) r(ZFIN:wnt3a)

decreases increases increases increases increases decreases increases includes increases decreases increases increases increases translatedTo decreases increases decreases includes decreases decreases decreases decreases translatedTo actsIn decreases increases translatedTo decreases translatedTo increases increases increases decreases translatedTo decreases decreases decreases decreases increases decreases increases increases decreases decreases decreases decreases

increases decreases

complex(SCOMP:"gamma p(ZFIN:notch1a) act(p(ZFIN:aanat2)) r(ZFIN:atg5)p(ZFIN:notch1b)p(ZFIN:pik3r1) r(ZFIN:nr4a2b) p(ZFIN:fn1a) r(ZFIN:eomesa)r(ZFIN:sim1a)p(ZFIN:rbpja)r(ZFIN:arxa) r(ZFIN:otpb)a(SCHEM:Cocaine)r(ZFIN:dlx2a)r(ZFIN:chd) r(ZFIN:otx2) p(ZFIN:emx3) p(ZFIN:usp39) r(ZFIN:pax6a) p(ZFIN:six3b)r(ZFIN:egr2b)p(ZFIN:eng2b) p(ZFIN:pax2b)p(ZFIN:foxi1) r(ZFIN:pax2a) r(ZFIN:)r(ZFIN:dkk1a)r(ZFIN:wnt8b)r(ZFIN:wnt8a)r(ZFIN:six3a) r(ZFIN:rx3) p(ZFIN:wnt3a)p(ZFIN:dvl1b) Secretase Complex")

decreases actsIn decreases decreases increases increases increases translatedTo actsIn actsIn translatedTo translatedTo actsIn actsIn actsIn translatedTo actsIn actsIn

act(p(ZFIN:notch1a)) a(CHEBI:melatonin) p(ZFIN:atg5) act(p(ZFIN:fn1a)) act(p(ZFIN:rbpja))p(ZFIN:picalma) p(ZFIN:otpb)p(ZFIN:ppargc1a)m(ZFIN:mir133b) p(ZFIN:otx2) p(ZFIN:fyna) act(p(ZFIN:usp39)) act(p(ZFIN:pax2b))act(p(ZFIN:foxi1)) p(ZFIN:pax2a) p(ZFIN:fxyd1) p(ZFIN:nlk1)act(p(ZFIN:wnt3a))act(p(ZFIN:dvl1b)) increases

decreases decreases increases decreases increases increases directlyDecreases decreases decreases increases actsIn increases actsIn increases decreases increases increases decreases decreases increases decreases increases increases actsIn increases decreases actsIn actsIn actsIn actsIn increases decreases decreases increases actsIn increases decreases decreases decreases decreasesdecreases

path(MESHD:"Neurogenic bp(GOBP:"somatic muscle bp(GOBP:"Notch signaling a(CHEBI:histamine)p(ZFIN:neurog1)bp(GOBP:sleep)r(ZFIN:park7)r(ZFIN:mul1a)r(ZFIN:pink1) act(p(ZFIN:atg5)) r(ZFIN:sncgb) r(ZFIN:myog)p(ZFIN:)p(ZFIN:pcdh8)r(ZFIN:tpma) act(p(ZFIN:picalma))a(CHEBI:clonidine)a(CHEMBLID:CHEMBL289480)a(CHEBI:"vitamina(CHEBI:"ammonium E") chloride") a(CHEBI:sirolimus) p(ZFIN:mtor)act(p(ZFIN:otpb))act(p(ZFIN:ppargc1a)) r(ZFIN:) p(ZFIN:tsc2) act(p(ZFIN:fyna))p(ZFIN:plxna2) r(ZFIN:cdkn1a)r(ZFIN:rb1)r(ZFIN:tshba)r(ZFIN:cga) r(ZFIN:)r(ZFIN:pomca)r(ZFIN:pou1f1)r(ZFIN:)r(ZFIN:ascl1a) r(ZFIN:prl) r(ZFIN:six1b) act(p(ZFIN:pax2a)) p(ZFIN:atp1a1a_1)act(p(ZFIN:fxyd1))composite(p(ZFIN:hmx2),p(ZFIN:hmx3a)) act(p(ZFIN:nlk1)) Inammation") development") decreasespathway")

