Systems Biology in Small Fish Models

Systems Biology in Small Fish ModelsD.L. VilleneuveThe McKim Conference on Predictive ToxicologySept. 25-27, 2007, Duluth, Minnesota

Systems Biology in Small Fish ModelsWhat is systems biology or the systems perspective?The systems perspective in ecotoxicology.An overview of a systems-oriented small fish research program.A case studyConclusions

The Systems Perspective The experimenter can draw a boundary around it, heuristically The experimenter can conduct defined perturbations within the boundaries The experimenter can, by reasoning generate sensible explanations for the changed behavior. Learning is assessed by how well the understanding enables prediction of changed system behavior in response to defined perturbations. A subset of the world whose behavior, and whose interaction with the world, we believe can be sensibly described.What is a system?Brent 2004, Nature Biotech. 22: 1211-1214Kuipers, B. 1994. MIT Press, Cambridge

www.oz.net/~geoffsi/bm2003-days/bm2003-20.htm Systems biology is the study of an organism, viewed as an integrated and interacting network of genes, proteins and biochemical reactions which give rise to life. Systems are comprised of parts which interact.

Interaction of these parts gives rise to "emergent properties".

Emergent properties cannot be attributed to any single parts of the system. Irreducible.

To understand systems, and to be able to fully understand a system's emergent properties, systems need be studied as a whole. http://www.systemsbiology.org/Intro_to_ISB_and_Systems_Biology/Why_Systems_MatterThe Systems Perspective

The Systems PerspectiveNon-equilibrium thermodynamics (1930s-40s) dealt with integration quantitatively aimed to discover general principles >> descriptive established connection to molecular mechanisms Investigation of biological self-organization (1950s) examination of how structures, oscillations, and/or waves arise in a steady or homogenous environment Feedback regulation in metabolism (late 1950s) Systems theory in biology (1960s) search for general biological laws governing behavior and evolution of living matter in a way analogous to the relation of physical laws and non-living matter Metabolic control analysis (1970s)Approaches to characterize properties of networks of interacting chemical reactions Convergence with mainstream molecular biology high throughput, genome-scale, data-richWesterhoff and Palsson, 2004 Nature Biotech. 22:1249-1252Wolkenhauer. 2001 Briefings in Bioinformatics. 2:258-270

The Systems Perspective Shift from reductionism to a holistic perspective Should reduce complexity rather than adding additional layers of complexity

Search for organizing principles over construction of predictive descriptions (models) that exactly describe the evolution of a system in space and time.

Identification of new concepts and hypotheses that provide a conceptual structure with logical coherence to rival chemistry and physics.Mesarovic et al. 2004. Syst. Biol. 1:19-27

The Systems Perspective in Ecotoxicology Shift from reductionism to a holistic perspective Historically, ecotoxicology and ecological risk assessment was holistic in focus Apical/integrative endpoints: Survival, growth, reproduction Not feasible to test every chemical, let alone every chemical mixture Increased emphasis on subtle, chronic impacts (e.g., development, behavior, etc.) with long-term population implications. Reductionist in the sense that testing of the universe of chemicals was the paradigm.

Search for organizing principles that underlie biological response to stressors.

Develop a conceptual structure with logical coherence that allows us to predict, with reasonable and quantitative certainty, integrated, ecologically-relevant impacts.

The Systems Perspective in Ecotoxicology

USEPA Cincinnati, OHD. Bencic, M. Kostich, I. Knoebl, D. Lattier, J. Lazorchak, G. Toth, R. Wang, USEPA Duluth, MN, and Grosse Isle, MIG. Ankley, E Durhan, M Kahl, K Jensen, E Makynen, D. Martinovic, D. Miller, D. Villeneuve, USEPA Athens, GAT. Collette, D. Ekman, J. Kenneke, T. Whitehead, Q. Teng USEPA-RTP, NCM. Breen, R. ConollyUSEPA STAR ProgramN. Denslow (Univ. of Florida), E. Orlando, (Florida Atlantic University), K. Watanabe (Oregon Health Sciences Univ.), M. Sepulveda (Purdue Univ.)USACE Vicksburg, MSE. PerkinsOther partnersJoint Genome Institute, DOE (Walnut Creek, CA)Sandia, DOE (Albuquerque, NM)Pacific Northwest National Laboratory (Richland, WA)C. Tyler (Univ. Exeter, UK)Linkage of Exposure and Effects Using Genomics, Proteomics, and Metabolomics in Small Fish Models

11HSDP45011.GnRHNeuronal SystemGABADopamineBrain

GonadotrophPituitaryD1 RD2 R??Y2 RNPYY1 RGnRH R?ActivinInhibinGABAA RGABAB RY2 RD2 RGnRH Circulating Sex Steroids / Steroid Hormone Binding GloblulinPACAPFollistatinActivinPAC1 RActivin RFSHbLHbGPaCirculating LH, FSH FSH RLH RCirculating LDL, HDLLDL RHDL ROuter mitochondrial membraneInner mitochondrial membraneStAR P450sccpregnenolone3bHSDP450c17androstenedionetestosteroneCholesterolestradiolGonad(Generalized, gonadal, steroidogenic cell)Androgen / Estrogen Responsive Tissues(e.g. liver, fatpad, gonads)ARER3456BloodBloodCompartment17HSDP450arom11-ketotestosteroneprogesterone17-hydroxyprogesterone17,20-P (MIS)20HSD

Fipronil* (-)Muscimol* (+)

Apomorphine (+)Haloperidol (-)

Trilostane (-)Ketoconazole (-)

Fadrozole (-)Prochloraz (-,-)

Vinclozolin (-)Flutamide (-)-trebolone (+)Ethynyl estradiol (+)78910111221Chemical Probes

Figure 1. Conceptual Overview of ResearchIncreasing Ecological RelevanceIncreasing Diagnostic (Screening) UtilityPhase 1.Fathead minnow 21 d reproduction test

Phase 2.Zebrafish genomics proteomics, metabalomicsPopulation modelingPhase 3. Fathead minnow molecular markers metabolomicsSystems modelingMolecular

mRNA, protein, Enzyme assaysCellular

Metabolite profilesOrganFunctional andStructural change(Pathology)Individual

Altered reproductionor developmentPopulation

Decreased numbersof animalsLevels of BiologicalOrganizationWell characterized genome, low eco / regulatory relevanceComputational modelingPoorly characterized genomehigh eco / regulatory relevances Depict the flow of information

A Case Study with FadrozoleAromatase

OvaryBrain

Chart1

105.67550015471.16001622921.1600162292

815.77180800828.35022964268.3502296426

1521.150972877432.967819434432.9678194344

2641.115170762472.568430940372.5684309403

3915.6434244345181.0148544948181.0148544948

4684.63776393561.038190912961.0381909129

IC50=8.82 +/- 1.58 M

A

*B*

*C*

*D*

*E*

*F*

fadrozole (uM)

fmol/h/mg

Fig1

A. Brain

B. Ovary

&A

&F

Fig1

5211.7958042402279.2917918464279.2917918464

3781.5648140955501.617257516501.617257516

3876.3664579737398.1876749747398.1876749747

ANOVA p=0.0593

brain fmol/h/mg

fmol/h/mg

Fig2

92.567078269817.040929608717.0409296087

17.61433272994.21165942574.2116594257

64.923946586826.759560242726.7595602427

A

AB

B

ANOVA p=0.0199

fmol/h/mg

Fig3

A. Brain

B. Ovary

&A

&F

Fig3

0.52182581730.12865458090.1286545809

0.55631737610.13913094450.1391309445

0.72898982040.20469249330.2046924933

ANOVA p=0.6284

pg P450aromB mRNA / ug total RNA

Fig4

69.6607717278.57367495528.5736749552

115.855689717829.04734423229.047344232

55.328877924517.284678991917.2846789919

ANOVA (log y) p=0.1352

pg P450aromB mRNA / ug total RNA

Fig5

A. Brain

B. Ovary

&A

&F

Fig5

105.67550015471.16001622921.1600162292

815.77180800828.35022964268.3502296426

1521.150972877432.967819434432.9678194344

2641.115170762472.568430940372.5684309403

3915.6434244345181.0148544948181.0148544948

4684.63776393561.038190912961.0381909129

IC50=8.82 +/- 1.58 M

A

*B*

*C*

*D*

*E*

*F*

fadrozole (uM)

fmol/h/mg

Fig6

2.18327194960.14883373870.1488337387

2.90690721950.10371134010.1037113401

4.60666491750.28372638360.2837263836

5.8648370230.101131210.10113121

6.4694631780.07793391910.0779339191

IC50 = 6.93 +/- 0.80 M

A

B

*C*

*D*

*E*

fadrozole (uM)

fmol/h/mg

Fig7

&A

&F

Fig7

0.04976453920.0685840784

0.12802174660.1345654982

0.26077568050.2648337828

0.50247712390.48457595

11

Ovary

Brain

nM [3H]-AD

AAo/AAmax

Fig8

&A

&F

Fig8

6238.69672043581189.97519258399.27288579079.2728857907163.3639139103163.3639139103

14779.15642915641319.06598297956.179350241556.1793502415298.2195570378298.2195570378

12595.69169960471345.642559555619.193451356319.1934513563112.9876512721112.9876512721

11456.29847238541367.716055976960.919357945160.9193579451493.7705441699493.7705441699

10637.79617562231433.2218780045134.5068291497134.5068291497596.6096776993596.6096776993

Brain

Ovary

mg/ml microsomal protein

fmol/h/mg

A.

B.