translatedTo decreases actsIn increases increases increases increases increases increases increases increasesdecreases increaseshasModi cation translatedTo translatedTo increases increases includes hasModi cation decreases decreases decreases decreases includes decreases increases decreases increases increases increases increases actsIn translatedTo translatedTo decreases increases directlyIncreases translatedTo actsIn translatedTo translatedTo actsIn increases Behavior increases increases Mechanistic hy- decreases decreases act(p(ZFIN:neurog1)) p(ZFIN:park7)p(ZFIN:mul1a) p(ZFIN:sncb) p(ZFIN:pink1) p(ZFIN:)p(ZFIN:map1lc3b) increases p(ZFIN:her6)p(ZFIN:map1lc3a) bp(GOBP:autophagy) p(ZFIN:mtor,pmod(P))act(p(ZFIN:mtor))r(ZFIN:oxt) p(ZFIN:rps6) p(ZFIN:pitx3) act(p(ZFIN:tsc2)) p(ZFIN:plxna2,pmod(P))p(ZFIN:sema6a)p(ZFIN:cdkn1a)p(ZFIN:rb1) p(ZFIN:e2f4)p(ZFIN:chmp1a) r(ZFIN:wnt1) p(ZFIN:six1b) r(ZFIN:eng2b) r(ZFIN:eng2a)r(ZFIN:fgf8a)act(p(ZFIN:atp1a1a_1))p(ZFIN:hmx3a)r(ZFIN:) r(ZFIN:fgf3)p(ZFIN:hmx2)r(ZFIN:fgf10a)

translatedTo decreases actsIn decreases decreases actsIn decreases increaseshasModi cation translatedTo increases increases increases decreases decreases decreases translatedTo decreases increases actsIn actsIn decreases actsIn decreasesincreasesdecreases actsIn actsIn actsIn translatedTo actsIn increases actsIn increases

a(CHEBI:pentetrazol)p(ZFIN:gsk3b) r(ZFIN:ebf2) act(p(ZFIN:sncb)) act(p(ZFIN:pink1)) a(CHEBI:rotenone) increases p(ZFIN:dld) p(ZFIN:insm1a) act(p(ZFIN:her6)) p(ZFIN:htt)p(ZFIN:maptb)p(ZFIN:mapta) p(ZFIN:rps6,pmod(P)) act(p(ZFIN:pitx3)) path(MESHD:Megalencephaly)r(ZFIN:rps6ka5)r(ZFIN:scn1lab)r(ZFIN:ctss2_1)r(ZFIN:qars) p(ZFIN:auts2a) p(ZFIN:mif) act(p(ZFIN:plxna2))act(p(ZFIN:sema6a)) act(p(ZFIN:rb1)) act(p(ZFIN:chmp1a))p(ZFIN:bmi1a)p(ZFIN:nlk2) p(ZFIN:wnt1) act(p(ZFIN:six1b)) p(ZFIN:cd74a) p(ZFIN:fgf8a)p(ZFIN:eng2a)r(ZFIN:rhoaa)act(p(ZFIN:hmx3a))p(ZFIN:mpp5a) p(ZFIN:crb2a) p(ZFIN:slc9a3r1) translatedTo potheses genera- directlyDecreases increases increases translatedTo increases increases hasModi cation directlyIncreasesactsIn decreases decreases decreases actsIn actsIn decreases increases increases actsIn increases decreases decreases increases actsIn increases increases decreases increases translatedTo decreases decreases increases increases increases decreases increases decreases decreases decreases decreases increases actsIn decreases increases actsIn actsIn decreases increases actsIn actsIn translatedTo actsIn actsIn actsIn decreases

decreases increases decreases decreases increases tion decreases a(CHEBI:"reactive oxygen r(ZFIN:ptgs1)a(CHEMBL:"6−Bromoindirubin")p(ZFIN:gsk3b,pmod(P)) p(ZFIN:ebf2) r(ZFIN:fezf2) r(ZFIN:reln)r(ZFIN:tigarb)r(ZFIN:) r(ZFIN:park2) r(ZFIN:th) r(ZFIN:th2) act(p(ZFIN:dld))act(p(ZFIN:insm1a)) p(ZFIN:ache)m(ZFIN:"mir9−2") p(ZFIN:aspm) a(CHEBI:"microcystin−LR") p(ZFIN:stil)act(p(ZFIN:rps6))p(ZFIN:kctd13)p(ZFIN:)r(ZFIN:drd2a)r(ZFIN:drd2b)p(ZFIN:met)p(ZFIN:ppp2ca)a(CHEBI:ethanol) act(p(ZFIN:auts2a))act(p(ZFIN:mif))r(ZFIN:shtn1)r(ZFIN:mmp2)r(ZFIN:rasl11b)r(ZFIN:dcdc2b)r(ZFIN:hdac1)r(ZFIN:rbbp4)r(ZFIN:rbl2) p(ZFIN:lef1)act(p(ZFIN:bmi1a)) act(p(ZFIN:nlk2))p(ZFIN:) r(ZFIN:eya1)act(p(ZFIN:wnt1))r(ZFIN:dacha) r(ZFIN:dachb)r(ZFIN:pax2b) act(p(ZFIN:cd74a))p(ZFIN:fgfr1a)act(p(ZFIN:fgf8a)) p(ZFIN:rhoaa) p(ZFIN:cldn5a)act(p(ZFIN:mpp5a))p(ZFIN:tjp1a)act(p(ZFIN:crb2a))p(ZFIN:prkci) p(ZFIN:vangl1) act(p(ZFIN:slc9a3r1)) species") actsIn increases directlyDecreases actsIn decreases actsIn translatedTo translatedTo increases increases increases directlyIncreases directlyDecreases increasestranslatedTo hasModi cationincreases actsIn decreases actsIn increases increases increases directlyIncreases translatedTo increases actsIn increases decreases increases directlyDecreases increases actsIn actsIn increases decreases increases decreases

increases increases increases increases actsIn increases translatedTo decreases actsIn translatedTo translatedTo translatedTo increases directlyDecreases actsIn actsIn actsIn decreases decreases translatedTo translatedTo directlyDecreases increases actsIn translatedTo translatedTo increases actsIn increases actsIn increases increases increases increases increases increases increases decreases increases decreases increases increases actsIn decreases increases increases increases increases

a(CHEBI:"SC−560")p(ZFIN:ptgs1) act(p(ZFIN:gsk3b))a(CHEBI:"retinoic acid") act(p(ZFIN:ebf2)) p(ZFIN:fezf2) p(ZFIN:tigarb)p(ZFIN:lgi1a) p(ZFIN:th)p(ZFIN:park2) p(ZFIN:th2) p(ZFIN:sncgb) r(ZFIN:) p(ZFIN:rbbp4) p(ZFIN:smo)p(ZFIN:elavl3)act(p(ZFIN:ache))r(ZFIN:ccna2)p(ZFIN:zic5)r(ZFIN:shha) act(p(ZFIN:aspm)) p(ZFIN:tp53)r(ZFIN:ache)r(ZFIN:lratb)r(ZFIN:pmela)p(ZFIN:rgrb)p(ZFIN:gap43)r(ZFIN:pmelb)r(ZFIN:manf)r(ZFIN:tuba1a)r(ZFIN:rpe65a)p(ZFIN:tuba1a)r(ZFIN:chrna7)p(ZFIN:inpp5kb)r(ZFIN:mbpa)r(ZFIN:gap43)r(ZFIN:stra6)r(ZFIN:rdh5)r(ZFIN:rlbp1b)r(ZFIN:dct) r(ZFIN:tyr)act(p(ZFIN:stil))act(p(ZFIN:kctd13))p(SFAM:"Histone H3act(p(ZFIN:foxe3)) Family") act(p(ZFIN:met))act(p(ZFIN:ppp2ca))r(ZFIN:agrn) p(ZFIN:pcna)p(ZFIN:) p(ZFIN:rasl11b)p(ZFIN:tsen54)p(ZFIN:wdr62)p(ZFIN:dcdc2b)p(ZFIN:rars2)p(ZFIN:hdac1)p(ZFIN:rbl2) r(ZFIN:cdkn2a_b) p(ZFIN:lef1,pmod(P))act(p(ZFIN:klf8)) p(ZFIN:eya1) p(ZFIN:dacha) a(CHEBI:acrylamide) p(ZFIN:dachb) p(ZFIN:olig2) p(ZFIN:nkx6_1) r(ZFIN:hmx3a)a(CHEMBLID:CHEMBL374400)act(p(ZFIN:fgfr1a))r(ZFIN:)act(p(ZFIN:rhoaa))act(p(ZFIN:cldn5a)) act(p(ZFIN:tjp1a))act(p(ZFIN:prkci)) p(ZFIN:celsr2)act(p(ZFIN:vangl1))complex(p(ZFIN:fzd4),p(ZFIN:vangl2))