&A

&F

3197.58059960179.721953274479.7219532744

2579.974640757951.926748744751.9267487447

3073.9914938098110.7724416262110.7724416262

2291.777651179429.612770045929.6127700459

1493.553444648227.334051897327.3340518973

a

a

ab

b

b

c

ANOVA (log x) p

PregnenoloneProgesterone17a-OH-Pregnenolone17a-OH-ProgesteroneCYP17 (hydroxylase)CYP11A3b-HSD3b-HSDDHEAAndrostenedioneTestosteroneCYP17 (lyase)3b-HSD17b-HSDCYP1917b-estradiolCholesterol20b-HSD1720-dihydroxy-4-pregnen-3-oneCYP17 (hydroxylase)CYP17 (lyase)estroneCYP1917b-HSD11-OH-TestosteroneCYP11B111-Ketotestosterone11HSD11-deoxycorticosteroneCYP21corticosteroneCYP11B1aldosteroneCYP11B211-deoxycortisolCYP21cortisolCYP11B2A Case Study with Fadrozole

25oC, 12 hRIAAndrostenedioneTestosterone17b-HSDCYP1917b-estradiolestroneCYP1917b-HSD

Chart1

0.31989224950.03820228980.0382022898

0.20519668820.02605756530.0260575653

0.15406868360.02440912480.0244091248

A

B

C

24 h-E2

Fadrozole (ug/L)

Ex vivo E2 production (ng/ml*12 h)

E2-RIA d1,16

Small Fish Comp Tox

Phase 3, Fadrozole

Ex vivo steroid production

24 h, d.16 females, E2

Extraction: Notebook 341:83

RIA: Notebook 341:85-90

DLV, NDM, JET

adjusted

E2 (ng/ml)log E2dpmdpmBo/BmBo/BmMeanSDCV

60.77815125041777.781750.930.10775106140.10451585460.1061334580.00228763662.1554340001

20.30102999572480.792664.480.19245805230.21459120420.20352462820.01565050187.6897336556

0.6666666667-0.17609125914608.974475.540.44888644420.43280921380.4408478290.01136831872.5787398599

0.2222222222-0.65321251386630.838484.190.69250414040.91581895610.80416154830.157907420519.6362809051

0.0740740741-1.130333768510303.59759.941.13503102361.06953646171.10228374270.04631164894.2014271877

0.024691358-1.607455023210961.211064.91.21427852781.22677353491.22052603140.00883530430.7238931442

MeanSDCVSigmoid fit; regression using GraphPad Prism

Bm9217.219148.469182.8352178.798099924223.7268566834

NS877.97889.07883.527.84888527120.8883653195

linearadj for dil

CT iddpmBo/Bmlog ng/mlng/mldil factorng/ml

580525117.730.510187889-0.27460080.53137265330.53333333330.2833987484

580546121.840.6311749825-0.43005850.37148518620.61538461540.2286062684

580564205.580.400281228-0.11684840.76410246370.50.3820512319

580587147.170.7547189135-0.58426740.26045494060.50.1302274703

580607565.580.8051339177-0.64890510.224437230.57142857140.1282498457

580626302.650.6529611179-0.45721570.34896695190.50.1744834759

580645166.540.5160690973-0.28246340.52183908020.50.2609195401

580664959.810.4911598126-0.24885560.56382509270.50.2819125464

580689853.261.0807807632-1.0940160.08053487710.50.0402674385

580709640.021.0550870765-1.0391030.0913896470.57142857140.0522226554

5807210390.81.1455499641-1.2635920.05450144290.53333333330.0290674362

580745956.390.6112396023-0.40509460.39346435990.50.19673218

580763764.210.3470997305-0.029176910.93502471390.50.467512357

580784729.250.4633792066-0.21025840.61622824440.50.3081141222

580804299.660.4116171033-0.13435810.73390847020.57142857140.4193762687

580824644.330.4531470368-0.19570950.63722161650.53333333330.3398515288

580846519.770.6791223131-0.48973850.32378855980.57142857140.1850220341

5808613511.71.52159304710.57142857140

580887453.060.7915761723-0.6313210.23371091730.53333333330.1246458226

580905139.150.5127688249-0.27807060.52714416060.53333333330.2811435523

5809211174.41.23996739490.53333333330

5809410215.21.1243915914-1.2018970.06282073310.57142857140.0358975618

5809610847.51.200578602-1.4760980.03341196360.57142857140.0190925506

5809811691.71.30229784020.50

583886036.970.6209488373-0.41726870.38258796110.57142857140.2186216921

583908790.110.9526798296-0.85737110.13887654390.57142857140.0793580251

583926065.930.624438282-0.42164160.3787550220.57142857140.2164314412

583946796.590.7124768731-0.53125110.29427197230.57142857140.1681554127

583964965.120.4917996244-0.24972620.56269596380.57142857140.3215405508

583988958.980.973027292-0.89000120.12882459920.57142857140.0736140567

584005236.030.5244420774-0.29359420.50863448310.57142857140.290648276

584024204.680.4001727853-0.1166780.76440232620.57142857140.4368013292

584044340.080.4164873848-0.14173360.72154994850.57142857140.4123142563

584065392.30.5432713423-0.31830250.48050454510.57142857140.2745740258

584085053.270.5024209829-0.26415910.54430321560.57142857140.3110304089

5841011336.51.25949912730.57142857140

584126282.380.6505187476-0.45417740.35141686490.57142857140.2008096371

5841410655.51.1774441626-1.3745880.04220967420.57142857140.0241198138

584167125.030.7520512235-0.5808960.26248470370.57142857140.1499912593

584186680.150.6984467995-0.51376960.30635882840.57142857140.1750621877

584207013.730.7386404782-0.56400140.27289689860.57142857140.1559410849

584226722.130.7035050483-0.5200660.30194928110.57142857140.1725424464

584244831.40.4756874513-0.22751850.59221785910.57142857140.3384102052

584265363.110.5397541845-0.31371920.48560237260.57142857140.2774870701

584284459.020.430818688-0.16320570.68674309280.57142857140.3924246244

584304406.40.4244784057-0.15377360.70182106610.57142857140.4010406092

584324334.960.4158704664-0.14079960.72310339410.52631578950.3805807337

584343477.20.31251735840.034193231.08191521920.52631578950.5694290627

QA3830.870.3551317187-0.043097130.90553005640.52631578950.4765947665

QA4305.260.4122918578-0.13538160.73218090760.52631578950.3853583724

QA4043.070.3807000939-0.085735130.82085201760.52631578950.4320273777

QA4607.430.4487008868-0.1893090.64668233830.52631578950.3403591254

Mean0.776311330.4085849105

SD0.1117027630.0587909279

CV14.388913140114.3889131401

E2-RIA d1,16

0

0

0

0

0

0

T-RIA d1,16

Small Fish Comp Tox

Phase 3, Fadrozole

Small Fish Comp Tox

Phase 3, Fadrozole


24 h, d.16 females, T



DLV, NDM, JET

T (ng/ml)log TdpmdpmBo/BmBo/BmMeanSDCV

60.7781512504769.79853.710.07044473850.10121092360.08582783110.021754978125.3472303948

20.30102999571043.31942.920.17072079830.13391649280.15231864560.02602457417.0856127958

0.6666666667-0.17609125911549.651706.360.35635175810.41380372260.38507774030.040624673710.5497330623

0.2222222222-0.65321251382536.682366.960.71821004740.65598844430.68709924590.219172590231.898243457

0.0740740741-1.13033376852819.152856.240.82176729590.83536498180.82856613890.00961501591.160440366

0.024691358-1.60745502323178.793212.420.95361608990.9659452940.95978069190.16645720917.3432545992

Sigmoid fit R^2 =0.9967; Regression conducted using GraphPad Prism

MeanSDCV

Bm3300.843309.783305.31410.51633345512.4199041377

NS568.75586.53577.6412.57235856952.1765041496

Bm-3SD2069.2909996350.546859040710.5=MDL

polynomialadj for dil


580522359.220.6531508577-0.62443230.23744755390.53333333330.1266386954

580543268.070.98634732210.61538461540

580562808.770.8179618502-0.98558960.10337378070.50.0516868903

580582468.550.6932326858-0.69866390.20014101590.50.100070508

580602550.650.7233316347-0.75839320.17442422420.57142857140.0996709853

580622877.090.8430088684-1.0630150.08649380440.50.0432469022

580642774.530.8054090121-0.95040710.11209671870.50.0560483593

580661770.590.4373512925-0.26710980.54061762460.50.2703088123

580682149.750.5763563774-0.49298310.32137855960.50.1606892798

580702136.890.5716417309-0.48519990.3271900590.57142857140.186965748

580721979.330.5138781451-0.39127760.40618361460.53333333330.2166312611

580742437.260.6817613568-0.67686420.21044363750.50.1052218188

580762880.740.8443470068-1.0675280.08559965220.50.0427998261

580782669.960.7670722631-0.85402370.13995109470.50.0699755474

580802964.090.8749042223-1.1817730.06580016760.57142857140.0376000958

580822842.620.8303717092-1.0226350.09492158910.53333333330.0506248475

580842588.930.7373655904-0.78779450.16300671670.57142857140.0931466953

580863538.441.08546855010.57142857140

580882536.740.7182320442-0.74800460.17864686530.53333333330.0952783281

580902819.140.8217636298-0.99667260.10076910470.53333333330.0537435225

580922624.60.7504426855-0.81623170.15267513050.53333333330.0814267363

580942263.510.6180623023-0.56302920.27350848250.57142857140.1562905614

580962318.40.6381857043-0.59788840.25241293110.57142857140.1442359606

580982350.160.6498293415-0.61849030.24071862830.50.1203593142

583882755.50.7984323617-0.9317560.11701566360.57142857140.0668660935

583903101.520.9252878831-1.4647370.03429754230.57142857140.0195985956

583923102.530.9256581625-1.4676940.03406481230.57142857140.019465607

583942637.440.7551499998-0.82672560.14903023970.57142857140.085160137

583962473.930.6952050651-0.70246950.19839489820.57142857140.1133685133

583982636.570.7548310463-0.82600440.14927792860.57142857140.0853016735

584002720.090.785450586-0.89844920.1263428880.57142857140.072195936

584022247.380.6121488303-5.53E-010.27994441010.57142857140.1599682344

584042706.770.7805672974-0.88632460.12991981690.57142857140.0742398954

584063008.320.8911195269-1.2551990.05556495920.57142857140.0317514053

584082700.020.7780926578-0.8803220.13172797030.57142857140.0752731259

584103295.10.99625687860.57142857140

584123066.90.9125957319-1.3750210.04216761130.57142857140.0240957779

584143055.740.9085043279-1.3495690.04471271070.57142857140.0255501204

584162893.350.84897-1.0832530.08255568780.57142857140.0471746787

584183006.830.8905732732-1.2525650.05590298520.57142857140.031944563

584202674.50.7687366874-0.85794330.13869368910.57142857140.0792535366

584222728.380.78848981-0.90608230.12414170330.57142857140.0709381162

584242827.980.825004491-1.0063240.09855439580.57142857140.0563167976

584262351.990.6505002438-0.61969050.24005430570.57142857140.137173889

584282528.630.7152588106-0.7419650.18114860750.57142857140.10351349

584302415.060.6736225423-0.66168090.21793104430.57142857140.1245320253

584322404.070.6695934626-0.65426870.22168244360.52631578950.1166749703

584342106.030.5603280455-0.46663540.3414794710.52631578950.1797260374

1

1

1

QA1922.260.4929555261-0.35752440.43901119990.52631578950.2310585263

QA2003.220.5226365359-0.40541780.39317165440.52631578950.2069324497

QA1885.690.4795484791-0.33586390.46146216580.52631578950.2428748241

QA1955.110.5049987718-0.37695340.41980402670.52631578950.2209494878

Mean0.42836226170.225453822

SD0.02898635170.0152559746

CV6.76678463926.7667846392

T-RIA d1,16

0

0

0

0

0

0

T (log ng/ml)