decreases decreases actsIn actsIn decreases increases decreases translatedTo increases actsIn actsIn actsIn hasComponent Transcriptomics increasesdecreases actsIn hasModi cation actsInactsIn increases increases hasComponent increases decreases actsIn decreases increases increases increases actsIn actsIn increases increases decreases increases increases increases actsIn directlyDecreases actsIn actsIn actsIn actsIn actsIn actsIn actsIn actsIn actsIn actsIn increases actsIn increases translatedTo decreases decreases actsIn increases translatedTo increases increases increases decreases increases actsIn decreases increases decreases actsIn decreases decreases increases increases directlyDecreases decreases increases increases increases increases increases

p(SFAM:"Histone H3 bp(GOBP:"cerebellum bp(GOBP:"apical junction act(p(ZFIN:ptgs1)) p(ZFIN:ctnnb1)r(ZFIN:gbx2)increases p(ZFIN:zc4h2) act(p(ZFIN:fezf2)) act(p(ZFIN:tigarb))act(p(ZFIN:lgi1a))p(ZFIN:isl1)act(p(ZFIN:th))act(p(ZFIN:park2))p(ZFIN:arxa)act(p(ZFIN:th2))p(ZFIN:)r(ZFIN:her5)act(p(ZFIN:sncgb))p(ZFIN:agrn)act(p(ZFIN:rbbp4))act(p(ZFIN:smo))act(p(ZFIN:elavl3)) p(ZFIN:chrna1)p(ZFIN:axin1)p(ZFIN:shha)a(CHEBI:acetylcholine)act(p(ZFIN:zic5))p(ZFIN:ccnd1) bp(GOBP:"exit fromact(p(ZFIN:tp53)) mitosis") p(ZFIN:odf2a) p(ZFIN:gabarapb)p(ZFIN:drd2a)p(ZFIN:drd2b) r(ZFIN:tp53)act(p(ZFIN:rasl11b))act(p(ZFIN:tsen54))act(p(ZFIN:wdr62))act(p(ZFIN:rars2))act(p(ZFIN:hdac1))act(p(ZFIN:mnx1))act(p(ZFIN:dcdc2b))p(ZFIN:cdkn2a_b)r(ZFIN:ptf1a) r(ZFIN:pvalb7)r(ZFIN:met)r(ZFIN:roraa)p(ZFIN:lgi1b)act(p(ZFIN:eya1))act(p(ZFIN:dacha))r(ZFIN:opn1lw1)r(ZFIN:syn2a)a(CHEBI:"3−Methoxytyramine")r(ZFIN:syt1a)a(SCHEM:Norepinephrine)a(SCHEM:Choline)r(ZFIN:nsfa)a(CHEBI:"L−dopa")act(p(ZFIN:dachb))a(CHEBI:tyrosine)a(CHEBI:phenylalanine)act(p(ZFIN:olig2))act(p(ZFIN:nkx6_1))r(ZFIN:opn1mw1) r(ZFIN:rho) p(ZFIN:pax8)r(ZFIN:atoh1a) p(ZFIN:tmem67)p(ZFIN:fzd3a)p(ZFIN:arhgef11)p(ZFIN:kcnb1)p(ZFIN:kcng4b)p(ZFIN:nphp3)act(p(ZFIN:celsr2))p(ZFIN:fzd4)p(ZFIN:vangl2) increases increasesFamily",pmod(P)) increases morphogenesis") assembly") increases increases decreases actsIn increases increases increases increases directlyIncreases translatedTo decreases actsIn decreases increases actsIn increases directlyIncreases decreases increases actsIn increases increases increases decreases increases increases increases increases increases increases increases actsIn directlyIncreases decreases directlyDecreases increases decreases increases increases decreases increases decreases decreases increases actsIn increases actsIn decreases actsIn actsIn actsIn translatedTo increases actsIn actsIn actsIn actsIn actsIn translatedTo actsIn actsIn actsIn actsIn actsIn actsIn actsIn

decreases translatedTo increases decreases decreases actsIn decreases decreases

increases increases decreases decreases decreases decreases

bp(GOBP:"neuron complex(SCOMP:"NADH bp(GOBP:"hair cell bp(GOBP:"spinal cord motor bp(GOBP:"cilium r(ZFIN:fosab) act(p(ZFIN:zc4h2)) act(p(ZFIN:isl1)) act(p(ZFIN:arxa)) act(p(ZFIN:vsx2))p(ZFIN:her5) act(p(ZFIN:agrn)) act(p(ZFIN:shha))act(p(ZFIN:chrna1))act(p(ZFIN:axin1)) act(p(ZFIN:ccnd1))bp(GOBP:"cell act(p(ZFIN:odf2a))death")bp(GOBP:"cell proliferation")act(p(ZFIN:gabarapb))act(p(ZFIN:drd2a))act(p(ZFIN:drd2b)) act(p(ZFIN:lef1))act(p(ZFIN:cdkn2a_b)) act(p(ZFIN:lgi1b))r(ZFIN:bmp4) bp(MESHPP:"Oxidative Stress")bp(MESHPP:Hearing) p(ZFIN:opn1mw1) p(ZFIN:rho)act(p(ZFIN:pax8)) bp(MESHPP:Permeability)act(p(ZFIN:tmem67))act(p(ZFIN:fzd3a))act(p(ZFIN:arhgef11))act(p(ZFIN:kcnb1))act(p(ZFIN:kcng4b))act(p(ZFIN:nphp3)) act(p(ZFIN:vangl2)) dierentiation") Dehydrogenase Complex I") dierentiation") neuron dierentiation") morphogenesis") increases increases decreases increases increases decreases decreases decreases increases increases increases increasesdecreases increases decreases increases decreases decreases increases decreases increases increases decreases decreases decreases actsIn increases decreases

decreases decreases actsIn increases decreases decreases translatedTo decreases increases decreases decreases decreases increases decreases increases decreases decreases

translatedTo increases decreases increases actsIn decreases decreases increases increases decreases increases increases decreases decreases increases increases increases increases decreases decreases increases increases increases increases increases increases increases increases decreases increases decreases decreases decreases increases

bp(GOBP:"dopaminergic bp(GOBP:"brain p(ZFIN:fosab) r(ZFIN:)r(ZFIN:vsx2)r(ZFIN:)r(ZFIN:gata2a)r(ZFIN:nkx6_1)r(ZFIN:)act(p(ZFIN:her5)) r(ZFIN:fgf19)r(ZFIN:slc17a7a) r(ZFIN:gabra6b) bp(MESHPP:"Cell Count") path(MESHD:Microphthalmos) path(MESHD:Microcephaly) r(ZFIN:axin2)r(ZFIN:zic2a)r(ZFIN:ccnd1) act(p(ZFIN:opn1mw1))r(ZFIN:sox9a)act(p(ZFIN:rho))r(ZFIN:sox9b) path(MESHD:Hydrocephalus) neuron dierentiation") morphogenesis")

decreases increases increases increases increases

translatedTo actsIn translatedTo translatedTo increases increases increases increases increases

Standard toxicology act(p(ZFIN:fosab)) r(ZFIN:neurog1) p(ZFIN:fgf19)a(CHEBI:dopamine) p(ZFIN:sox9a)bp(GOBP:phototaxis)p(ZFIN:sox9b)p(ZFIN:atoh1a)

increases increases actsIn actsIn increases actsIn increases

path(MESHD:Seizures) act(p(ZFIN:fgf19))bp(GOBP:locomotion) act(p(ZFIN:sox9a)) act(p(ZFIN:atoh1a))

actsIn increases increases increasesincreases increases increases

r(ZFIN:gad1b) actsIn r(ZFIN:dlx3b)r(ZFIN:tlx2)r(ZFIN:eya2)

translatedTo translatedTo translatedTo

p(ZFIN:dlx3b)p(ZFIN:tlx2)p(ZFIN:eya2)

actsIn actsIn actsIn

act(p(ZFIN:dlx3b))act(p(ZFIN:tlx2))act(p(ZFIN:eya2))

increases

increases increases

bp(GOBP:"otic vesicle Eye size formation") Zebrafish embryos were exposed to imidaclo- Pathological endpoints represented in the model: act(p(ZFIN:sox9a)) act(p(ZFIN:atoh1a)) 120000 prid at EC5 (126.2 mg/L) and EC30 (222.7 n.s. n.s. Microcephaly increases Megalencephaly increases mg/L) as determined by the OECD fish increases increases 90000 increases )