Bo/Bm

forSAS

CT idfadtime pointtissueex_vivo_wtE2_ng_mlT_ng_ml

5805201y28.30.28339874840.1266386954

5805401y33.30.22860626840

5805601y30.10.38205123190.0516868903

5805801y43.30.13022747030.100070508

5806031y61.30.12824984570.0996709853

5806231y27.40.17448347590.0432469022

5806431y33.50.26091954010.0560483593

5806631y28.50.28191254640.2703088123

58068301y17.70.04026743850.1606892798

58070301y300.05222265540.186965748

58072301y24.40.02906743620.2166312611

58074301y13.80.196732180.1052218188

5807601y13.30.4675123570.0427998261

5807801y22.50.30811412220.0699755474

5808001y21.50.41937626870.0376000958

5808201y33.50.33985152880.0506248475

5808431y23.20.18502203410.0931466953

5808631blankblank00

5808831y30.20.12464582260.0952783281

5809031y26.70.28114355230.0537435225

58092301y43.100.0814267363

58094301y9.70.03589756180.1562905614

58096301y35.20.01909255060.1442359606

58098301y42.100.1203593142

58388016y19.70.21862169210.0668660935

58390016y8.40.07935802510.0195985956

58392016y12.80.21643144120.019465607

58394016y7.50.16815541270.085160137

58396316y9.10.32154055080.1133685133

58398316y7.30.07361405670.0853016735

58400316y15.40.2906482760.072195936

58402316y90.43680132920.1599682344

584043016y10.30.41231425630.0742398954

584063016y150.27457402580.0317514053

584083016y130.31103040890.0752731259

584103016y3.300

58412016y140.20080963710.0240957779

58414016y1.70.02411981380.0255501204

58416016y8.30.14999125930.0471746787

58418016y12.70.17506218770.031944563

58420316y6.40.15594108490.0792535366

58422316y11.90.17254244640.0709381162

58424316y7.20.33841020520.0563167976

58426316y13.10.27748707010.137173889

584283016y120.39242462440.10351349

584303016y8.80.40104060920.1245320253

584323016y15.70.38058073370.1166749703

584343016y9.60.56942906270.1797260374

figures

CT idfadtime pointtissueex_vivo_wtE2_ng_mlT_ng_mlE2_relT_rel24 h-E224 h-T24 h-wts

5808631blankblank00Fad ug/LMeanSDcountSECVFad ug/LMeanSDcountSECVFad ug/LMeanSDcountSECV

5805201y28.30.28339874840.12663869540.8859193962.113302355200.31989224950.108052392680.038202289833.777746346700.05992455130.039097735280.013823136965.2449361585028.2259.142639506383.232411196432.3919911651

5805401y33.30.228606268400.7146352212030.20519668820.068941837770.026057565333.59792903230.10163480070.077833236670.029418198276.5812851741332.971428571412.886131341674.870499841639.0827207067

5805601y30.10.38205123190.05168689031.19431224890.8625327885300.15406868360.069039430780.024409124844.8108136597300.1464775850.043882857980.015514933229.9587529836302712.706803576984.492533488247.0622354701

5805801y43.30.13022747030.1000705080.40709792291.6699417148

5807601y13.30.4675123570.04279982611.46146822180.7142285617

5807801y22.50.30811412220.06997554740.96318095451.1677275147

5808001y21.50.41937626870.03760009581.31099227750.627457277

5808201y33.50.33985152880.05062484751.06239375730.8448097881

5806031y61.30.12824984570.09967098530.40091576441.6632746191

5806231y27.40.17448347590.04324690220.54544452460.7216892121

5806431y33.50.26091954010.05604835930.81564820820.9353154611

5806631y28.50.28191254640.27030881230.88127345024.5108191286

5808431y23.20.18502203410.09314669530.5783886121.5543995448

5808831y30.20.12464582260.09527832810.38964939841.5899714902

5809031y26.70.28114355230.05374352250.87886953430.8968531483

58068301y17.70.04026743850.16068927980.12587813112.6815266249

58070301y300.05222265540.1869657480.16325076813.1200191556

58072301y24.40.02906743620.21663126110.09086633473.6150668861

58074301y13.80.196732180.10522181880.61499514381.7559049916

58092301y43.100.081426736301.3588209592Normality ok except 30 ug/LNormality ok except 3 ug/LLevene's test okNormality ok except 3 ug/LLevene's ok

58094301y9.70.03589756180.15629056140.11221766652.6081223478Levene's test okKW p=0.0076KW p=0.8063

58096301y35.20.01909255060.14423596060.05968431772.4069593759KW p=0.0005

58098301y42.100.120359314202.0085142321

58388016y19.70.21862169210.06686609351.41898851181.6724071518d. 16-E2d. 16-Td. 16-wts

58390016y8.40.07935802510.01959859560.51508212580.4901861272Fad ug/LMeanSDcountSECVFad ug/LMeanSDcountSECVFad ug/LMeanSDcountSECV

58392016y12.80.21643144120.0194656071.40477244360.486859911800.15406868360.069039430780.024409124844.810813659700.03998194660.02442835180.008636726361.0984532737010.63755.379308904181.901872902150.5692963958

58394016y7.50.16815541270.0851601371.09143148882.129964749930.25837312740.11688507980.04132511645.238868349930.09681458710.036347464580.012850769337.543376070339.9253.212364505281.135742362632.3663929995

58412016y140.20080963710.02409577791.30337737950.6026664511300.34267421510.163292342480.057732561347.6523575945300.08821386870.056150345980.019852145263.65251486013010.96253.957971161481.39935412436.1046400126

58414016y1.70.02411981380.02555012040.15655234580.6390414316

58416016y8.30.14999125930.04717467870.97353502191.1798994973

58418016y12.70.17506218770.0319445631.13626068290.7989746793

58396316y9.10.32154055080.11336851332.08699485982.8354925861

58398316y7.30.07361405670.08530167350.47780025742.1335047598

58400316y15.40.2906482760.0721959361.88648510021.80571338

58402316y90.43680132920.15996823442.83510781684.0010116513

58420316y6.40.15594108490.07925353661.01215302961.9822330645

58422316y11.90.17254244640.07093811621.11990602061.7742536858

58424316y7.20.33841020520.05631679762.19648923641.4085556697

58426316y13.10.27748707010.1371738891.80106082273.4308957038

584043016y10.30.41231425630.07423989542.67617173451.8568354379

584063016y150.27457402580.03175140531.78215338330.7941435558

584083016y130.31103040890.07527312592.01877761011.882677863

584103016y3.30000

584283016y120.39242462440.103513492.54707585762.5890057566

584303016y8.80.40104060920.12453202532.6029988693.1147064061

584323016y15.70.38058073370.11667497032.47020176192.9181913367Normality okLevene's test okNormality okLevene's test okNormality okLevene's test ok

584343016y9.60.56942906270.17972603743.69594293514.4951797619ANOVA p=0.0199ANOVA p=0.0246ANOVA p=0.8848

&A

&F

figures

00.03820228980.0382022898

00.02605756530.0260575653

00.02440912480.0244091248

A

B

C

24 h-E2

Fadrozole (ug/L)

Ex vivo E2 production (ng/ml)

00.01382313690.0138231369

00.02941819820.0294181982

00.01551493320.0155149332

A

AB

B

24 h-T

Fadrozole (ug/L)

Ex vivo T production (ng/ml)

00.02440912480.0244091248

00.0413251160.041325116

00.05773256130.0577325613

A

AB

B

d. 16-E2

Fadrozole (ug/L)


00.00863672630.0086367263

00.01285076930.0128507693

00.01985214520.0198521452

A

B

B

d. 16-T

Fadrozole (ug/L)


03.23241119643.2324111964

04.87049984164.8704998416

04.49253348824.4925334882

24 h-wts

Fadrozole (ug/L)

Ex vivo tissue wt (mg)

01.90187290211.9018729021

01.13574236261.1357423626

01.3993541241.399354124

d. 16-wts

Fadrozole (ug/L)


MBD0023823A.bin

MBD0022DDB4.bin

Chart2

0.05992455130.01382313690.0138231369

0.10163480070.02941819820.0294181982

0.1464775850.01551493320.0155149332

A

AB

B

24 h-T

Fadrozole (ug/L)

Ex vivo T production (ng/ml*12 h)

E2-RIA d1,16

Small Fish Comp Tox

Phase 3, Fadrozole


24 h, d.16 females, E2



DLV, NDM, JET

adjusted

E2 (ng/ml)log E2dpmdpmBo/BmBo/BmMeanSDCV

60.77815125041777.781750.930.10775106140.10451585460.1061334580.00228763662.1554340001

20.30102999572480.792664.480.19245805230.21459120420.20352462820.01565050187.6897336556

0.6666666667-0.17609125914608.974475.540.44888644420.43280921380.4408478290.01136831872.5787398599

0.2222222222-0.65321251386630.838484.190.69250414040.91581895610.80416154830.157907420519.6362809051

0.0740740741-1.130333768510303.59759.941.13503102361.06953646171.10228374270.04631164894.2014271877

0.024691358-1.607455023210961.211064.91.21427852781.22677353491.22052603140.00883530430.7238931442