2 Microphthalmos embryo toxicity test. At 120 hours of exposure Seizures 60000 r(ZFIN:dlx3b) r(ZFIN:tlx2) r(ZFIN:eya2) the below morphological, cardiological, and Hydrocephalus translatedTo translatedTo translatedTo behavioral endpoints were measured. (µm Area Neurogenic inflammation 30000 p(ZFIN:dlx3b) p(ZFIN:tlx2) p(ZFIN:eya2) Biological processes represented in the model: 0 actsIn actsIn actsIn Representative image of morphological measurements Control EC5 EC30 Neuron differentiation Autophagy act(p(ZFIN:dlx3b)) act(p(ZFIN:tlx2))act(p(ZFIN:eya2)) Body length Pericardial area Heart rate Blood flow Locomotion Phototaxis increases increases 5000 40000 200 n.s. n.s. 60 n.s. n.s. Oxidative stress bp(GOBP:"otic vesicle *** n.s. n.s. formation") 4000 30000 150 Notch signaling pathway cytoscape.org )

2 40

3000 m µ 20000 100 2000 BPM Area ( Area Activity (%) Activity Length (µm) Length 20 10000 50 1000 Network Scoring

0 0 0 0 Control EC5 EC30 Control EC5 EC30 Control EC5 EC30 Control EC5 EC30 The above model constitutes the functional layer (yellow spheres in this panel), Light/dark response where each node represents a biological entity. 0.3 Control Light/dark assay tests the abili- EC5 ty of the fish to detect light, Additionally, the model contains a downstream transcript layer (black spheres), EC30 escape danger and swim. Dark which are transcripts that have been described in the literature to be regulated by 0.2 10-minute periods are indicat- a given molecular entity. ed by shaded rectangles.

0.1 By measuring transcript abundance in transcriptomic datasets, activity of the up- Velocity (cm/s) Velocity ANOVA stream node in the functional layer can be quantified. Control vs. EC5 *** 0.0 Control vs. EC30 *** The most affected nodes can provide a mechanistic understanding of the molec- Martin, F., Sewer, A., Talikka, M., Xiang, Time (10 min intervals) 40 ular events that occur upon experimental treatment. Y., Hoeng, J., & Peitsch, M. C. (2014).

) Quantification of biological network per- 2 turbations for mechanistic insight and 30 Overall changes in the functional layer can be used to calculate the network per- diagnostics using two-layer causal turbation amplitudes (NPA). models. BMC Bioinformatics, 15, 238. doi: 10.1186/1471-2105-15-238 20

ANOVA 10 Control vs. EC5 n.s. Max acceleration (cm/s acceleration Max Control vs. EC30 **** 0 Conclusions Time (10 min intervals)

We have constructed a zebrafish specific biological network. After testing the acute toxicity of imidacloprid, we are cur- Startle response Startle response assay rently sequencing the transcriptomes of exposed zebrafish embryos. Vibration stimulus response Sudden darkness response tests motor function and 0.8 0.4 Control Control sensory physiology. Transcriptomics data will be used to quantify the NPA and to generate mechanistic hypotheses into imidacloprid toxicity. EC5 EC5 EC30 EC30 0.6 0.3 Vibration Together with the standard toxicology approaches, which describe phenotypical changes, and systems toxicology meth- Control vs. EC5 n.s. ods, which explore the molecular detail underlying such phenotypes, we aim to gain mechanistic insights into causes of 0.4 0.2 Control vs. EC30 n.s. adverse outcomes induced by imidacloprid as well as other chemicals. Velocity (cm/s) Velocity 0.2 (cm/s) Velocity 0.1 Darkness Control vs. EC5 **** This method may be applied to toxicological evaluation of chemicals of environmental concern and chemicals of interest 0.0 0.0 Control vs. EC30 ** in human health as well as chemical mixtures. Time (secs) Time (secs)

SOT, Baltimore | 10-14 March, 2019 Competing Financial Interest – The research described in this poster was sponsored by Philip Morris International