MeanSDCVSigmoid fit; regression using GraphPad Prism

Bm9217.219148.469182.8352178.798099924223.7268566834

NS877.97889.07883.527.84888527120.8883653195

linearadj for dil


580525117.730.510187889-0.27460080.53137265330.53333333330.2833987484

580546121.840.6311749825-0.43005850.37148518620.61538461540.2286062684

580564205.580.400281228-0.11684840.76410246370.50.3820512319

580587147.170.7547189135-0.58426740.26045494060.50.1302274703

580607565.580.8051339177-0.64890510.224437230.57142857140.1282498457

580626302.650.6529611179-0.45721570.34896695190.50.1744834759

580645166.540.5160690973-0.28246340.52183908020.50.2609195401

580664959.810.4911598126-0.24885560.56382509270.50.2819125464

580689853.261.0807807632-1.0940160.08053487710.50.0402674385

580709640.021.0550870765-1.0391030.0913896470.57142857140.0522226554

5807210390.81.1455499641-1.2635920.05450144290.53333333330.0290674362

580745956.390.6112396023-0.40509460.39346435990.50.19673218

580763764.210.3470997305-0.029176910.93502471390.50.467512357

580784729.250.4633792066-0.21025840.61622824440.50.3081141222

580804299.660.4116171033-0.13435810.73390847020.57142857140.4193762687

580824644.330.4531470368-0.19570950.63722161650.53333333330.3398515288

580846519.770.6791223131-0.48973850.32378855980.57142857140.1850220341

5808613511.71.52159304710.57142857140

580887453.060.7915761723-0.6313210.23371091730.53333333330.1246458226

580905139.150.5127688249-0.27807060.52714416060.53333333330.2811435523

5809211174.41.23996739490.53333333330

5809410215.21.1243915914-1.2018970.06282073310.57142857140.0358975618

5809610847.51.200578602-1.4760980.03341196360.57142857140.0190925506

5809811691.71.30229784020.50

583886036.970.6209488373-0.41726870.38258796110.57142857140.2186216921

583908790.110.9526798296-0.85737110.13887654390.57142857140.0793580251

583926065.930.624438282-0.42164160.3787550220.57142857140.2164314412

583946796.590.7124768731-0.53125110.29427197230.57142857140.1681554127

583964965.120.4917996244-0.24972620.56269596380.57142857140.3215405508

583988958.980.973027292-0.89000120.12882459920.57142857140.0736140567

584005236.030.5244420774-0.29359420.50863448310.57142857140.290648276

584024204.680.4001727853-0.1166780.76440232620.57142857140.4368013292

584044340.080.4164873848-0.14173360.72154994850.57142857140.4123142563

584065392.30.5432713423-0.31830250.48050454510.57142857140.2745740258

584085053.270.5024209829-0.26415910.54430321560.57142857140.3110304089

5841011336.51.25949912730.57142857140

584126282.380.6505187476-0.45417740.35141686490.57142857140.2008096371

5841410655.51.1774441626-1.3745880.04220967420.57142857140.0241198138

584167125.030.7520512235-0.5808960.26248470370.57142857140.1499912593

584186680.150.6984467995-0.51376960.30635882840.57142857140.1750621877

584207013.730.7386404782-0.56400140.27289689860.57142857140.1559410849

584226722.130.7035050483-0.5200660.30194928110.57142857140.1725424464

584244831.40.4756874513-0.22751850.59221785910.57142857140.3384102052

584265363.110.5397541845-0.31371920.48560237260.57142857140.2774870701

584284459.020.430818688-0.16320570.68674309280.57142857140.3924246244

584304406.40.4244784057-0.15377360.70182106610.57142857140.4010406092

584324334.960.4158704664-0.14079960.72310339410.52631578950.3805807337

584343477.20.31251735840.034193231.08191521920.52631578950.5694290627

QA3830.870.3551317187-0.043097130.90553005640.52631578950.4765947665

QA4305.260.4122918578-0.13538160.73218090760.52631578950.3853583724

QA4043.070.3807000939-0.085735130.82085201760.52631578950.4320273777

QA4607.430.4487008868-0.1893090.64668233830.52631578950.3403591254

Mean0.776311330.4085849105

SD0.1117027630.0587909279

CV14.388913140114.3889131401

E2-RIA d1,16

0

0

0

0

0

0

T-RIA d1,16

Small Fish Comp Tox

Phase 3, Fadrozole

Small Fish Comp Tox

Phase 3, Fadrozole


24 h, d.16 females, T



DLV, NDM, JET

T (ng/ml)log TdpmdpmBo/BmBo/BmMeanSDCV

60.7781512504769.79853.710.07044473850.10121092360.08582783110.021754978125.3472303948

20.30102999571043.31942.920.17072079830.13391649280.15231864560.02602457417.0856127958

0.6666666667-0.17609125911549.651706.360.35635175810.41380372260.38507774030.040624673710.5497330623

0.2222222222-0.65321251382536.682366.960.71821004740.65598844430.68709924590.219172590231.898243457

0.0740740741-1.13033376852819.152856.240.82176729590.83536498180.82856613890.00961501591.160440366

0.024691358-1.60745502323178.793212.420.95361608990.9659452940.95978069190.16645720917.3432545992

Sigmoid fit R^2 =0.9967; Regression conducted using GraphPad Prism

MeanSDCV

Bm3300.843309.783305.31410.51633345512.4199041377

NS568.75586.53577.6412.57235856952.1765041496

Bm-3SD2069.2909996350.546859040710.5=MDL

polynomialadj for dil


580522359.220.6531508577-0.62443230.23744755390.53333333330.1266386954

580543268.070.98634732210.61538461540

580562808.770.8179618502-0.98558960.10337378070.50.0516868903

580582468.550.6932326858-0.69866390.20014101590.50.100070508

580602550.650.7233316347-0.75839320.17442422420.57142857140.0996709853

580622877.090.8430088684-1.0630150.08649380440.50.0432469022

580642774.530.8054090121-0.95040710.11209671870.50.0560483593

580661770.590.4373512925-0.26710980.54061762460.50.2703088123

580682149.750.5763563774-0.49298310.32137855960.50.1606892798

580702136.890.5716417309-0.48519990.3271900590.57142857140.186965748

580721979.330.5138781451-0.39127760.40618361460.53333333330.2166312611

580742437.260.6817613568-0.67686420.21044363750.50.1052218188

580762880.740.8443470068-1.0675280.08559965220.50.0427998261

580782669.960.7670722631-0.85402370.13995109470.50.0699755474

580802964.090.8749042223-1.1817730.06580016760.57142857140.0376000958

580822842.620.8303717092-1.0226350.09492158910.53333333330.0506248475

580842588.930.7373655904-0.78779450.16300671670.57142857140.0931466953

580863538.441.08546855010.57142857140

580882536.740.7182320442-0.74800460.17864686530.53333333330.0952783281

580902819.140.8217636298-0.99667260.10076910470.53333333330.0537435225

580922624.60.7504426855-0.81623170.15267513050.53333333330.0814267363

580942263.510.6180623023-0.56302920.27350848250.57142857140.1562905614

580962318.40.6381857043-0.59788840.25241293110.57142857140.1442359606

580982350.160.6498293415-0.61849030.24071862830.50.1203593142

583882755.50.7984323617-0.9317560.11701566360.57142857140.0668660935

583903101.520.9252878831-1.4647370.03429754230.57142857140.0195985956

583923102.530.9256581625-1.4676940.03406481230.57142857140.019465607

583942637.440.7551499998-0.82672560.14903023970.57142857140.085160137

583962473.930.6952050651-0.70246950.19839489820.57142857140.1133685133

583982636.570.7548310463-0.82600440.14927792860.57142857140.0853016735

584002720.090.785450586-0.89844920.1263428880.57142857140.072195936

584022247.380.6121488303-5.53E-010.27994441010.57142857140.1599682344

584042706.770.7805672974-0.88632460.12991981690.57142857140.0742398954

584063008.320.8911195269-1.2551990.05556495920.57142857140.0317514053

584082700.020.7780926578-0.8803220.13172797030.57142857140.0752731259

584103295.10.99625687860.57142857140

584123066.90.9125957319-1.3750210.04216761130.57142857140.0240957779

584143055.740.9085043279-1.3495690.04471271070.57142857140.0255501204

584162893.350.84897-1.0832530.08255568780.57142857140.0471746787

584183006.830.8905732732-1.2525650.05590298520.57142857140.031944563

584202674.50.7687366874-0.85794330.13869368910.57142857140.0792535366

584222728.380.78848981-0.90608230.12414170330.57142857140.0709381162

584242827.980.825004491-1.0063240.09855439580.57142857140.0563167976

584262351.990.6505002438-0.61969050.24005430570.57142857140.137173889

584282528.630.7152588106-0.7419650.18114860750.57142857140.10351349

584302415.060.6736225423-0.66168090.21793104430.57142857140.1245320253

584322404.070.6695934626-0.65426870.22168244360.52631578950.1166749703

584342106.030.5603280455-0.46663540.3414794710.52631578950.1797260374

1

1

1

QA1922.260.4929555261-0.35752440.43901119990.52631578950.2310585263

QA2003.220.5226365359-0.40541780.39317165440.52631578950.2069324497

QA1885.690.4795484791-0.33586390.46146216580.52631578950.2428748241

QA1955.110.5049987718-0.37695340.41980402670.52631578950.2209494878

Mean0.42836226170.225453822

SD0.02898635170.0152559746

CV6.76678463926.7667846392

T-RIA d1,16

0

0

0

0

0

0

T (log ng/ml)

Bo/Bm

forSAS

CT idfadtime pointtissueex_vivo_wtE2_ng_mlT_ng_ml

5805201y28.30.28339874840.1266386954

5805401y33.30.22860626840

5805601y30.10.38205123190.0516868903

5805801y43.30.13022747030.100070508

5806031y61.30.12824984570.0996709853

5806231y27.40.17448347590.0432469022

5806431y33.50.26091954010.0560483593

5806631y28.50.28191254640.2703088123

58068301y17.70.04026743850.1606892798

58070301y300.05222265540.186965748

58072301y24.40.02906743620.2166312611

58074301y13.80.196732180.1052218188

5807601y13.30.4675123570.0427998261

5807801y22.50.30811412220.0699755474

5808001y21.50.41937626870.0376000958

5808201y33.50.33985152880.0506248475

5808431y23.20.18502203410.0931466953

5808631blankblank00

5808831y30.20.12464582260.0952783281

5809031y26.70.28114355230.0537435225

58092301y43.100.0814267363

58094301y9.70.03589756180.1562905614

58096301y35.20.01909255060.1442359606

58098301y42.100.1203593142

58388016y19.70.21862169210.0668660935

58390016y8.40.07935802510.0195985956

58392016y12.80.21643144120.019465607

58394016y7.50.16815541270.085160137

58396316y9.10.32154055080.1133685133

58398316y7.30.07361405670.0853016735

58400316y15.40.2906482760.072195936

58402316y90.43680132920.1599682344

584043016y10.30.41231425630.0742398954

584063016y150.27457402580.0317514053

584083016y130.31103040890.0752731259

584103016y3.300

58412016y140.20080963710.0240957779

58414016y1.70.02411981380.0255501204

58416016y8.30.14999125930.0471746787

58418016y12.70.17506218770.031944563

58420316y6.40.15594108490.0792535366

58422316y11.90.17254244640.0709381162

58424316y7.20.33841020520.0563167976

58426316y13.10.27748707010.137173889

584283016y120.39242462440.10351349

584303016y8.80.40104060920.1245320253

584323016y15.70.38058073370.1166749703

584343016y9.60.56942906270.1797260374

figures

CT idfadtime pointtissueex_vivo_wtE2_ng_mlT_ng_mlE2_relT_rel24 h-E224 h-T24 h-wts

5808631blankblank00Fad ug/LMeanSDcountSECVFad ug/LMeanSDcountSECVFad ug/LMeanSDcountSECV

5805201y28.30.28339874840.12663869540.8859193962.113302355200.31989224950.108052392680.038202289833.777746346700.05992455130.039097735280.013823136965.2449361585028.2259.142639506383.232411196432.3919911651

5805401y33.30.228606268400.7146352212030.20519668820.068941837770.026057565333.59792903230.10163480070.077833236670.029418198276.5812851741332.971428571412.886131341674.870499841639.0827207067

5805601y30.10.38205123190.05168689031.19431224890.8625327885300.15406868360.069039430780.024409124844.8108136597300.1464775850.043882857980.015514933229.9587529836302712.706803576984.492533488247.0622354701

5805801y43.30.13022747030.1000705080.40709792291.6699417148

5807601y13.30.4675123570.04279982611.46146822180.7142285617

5807801y22.50.30811412220.06997554740.96318095451.1677275147

5808001y21.50.41937626870.03760009581.31099227750.627457277

5808201y33.50.33985152880.05062484751.06239375730.8448097881

5806031y61.30.12824984570.09967098530.40091576441.6632746191

5806231y27.40.17448347590.04324690220.54544452460.7216892121

5806431y33.50.26091954010.05604835930.81564820820.9353154611

5806631y28.50.28191254640.27030881230.88127345024.5108191286

5808431y23.20.18502203410.09314669530.5783886121.5543995448

5808831y30.20.12464582260.09527832810.38964939841.5899714902

5809031y26.70.28114355230.05374352250.87886953430.8968531483

58068301y17.70.04026743850.16068927980.12587813112.6815266249

58070301y300.05222265540.1869657480.16325076813.1200191556

58072301y24.40.02906743620.21663126110.09086633473.6150668861

58074301y13.80.196732180.10522181880.61499514381.7559049916

58092301y43.100.081426736301.3588209592Normality ok except 30 ug/LNormality ok except 3 ug/LLevene's test okNormality ok except 3 ug/LLevene's ok

58094301y9.70.03589756180.15629056140.11221766652.6081223478Levene's test okKW p=0.0076KW p=0.8063

58096301y35.20.01909255060.14423596060.05968431772.4069593759KW p=0.0005

58098301y42.100.120359314202.0085142321

58388016y19.70.21862169210.06686609351.41898851181.6724071518d. 16-E2d. 16-Td. 16-wts

58390016y8.40.07935802510.01959859560.51508212580.4901861272Fad ug/LMeanSDcountSECVFad ug/LMeanSDcountSECVFad ug/LMeanSDcountSECV

58392016y12.80.21643144120.0194656071.40477244360.486859911800.15406868360.069039430780.024409124844.810813659700.03998194660.02442835180.008636726361.0984532737010.63755.379308904181.901872902150.5692963958

58394016y7.50.16815541270.0851601371.09143148882.129964749930.25837312740.11688507980.04132511645.238868349930.09681458710.036347464580.012850769337.543376070339.9253.212364505281.135742362632.3663929995

58412016y140.20080963710.02409577791.30337737950.6026664511300.34267421510.163292342480.057732561347.6523575945300.08821386870.056150345980.019852145263.65251486013010.96253.957971161481.39935412436.1046400126

58414016y1.70.02411981380.02555012040.15655234580.6390414316

58416016y8.30.14999125930.04717467870.97353502191.1798994973

58418016y12.70.17506218770.0319445631.13626068290.7989746793

58396316y9.10.32154055080.11336851332.08699485982.8354925861

58398316y7.30.07361405670.08530167350.47780025742.1335047598

58400316y15.40.2906482760.0721959361.88648510021.80571338

58402316y90.43680132920.15996823442.83510781684.0010116513

58420316y6.40.15594108490.07925353661.01215302961.9822330645

58422316y11.90.17254244640.07093811621.11990602061.7742536858

58424316y7.20.33841020520.05631679762.19648923641.4085556697

58426316y13.10.27748707010.1371738891.80106082273.4308957038

584043016y10.30.41231425630.07423989542.67617173451.8568354379

584063016y150.27457402580.03175140531.78215338330.7941435558

584083016y130.31103040890.07527312592.01877761011.882677863

584103016y3.30000

584283016y120.39242462440.103513492.54707585762.5890057566

584303016y8.80.40104060920.12453202532.6029988693.1147064061

584323016y15.70.38058073370.11667497032.47020176192.9181913367Normality okLevene's test okNormality okLevene's test okNormality okLevene's test ok

584343016y9.60.56942906270.17972603743.69594293514.4951797619ANOVA p=0.0199ANOVA p=0.0246ANOVA p=0.8848

&A

&F

figures

00.03820228980.0382022898

00.02605756530.0260575653

00.02440912480.0244091248

A

B

C

24 h-E2

Fadrozole (ug/L)


00.01382313690.0138231369

00.02941819820.0294181982

00.01551493320.0155149332

A

AB

B

24 h-T

Fadrozole (ug/L)


00.02440912480.0244091248

00.0413251160.041325116

00.05773256130.0577325613

A

AB

B

d. 16-E2

Fadrozole (ug/L)


00.00863672630.0086367263

00.01285076930.0128507693

00.01985214520.0198521452

A

B

B

d. 16-T

Fadrozole (ug/L)


03.23241119643.2324111964

04.87049984164.8704998416

04.49253348824.4925334882

24 h-wts

Fadrozole (ug/L)


01.90187290211.9018729021

01.13574236261.1357423626

01.3993541241.399354124

d. 16-wts

Fadrozole (ug/L)


MBD0023823A.bin

MBD0022DDB4.bin

Chart1

14.361.86326518321.8632651832

5.9591.54101529611.5410152961

3.068750.50539036360.5053903636

a

b

b

z

Female 24 h

Fadrozole (ug/L-nominal)

VTG (mg/ml)

Fadrozole, Female Vtg

Vtg

Fadrozole 24 h and 7 d exposures

FHM exposures in support of array validation

DLV, KJ, MK, LD

VTG ELISAS conducted by KJ

24-hr Fadrozole Exposure

Fad ug/LSexIDVtg (mg/ml)TreatSexIDVtg (mg/ml)Female 24 h

0F219.60M100550

0F49.040M30.004Mean14.365.9593.06875

0F1419.60M130.003SD5.27010978474.35864946321.4294598131

0F167.440M150.003SE1.86326518321.54101529610.5053903636

0F2610.20M250.004CV36.699928862973.143974881346.5811751709

0F2813.60M270.003

0F3814.30M370.002

0F4021.10M390.006

5F68.545M50.003

5F81.325M70.008

5F189.965M170.002

5F205.095M190.004

5F301.475M290.012

5F320.7825M310.004Male 24 h

5F428.815M410.0050550

5F4411.75M430.005Mean0.0031250.0053750.01775

50F106.0750M90.005SD0.00172688820.00320434970.0032043497

50F122.9550M110.004SE0.00061054720.00113290870.0011329087

50F221.5750M210.004CV55.260422417159.615808787118.0526744919

50F242.7850M230.111

50F343.8650M330.004

50F361.6950M350.002

50F463.2050M450

50F482.4350M470.012

7-d Fadrozole Exposure

TreatSexIDVtg (mg/ml)TreatSexIDVtg (mg/ml)Female 7d

0F218.40M10.0100550

0F420.40M30.004Mean19.3755.970.03175

0F1424.90M130.004SD4.49563280152.89367125520.0425768213

0F1617.90M150.003SE1.58944621981.0937049310.0150531795

0F2618.90M250.010CV23.203266072148.4702052802134.100224651

0F2816.50M270.003

0F3826.00M370.002

0F4012.00M390.004

5F65.495M50.004

5F88.035M70.002

5F1810.15M170.003

5F208.225M190

5F302.335M290Male 7d

5F323.085M3100550

5F424.545M410.011Mean0.0050.00250.0035

5F44no sample5M430SD0.00316227770.00377964470.0065683222

50F100.00750M90SE0.0011180340.00133630620.0023222526

50F120.02150M110CV63.2455532034151.1857892037187.6663499196

50F220.13550M210.012

50F240.00850M230

50F340.02550M330

50F360.03150M350

50F460.01750M450

50F480.01050M470.016

&A

&F

Figures

Fadrozole 24 h and 7 d exposures

FHM exposures in support of array validation

DLV, KJ, MK, LDStatistics

VTG ELISAS conducted by KJ

Female, 24 h

Kolmogorov-Smirnov p=0.150 (normal)

Female 24 hLevene's test p= 0.0046 (non-trans); p=0.0013 (log trans) - violated homogeneity

0550Non-parametric one-way ANOVA p=0.0017

Mean14.365.9593.06875Welch's one-way ANOVA p=.0005

SD5.27010978474.35864946321.4294598131Dunnett's test = both treatments different from control

SE1.86326518321.54101529610.5053903636Fishers LSD = (0-A, 5-B, 50-B)

CV36.699928862973.143974881346.5811751709

Female, 7d

Non-transformed data was not normally distributed

Male 24 hLog trans Kolm-Smir p>0.150 (normal)

0550Log trans Levene's test p=0.1547

Mean0.0031250.0053750.01775Log trans one-way ANOVA p

BrainsGnRHneuronscGnRHneuronsdopaminergicneuronssGnRH releasecGnRH releasedopamine releaseGnRHPituitaryFSH gonadotrophsLH gonadotrophs TestisOvariesLeydigcellsSertoli cellsSperm-atocytesThecacellsGranulosacellsOocytesFSHLHLiverSperm mat.oocyte mat.steroidssteroidsSteroid metab.vitellogenesisBlood supplyBlood supply

state transitionactivated proteinFigure Key

Inner mitochondrial membraneFadrozole

Evidence for compensatory/feedback response to fadrozole.exposureexposurepost-exposurepost-exposure

Chart1

10.64145564170.14586154520.07006656710.07006656710.11942236750.11942236750.081457320.08145732

10.53099060980.20668239950.06325857470.06325857470.14333392830.14333392830.15518425060.1551842506

10.59229788610.30946009320.05299525550.05299525550.17950254390.17950254390.09471836430.0947183643

10.96481614430.87126656920.25900102660.25900102660.21613330920.21613330920.1263969650.126396965

10.89354557341.81310187730.44434440540.44434440540.2364491670.2364491670.21820686290.2182068629

12.47728187023.01296882721.03462874651.03462874650.18095712190.18095712190.48690591810.4869059181

11.95546663281.61361971330.33879169720.33879169720.23921811950.23921811950.21922348270.2192234827

11.67699964292.22416526890.37471963770.37471963770.1584301510.1584301510.26822528110.2682252811

#

#

#

#

#

*

*

*

*

0

3

30

Time point

Ex vivo E2 production (relative to control)

Sheet1

CT idfadtime pointE2_relT_rel

58052010.8859193962.113302355224 hMeanSDcountSE24 hMeanSDcountSE

58054010.71463522120010.337777463580.1194223675010.652449361680.230675684

58056011.19431224890.862532788530.64145564170.215515811370.0814573231.69604608631.2988538970.4909206261

58058010.40709792291.6699417148300.14586154520.19817817980.0700665671302.44436682170.732301818180.2589077907

58076011.46146822180.7142285617


58080011.31099227750.627457277010.405409570780.1433339283012.599367829180.9190153094

58082011.06239375730.844809788130.53099060980.438927343780.155184250631.53146446483.201770441981.1319967956

58060310.40091576441.6632746191300.20668239950.178922268680.0632585747307.80939420214.031428156281.4253250936

58062310.54544452460.7216892121

58064310.81564820820.93531546114 dMeanSDcountSE4 dMeanSDcountSE

58066310.88127345024.5108191286010.507709864380.1795025439010.449878228180.1590559729

58084310.5783886121.554399544830.59229788610.267903990980.094718364330.832015360.477066830480.1686685954

58088310.38964939841.5899714902300.30946009320.140212266870.0529952555302.15938457480.574971068370.2173186368

58090310.87886953430.8968531483


580703010.16325076813.1200191556010.571834986270.2161333092010.794780176170.3003986704

580723010.09086633473.615066886130.96481614430.357504604280.12639696531.74401530281.779858235180.6292749138

580743010.61499514381.7559049916300.87126656920.685252305870.2590010266306.85463325914.157058620971.5712204709

5809230101.3588209592


580963010.05968431772.4069593759010.668779237780.236449167011.016475565780.3593783827

5809830102.008514232130.89354557340.617182209880.218206862931.79445136781.261639283780.4460568465

58100021.71605163110301.81310187731.256795768980.4443444054302.94205177442.207579791380.7804973202

58102021.42032194730

58104020.6783947327010 dMeanSDcountSE10 dMeanSDcountSE

58106021.21729454190010.51182403280.1809571219010.451297209280.1595576585

58124020.63376734877.412989289232.47728187021.37717790680.486905918131.09665294591.000009533480.3535567611

58126020.90284214630303.01296882722.926372010781.0346287465302.58386779341.393396890280.492640195

58128020.76660974590


58108320.23043089490010.676611017880.2392181195010.878360812880.3105474435

58110320.6827012862031.95546663280.620057644980.219223482730.3785333640.42759575680.1511779293

58112320.45773391323.3446588395301.61361971330.958247625980.3387916972300.86425836840.984991247380.3482469952

58114321.54096400210


58134320.45607100630010.448108136680.158430151010.610984532780.2160156531

58136320.3568149942031.67699964290.758655660780.268225281132.42145756260.909096919180.3214142981

58138320.14171389720302.22416526891.059867187480.3747196377302.20634251471.404392497180.4965277291

581163020.15506631010.8359186709

581183020.62121881258.817714348

581203020.14861856826.5094939192

581223020.07808292287.6837057603

581403020.063377617615.0685743617

581423020.12147300968.2488788577

581443020.21674783179.8324766934

581463020.24887412365.4783910057

58148041.16496360950.7923813134

58150040.66956030620.3969951719

58152041.80328490081.2091583299

58154040.77185131130.7644784932

58172040.79403646330.8050046186

58174041.52296182141.2786626575

58176040.19466568370.8710047534

58178041.07867590391.882314662

58156340.182561881.2172883485

58158340.8074837650.7046476779

58160340.88608337721.4939078698

58162340.47663255181.2780671875

58180340.55671932810.3281136705

58182340.62004184420.1234123508

58184340.29930646610.8640514933

58186340.90955387680.6466342815

581643040.44609545583.1722537914

581663040.47863427951.7795378312

581683040.19610397611.9234332756

581883040.30131003741.3352961447

581903040.14847686852.3892897782

581923040.17217317942.2018824255

581943040.42342685552.3139987772

58196080.73202679920.579926722

58198080.73362518870.1589241651

58200081.55964973340.4647927377

58220081.33942969551.8651909446

58222081.82584268881.6628373958

58224080.40044137390.298751698T relativeMeanMeanMeanSESESE

58226080.40898452061.9695763367E2 relativeMeanMeanMeanSESESEdays03300330

58204380.44528937150.2421837166days0330033024 h11.69604608632.44436682170.2306756840.49092062610.2589077907

58206380.78028570160.29497316124 h10.64145564170.14586154520.11942236750.081457320.070066567148 h11.53146446487.80939420210.91901530941.13199679561.4253250936

58208381.18373194321.554991816548 h10.53099060980.20668239950.14333392830.15518425060.06325857474 d10.832015362.15938457480.15905597290.16866859540.2173186368

58210380.51697747120.14449520374 d10.59229788610.30946009320.17950254390.09471836430.05299525558 d11.74401530286.85463325910.30039867040.62927491381.5712204709

58228381.11814589834.89333674488 d10.96481614430.87126656920.21613330920.1263969650.25900102669 d11.79445136782.94205177440.35937838270.44605684650.7804973202

58230380.94651675333.91411118099 d10.89354557341.81310187730.2364491670.21820686290.444344405410 d11.09665294592.58386779340.15955765850.35355676110.492640195

58232381.36088639490.990275057810 d12.47728187023.01296882720.18095712190.48690591811.034628746512 d10.3785333640.86425836840.31054744350.15117792930.3482469952

58234381.36669562061.917755541312 d11.95546663281.61361971330.23921811950.21922348270.338791697216 d12.42145756262.20634251470.21601565310.32141429810.4965277291

582123081.93346858519.223973655916 d11.67699964292.22416526890.1584301510.26822528110.3747196377

582143080.32106662428.1566457122

582163080.74046528964.9391328698

582363080.10347622221.7491969416

582383080.481341944614.444714409

582403080.85561036894.8809207543

582423081.66343695014.5878484711

58244090.85443928330.5797096217

58246092.31574762030.8797289579

58248091.4531346763.4279996944

58250090.79170699250.2628597465

58268090.41896184590.6753578084

58270090.27024054740.3193272718

58272090.59138998360.8187135305

58274091.3043790511.0363033688

58252391.20249771572.4785770542

58254390.22042378652.0895620606

58256390.13050298160.9811789697

58258391.30315236354.5499086065

58276390.47273218650.9356139639

58278391.94772477061.4507268925

58280390.74083824940.9411042543

58282391.13049253360.9289391411

582603092.1931362283.2176226678

582623092.00300553992.4116925427

582643090.01259521297.0803043862

582663092.58600693062.4101458099

582843091.40526757011.1641742142

582863093.68352025935.2303497615

582883090.11328477340.5140791555

582903092.50799850421.5080456576

582920101.04296163931.9913261875

582940100.51883735320.9579141865

582960101.19255587191.3145291014

582980101.10908591050.8018020423

583160101.86615290480.7712967566

583180100.32364233420.719988558

583200100.55967400410.8143563836

583220101.3870899820.6287867841

583003100.68417001280.5794224326

583023101.67760702140.2468632857

583043103.58975152080.5226964592

583063103.78846635913.3010115332

583243103.09026508531.3205705416

583263104.30193788261.4191121066

583283101.03755334561.1040036395

583303101.64850373380.2795435687

5830830103.46710133443.8767238411

5831030104.65330945652.2911520153

58312301004.7121391322

5831430100.97388580361.2281966404

5833230101.23558033650.9860718437

5833430108.78154413053.8236361429

5833630100.64643049391.4431188687

5833830104.34589906182.3099038627

583400120.84404803492.0154109771

583420120.91767509021.1817282552

583440120.20892916450.2751245338

583460121.75495531760

583640120.70495723890.7872604238

583660122.26061914932.5289195238

583680120.47736315910.8480798011

583700120.83145284540.3634764851

583483121.9113584970.5288914175

583503121.43915420750

583523121.58101310

583543122.79447193530.7918777121

583723120.99613634720

583743122.67502013580

583763122.32850902271.0292427575

583783121.91806981710.6782550245

5835630122.3538733481.9097834245

5835830121.95227943690.9631825806

5836030121.10956845120

5836230121.91585828590

5838030120.14811509180.1472218845

5838230122.92953443522.4897008793

5838430122.04849333991.4041781786

5838630120.45123531740

583880161.41898851181.6724071518

583900160.51508212580.4901861272

583920161.40477244360.4868599118

583940161.09143148882.1299647499

584120161.30337737950.6026664511

584140160.15655234580.6390414316

584160160.97353502191.1798994973

584180161.13626068290.7989746793

583963162.08699485982.8354925861

583983160.47780025742.1335047598

584003161.88648510021.80571338

584023162.83510781684.0010116513

584203161.01215302961.9822330645

584223161.11990602061.7742536858

584243162.19648923641.4085556697

584263161.80106082273.4308957038

5840430162.67617173451.8568354379

5840630161.78215338330.7941435558

5840830162.01877761011.882677863

58410301600

5842830162.54707585762.5890057566

5843030162.6029988693.1147064061

5843230162.47020176192.9181913367

5843430163.69594293514.4951797619

Sheet1

0.11942236750.11942236750.14333392830.14333392830.17950254390.17950254390.21613330920.21613330920.2364491670.2364491670.18095712190.18095712190.23921811950.23921811950.1584301510.158430151

0.081457320.081457320.15518425060.15518425060.09471836430.09471836430.1263969650.1263969650.21820686290.21820686290.48690591810.48690591810.21922348270.21922348270.26822528110.2682252811

0.07006656710.07006656710.06325857470.06325857470.05299525550.05299525550.25900102660.25900102660.44434440540.44434440541.03462874651.03462874650.33879169720.33879169720.37471963770.3747196377

a

a

a

a

a

a

a

a

b

b

b

a

a

ab

b

b

b

ab

b

ab

b

c

a

a

24 h

48 h

4 d

8 d

9 d

10 d

12 d

16 d

Fadrozole (ug/L)

E2 production (relative to control)

Sheet2

0.2306756840.2306756840.91901530940.91901530940.15905597290.15905597290.30039867040.30039867040.35937838270.35937838270.15955765850.15955765850.31054744350.31054744350.21601565310.2160156531

0.49092062610.49092062611.13199679561.13199679560.16866859540.16866859540.62927491380.62927491380.44605684650.44605684650.35355676110.35355676110.15117792930.15117792930.32141429810.3214142981

0.25890779070.25890779071.42532509361.42532509360.21731863680.21731863681.57122047091.57122047090.78049732020.78049732020.4926401950.4926401950.34824699520.34824699520.49652772910.4965277291

a

a

b

a

b

a

a

b

a

a

b

a

a

a

a

b

b

a

ab

b

a

a

ab

b

24 h

48 h

4 d

8 d

9 d

10 d

12 d

16 d

Fadrozole (ug/L)

T production (relative to control)

Sheet3

0.11942236750.11942236750.081457320.081457320.07006656710.0700665671

0.14333392830.14333392830.15518425060.15518425060.06325857470.0632585747

0.17950254390.17950254390.09471836430.09471836430.05299525550.0529952555

0.21613330920.21613330920.1263969650.1263969650.25900102660.2590010266

0.2364491670.2364491670.21820686290.21820686290.44434440540.4443444054

0.18095712190.18095712190.48690591810.48690591811.03462874651.0346287465

0.23921811950.23921811950.21922348270.21922348270.33879169720.3387916972

0.1584301510.1584301510.26822528110.26822528110.37471963770.3747196377

&F

*

*

*

*

#

#

#

#

#

0

3

30

Time point


0.2306756840.2306756840.49092062610.49092062610.25890779070.2589077907

0.91901530940.91901530941.13199679561.13199679561.42532509361.4253250936

0.15905597290.15905597290.16866859540.16866859540.21731863680.2173186368

0.30039867040.30039867040.62927491380.62927491381.57122047091.5712204709

0.35937838270.35937838270.44605684650.44605684650.78049732020.7804973202

0.15955765850.15955765850.35355676110.35355676110.4926401950.492640195

0.31054744350.31054744350.15117792930.15117792930.34824699520.3482469952

0.21601565310.21601565310.32141429810.32141429810.49652772910.4965277291

&F

#

#

#

#

#

#

#

*

0

3

30

Time point


0.07006656710.07006656710.11942236750.11942236750.081457320.08145732

0.06325857470.06325857470.14333392830.14333392830.15518425060.1551842506

0.05299525550.05299525550.17950254390.17950254390.09471836430.0947183643

0.25900102660.25900102660.21613330920.21613330920.1263969650.126396965

0.44434440540.44434440540.2364491670.2364491670.21820686290.2182068629

1.03462874651.03462874650.18095712190.18095712190.48690591810.4869059181

0.33879169720.33879169720.23921811950.23921811950.21922348270.2192234827

0.37471963770.37471963770.1584301510.1584301510.26822528110.2682252811

#

#

#

#

#

*

*

*

*

0

3

30

Time point


0.07006656710.07006656710.11942236750.11942236750.081457320.08145732

0.06325857470.06325857470.14333392830.14333392830.15518425060.1551842506

0.05299525550.05299525550.17950254390.17950254390.09471836430.0947183643

0.25900102660.25900102660.21613330920.21613330920.1263969650.126396965

0.44434440540.44434440540.2364491670.2364491670.21820686290.2182068629

1.03462874651.03462874650.18095712190.18095712190.48690591810.4869059181

0.33879169720.33879169720.23921811950.23921811950.21922348270.2192234827

0.37471963770.37471963770.1584301510.1584301510.26822528110.2682252811

#

#

#

#

#

*

#

#

0

3

30

Time point

Ex vivo T production (relative to control)

Chart2

11.69604608632.44436682170.07006656710.07006656710.11942236750.11942236750.081457320.08145732

11.53146446487.80939420210.06325857470.06325857470.14333392830.14333392830.15518425060.1551842506

10.832015362.15938457480.05299525550.05299525550.17950254390.17950254390.09471836430.0947183643

11.74401530286.85463325910.25900102660.25900102660.21613330920.21613330920.1263969650.126396965

11.79445136782.94205177440.44434440540.44434440540.2364491670.2364491670.21820686290.2182068629

11.09665294592.58386779341.03462874651.03462874650.18095712190.18095712190.48690591810.4869059181

10.3785333640.86425836840.33879169720.33879169720.23921811950.23921811950.21922348270.2192234827

12.42145756262.20634251470.37471963770.37471963770.1584301510.1584301510.26822528110.2682252811

#

#

#

#

#

*

#

#

0

3

30

Time point


Sheet1

CT idfadtime pointE2_relT_rel


58054010.71463522120010.337777463580.1194223675010.652449361680.230675684

58056011.19431224890.862532788530.64145564170.215515811370.0814573231.69604608631.2988538970.4909206261

58058010.40709792291.6699417148300.14586154520.19817817980.0700665671302.44436682170.732301818180.2589077907

58076011.46146822180.7142285617


58080011.31099227750.627457277010.405409570780.1433339283012.599367829180.9190153094

58082011.06239375730.844809788130.53099060980.438927343780.155184250631.53146446483.201770441981.1319967956

58060310.40091576441.6632746191300.20668239950.178922268680.0632585747307.80939420214.031428156281.4253250936

58062310.54544452460.7216892121


58066310.88127345024.5108191286010.507709864380.1795025439010.449878228180.1590559729

58084310.5783886121.554399544830.59229788610.267903990980.094718364330.832015360.477066830480.1686685954

58088310.38964939841.5899714902300.30946009320.140212266870.0529952555302.15938457480.574971068370.2173186368

58090310.87886953430.8968531483


580703010.16325076813.1200191556010.571834986270.2161333092010.794780176170.3003986704

580723010.09086633473.615066886130.96481614430.357504604280.12639696531.74401530281.779858235180.6292749138

580743010.61499514381.7559049916300.87126656920.685252305870.2590010266306.85463325914.157058620971.5712204709

5809230101.3588209592


580963010.05968431772.4069593759010.668779237780.236449167011.016475565780.3593783827

5809830102.008514232130.89354557340.617182209880.218206862931.79445136781.261639283780.4460568465

58100021.71605163110301.81310187731.256795768980.4443444054302.94205177442.207579791380.7804973202

58102021.42032194730

58104020.6783947327010 dMeanSDcountSE10 dMeanSDcountSE

58106021.21729454190010.51182403280.1809571219010.451297209280.1595576585

58124020.63376734877.412989289232.47728187021.37717790680.486905918131.09665294591.000009533480.3535567611

58126020.90284214630303.01296882722.926372010781.0346287465302.58386779341.393396890280.492640195

58128020.76660974590


58108320.23043089490010.676611017880.2392181195010.878360812880.3105474435

58110320.6827012862031.95546663280.620057644980.219223482730.3785333640.42759575680.1511779293

58112320.45773391323.3446588395301.61361971330.958247625980.3387916972300.86425836840.984991247380.3482469952

58114321.54096400210


58134320.45607100630010.448108136680.158430151010.610984532780.2160156531

58136320.3568149942031.67699964290.758655660780.268225281132.42145756260.909096919180.3214142981

58138320.14171389720302.22416526891.059867187480.3747196377302.20634251471.404392497180.4965277291

581163020.15506631010.8359186709

581183020.62121881258.817714348

581203020.14861856826.5094939192

581223020.07808292287.6837057603

581403020.063377617615.0685743617

581423020.12147300968.2488788577

581443020.21674783179.8324766934

581463020.24887412365.4783910057

58148041.16496360950.7923813134

58150040.66956030620.3969951719

58152041.80328490081.2091583299

58154040.77185131130.7644784932

58172040.79403646330.8050046186

58174041.52296182141.2786626575

58176040.19466568370.8710047534

58178041.07867590391.882314662

58156340.182561881.2172883485

58158340.8074837650.7046476779

58160340.88608337721.4939078698

58162340.47663255181.2780671875

58180340.55671932810.3281136705

58182340.62004184420.1234123508

58184340.29930646610.8640514933

58186340.90955387680.6466342815

581643040.44609545583.1722537914

581663040.47863427951.7795378312

581683040.19610397611.9234332756

581883040.30131003741.3352961447

581903040.14847686852.3892897782

581923040.17217317942.2018824255

581943040.42342685552.3139987772

58196080.73202679920.579926722

58198080.73362518870.1589241651

58200081.55964973340.4647927377

58220081.33942969551.8651909446

58222081.82584268881.6628373958

58224080.40044137390.298751698T relativeMeanMeanMeanSESESE

58226080.40898452061.9695763367E2 relativeMeanMeanMeanSESESEdays03300330

58204380.44528937150.2421837166days0330033024 h11.69604608632.44436682170.2306756840.49092062610.2589077907

58206380.78028570160.29497316124 h10.64145564170.14586154520.11942236750.081457320.070066567148 h11.53146446487.80939420210.91901530941.13199679561.4253250936

58208381.18373194321.554991816548 h10.53099060980.20668239950.14333392830.15518425060.06325857474 d10.832015362.15938457480.15905597290.16866859540.2173186368

58210380.51697747120.14449520374 d10.59229788610.30946009320.17950254390.09471836430.05299525558 d11.74401530286.85463325910.30039867040.62927491381.5712204709

58228381.11814589834.89333674488 d10.96481614430.87126656920.21613330920.1263969650.25900102669 d11.79445136782.94205177440.35937838270.44605684650.7804973202

58230380.94651675333.91411118099 d10.89354557341.81310187730.2364491670.21820686290.444344405410 d11.09665294592.58386779340.15955765850.35355676110.492640195

58232381.36088639490.990275057810 d12.47728187023.01296882720.18095712190.48690591811.034628746512 d10.3785333640.86425836840.31054744350.15117792930.3482469952

58234381.36669562061.917755541312 d11.95546663281.61361971330.23921811950.21922348270.338791697216 d12.42145756262.20634251470.21601565310.32141429810.4965277291

582123081.93346858519.223973655916 d11.67699964292.22416526890.1584301510.26822528110.3747196377

582143080.32106662428.1566457122

582163080.74046528964.9391328698

582363080.10347622221.7491969416

582383080.481341944614.444714409

582403080.85561036894.8809207543

582423081.66343695014.5878484711

58244090.85443928330.5797096217

58246092.31574762030.8797289579

58248091.4531346763.4279996944

58250090.79170699250.2628597465

58268090.41896184590.6753578084

58270090.27024054740.3193272718

58272090.59138998360.8187135305

58274091.3043790511.0363033688

58252391.20249771572.4785770542

58254390.22042378652.0895620606

58256390.13050298160.9811789697

58258391.30315236354.5499086065

58276390.47273218650.9356139639

58278391.94772477061.4507268925

58280390.74083824940.9411042543

58282391.13049253360.9289391411

582603092.1931362283.2176226678

582623092.00300553992.4116925427

582643090.01259521297.0803043862

582663092.58600693062.4101458099

582843091.40526757011.1641742142

582863093.68352025935.2303497615

582883090.11328477340.5140791555

582903092.50799850421.5080456576

582920101.04296163931.9913261875

582940100.51883735320.9579141865

582960101.19255587191.3145291014

582980101.10908591050.8018020423

583160101.86615290480.7712967566

583180100.32364233420.719988558

583200100.55967400410.8143563836

583220101.3870899820.6287867841

583003100.68417001280.5794224326

583023101.67760702140.2468632857

583043103.58975152080.5226964592

583063103.78846635913.3010115332

583243103.09026508531.3205705416

583263104.30193788261.4191121066

583283101.03755334561.1040036395

583303101.64850373380.2795435687

5830830103.46710133443.8767238411

5831030104.65330945652.2911520153

58312301004.7121391322

5831430100.97388580361.2281966404

5833230101.23558033650.9860718437

5833430108.78154413053.8236361429

5833630100.64643049391.4431188687

5833830104.34589906182.3099038627

583400120.84404803492.0154109771

583420120.91767509021.1817282552

583440120.20892916450.2751245338

583460121.75495531760

583640120.70495723890.7872604238

583660122.26061914932.5289195238

583680120.47736315910.8480798011

583700120.83145284540.3634764851

583483121.9113584970.5288914175

583503121.43915420750

583523121.58101310

583543122.79447193530.7918777121

583723120.99613634720

583743122.67502013580

583763122.32850902271.0292427575

583783121.91806981710.6782550245

5835630122.3538733481.9097834245

5835830121.95227943690.9631825806

5836030121.10956845120

5836230121.91585828590

5838030120.14811509180.1472218845

5838230122.92953443522.4897008793

5838430122.04849333991.4041781786

5838630120.45123531740

583880161.41898851181.6724071518

583900160.51508212580.4901861272

583920161.40477244360.4868599118

583940161.09143148882.1299647499

584120161.30337737950.6026664511

584140160.15655234580.6390414316

584160160.97353502191.1798994973

584180161.13626068290.7989746793

583963162.08699485982.8354925861

583983160.47780025742.1335047598

584003161.88648510021.80571338

584023162.83510781684.0010116513

584203161.01215302961.9822330645

584223161.11990602061.7742536858

584243162.19648923641.4085556697

584263161.80106082273.4308957038

5840430162.67617173451.8568354379

5840630161.78215338330.7941435558

5840830162.01877761011.882677863

58410301600

5842830162.54707585762.5890057566

5843030162.6029988693.1147064061

5843230162.47020176192.9181913367

5843430163.69594293514.4951797619

Sheet1

0.11942236750.11942236750.14333392830.14333392830.17950254390.17950254390.21613330920.21613330920.2364491670.2364491670.18095712190.18095712190.23921811950.23921811950.1584301510.158430151

0.081457320.081457320.15518425060.15518425060.09471836430.09471836430.1263969650.1263969650.21820686290.21820686290.48690591810.48690591810.21922348270.21922348270.26822528110.2682252811

0.07006656710.07006656710.06325857470.06325857470.05299525550.05299525550.25900102660.25900102660.44434440540.44434440541.03462874651.03462874650.33879169720.33879169720.37471963770.3747196377

a

a

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24 h

48 h

4 d

8 d

9 d

10 d

12 d

16 d

Fadrozole (ug/L)


Sheet2

0.2306756840.2306756840.91901530940.91901530940.15905597290.15905597290.30039867040.30039867040.35937838270.35937838270.15955765850.15955765850.31054744350.31054744350.21601565310.2160156531

0.49092062610.49092062611.13199679561.13199679560.16866859540.16866859540.62927491380.62927491380.44605684650.44605684650.35355676110.35355676110.15117792930.15117792930.32141429810.3214142981

0.25890779070.25890779071.42532509361.42532509360.21731863680.21731863681.57122047091.57122047090.78049732020.78049732020.4926401950.4926401950.34824699520.34824699520.49652772910.4965277291

a

a

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a

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24 h

48 h

4 d

8 d

9 d

10 d

12 d

16 d

Fadrozole (ug/L)


Sheet3

0.11942236750.11942236750.081457320.081457320.07006656710.0700665671

0.14333392830.14333392830.15518425060.15518425060.06325857470.0632585747

0.17950254390.17950254390.09471836430.09471836430.05299525550.0529952555

0.21613330920.21613330920.1263969650.1263969650.25900102660.2590010266

0.2364491670.2364491670.21820686290.21820686290.44434440540.4443444054

0.18095712190.18095712190.48690591810.48690591811.03462874651.0346287465

0.23921811950.23921811950.21922348270.21922348270.33879169720.3387916972

0.1584301510.1584301510.26822528110.26822528110.37471963770.3747196377

&F

*

*

*

*

#

#

#

#

#

0

3

30

Time point


0.2306756840.2306756840.49092062610.49092062610.25890779070.2589077907

0.91901530940.91901530941.13199679561.13199679561.42532509361.4253250936

0.15905597290.15905597290.16866859540.16866859540.21731863680.2173186368

0.30039867040.30039867040.62927491380.62927491381.57122047091.5712204709

0.35937838270.35937838270.44605684650.44605684650.78049732020.7804973202

0.15955765850.15955765850.35355676110.35355676110.4926401950.492640195

0.31054744350.31054744350.15117792930.15117792930.34824699520.3482469952

0.21601565310.21601565310.32141429810.32141429810.49652772910.4965277291

&F

#

#

#

#

#

#

#

*

0

3

30

Time point


0.07006656710.07006656710.11942236750.11942236750.081457320.08145732

0.06325857470.06325857470.14333392830.14333392830.15518425060.1551842506

0.05299525550.05299525550.17950254390.17950254390.09471836430.0947183643

0.25900102660.25900102660.21613330920.21613330920.1263969650.126396965

0.44434440540.44434440540.2364491670.2364491670.21820686290.2182068629

1.03462874651.03462874650.18095712190.18095712190.48690591810.4869059181

0.33879169720.33879169720.23921811950.23921811950.21922348270.2192234827

0.37471963770.37471963770.1584301510.1584301510.26822528110.2682252811

#

#

#

#

#

*

*

*

*

0

3

30

Time point


0.07006656710.07006656710.11942236750.11942236750.081457320.08145732

0.06325857470.06325857470.14333392830.14333392830.15518425060.1551842506

0.05299525550.05299525550.17950254390.17950254390.09471836430.0947183643

0.25900102660.25900102660.21613330920.21613330920.1263969650.126396965

0.44434440540.44434440540.2364491670.2364491670.21820686290.2182068629

1.03462874651.03462874650.18095712190.18095712190.48690591810.4869059181

0.33879169720.33879169720.23921811950.23921811950.21922348270.2192234827

0.37471963770.37471963770.1584301510.1584301510.26822528110.2682252811

#

#

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#

*

#

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0

3

30

Time point


Aromatase A (CYP19A) gene expressionexposurepost-exposure

Chart1

1278.58003333332470.72842825.8473428571547.6822690753547.6822690753980.5975400569980.5975400569641.229579791641.229579791

975.9153754940.35622513618.9757428571124.2794187459124.27941874591023.62192927471023.62192927472105.39382987842105.3938298784

1345.78477142865617.206255525.0565142857304.2239148383304.2239148383689.9761769474689.97617694741089.5953607861089.595360786

1050.209352757.39706666672481.59025216.537382967216.537382967578.9286455984578.9286455984546.0749489434546.0749489434

982.90881849.9924252790.015125236.1028913768236.1028913768262.3420559405262.3420559405477.6438439085477.6438439085

1168.462351108.833952394.2108857143224.8358402555224.8358402555209.874045623209.874045623453.6921081955453.6921081955

a

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0

3

30

Time point (d)

CYP19A mRNA (copies/ng total RNA)

Fadrozole Phase 3 ovary, Arom-A

Document Name: Fadrozole Phase 3 ovaryArom-A8-3-07.sds

Plate Type: Absolute Quantification

User: dvillene

Document Information

Operator: JET

Run Date: FridayAugust 032007 17:35:10

Last Modified: FridayAugust 032007 18:58:42

Instrument Type: Applied Biosystems 7500 Real-Time PCR System

Comments:

Notebook 345p. 41

AromA mRNAlog copiesCtCtMeanSDCV

5.00E+077.698970004315.6515.4715.560.12727922060.8179898497

5.00E+066.698970004319.3919.2219.3050.12020815280.6226788542

500004.698970004326.6526.5826.6150.04949747470.1859758583

50003.698970004330.1730.1530.160.01414213560.0468903701

5002.698970004333.0833.3233.20.16970562750.5111615286

Fad (ug/L)TimeSample NameCtQtycopies/nglog ratio24 h

015105226.4448629.3972.586-0.1187999043Fad (ug/L)MeanSDcountSECV

015105427.2129426.68588.5336-0.336956655901278.58003333331341.54210040416547.6822690753104.9243743394

015105828.2714855.42297.1084-0.633812987532470.72842594.41722723227980.5975400569105.0061685142

015107624.32191599.633831.99260.4766967437302825.84734285711696.53400142537641.22957979160.036293387

015107827.9418309.57366.1914-0.5430197774

015108825.6580753.411615.06820.1014629481

315106025.4393170.411863.40820.1635800842

315106225.7177924.611558.49220.085976715

315106423.134125808251.60.8098102491

315106625.495104.521902.09040.1725032356

315108426.1459050.441181.0088-0.0344747844

315108627.1830058.01601.1602-0.3277376978

315109025.3796866.951937.3390.1804777031

3015106824.81138775.72775.5140.3366155042

3015107024.54165392.843307.85680.4128187822

3015107424.49170950.283419.00560.427171894

3015109226.3651124.551022.491-0.0970684241

3015109425.9964917.911298.35820.0066666071

3015109623.61301646.726032.93440.6738006849

3015109825.3896238.571924.77140.1776512388

025110025.28102887.62057.7520.100455864948 h

025110425.495106.291902.12580.0663020687Fad (ug/L)MeanSDcountSECV

025112426.3352082.441041.6488-0.195215849701632.8157452.10709498074226.053547490327.688801313

025112825.7476486.811529.7362-0.02832062363618.9072436.84670071674218.423350358470.5835544839

325110826.546628.19932.5638-0.24325861453017626.9649521103.4958004042410551.7479002021119.7227989065

325111226.546734.38934.6876-0.24227068643-out822.7836191.98708557833110.843795539523.3338493351

325113227.1830054.97601.0994-0.4339908731

325113634363.97.278-2.3509251163

3025111620.382.45E+06490001.4772589124

3025112023.03440708.228814.16440.732243979

3025114022.92474097.59481.950.7639604933

3025114424.59160587.273211.74540.2938039432

045114826.0861171.51223.430.09816696564 d

045115026.2853707.191074.14380.0416502659Fad (ug/L)MeanSDcountSECV

045115225.7675413.041508.26080.18906428370975.915375351.51527902858124.279418745936.0190328007

045115426.7639467.27789.3454-0.092145077734940.3562252895.240030245681023.621929274758.6038718341

045117226.4249175.29983.50580.00336476533013618.97574285715570.348485708172105.393829878440.9013760718

045117426.8736760.55735.211-0.1230001639

045117628.0117563.04351.2608-0.443782474

045117826.1957108.271142.16540.0683168397

345115626.1657982.431159.64860.0749142479

345115823.35357657.387153.14760.8650850264

345116024.3193969.733879.39460.5993517968

345116225.5884810.291696.20580.2400663838

345118022.92474412.419488.24820.9877738766

345118224.02231780.754635.6150.6766951994

345118424.23203117.564062.35120.6193653064

345118623.29372411.947448.23880.8826414321

3045116423.16405379.038107.58060.9194791147

3045116622.72538322.0610766.44121.0426600128

3045116822.4664034.8813280.69761.1338087279

3045118821.491.19E+06238001.3871647969

3045119022.42655338.3813106.76761.1280834388

3045119221.94889063.1917781.26381.260550465

3045119423.09424503.978490.07940.9394995917

085119626.0363200.191264.0038-0.02722723

085119826.3351931.661038.6332-0.11251340898 d

085120026.2654553.921091.0784-0.0911196515Fad (ug/L)MeanSDcountSECV

085122025.9666064.411321.2882-0.007978053201345.7847714286860.47517272528304.223914838363.9385428483

085122226.7140711.04814.2208-0.21823341735617.206251951.54733430678689.976176947434.7423122359

085122424.58161252.423225.04840.3795606271305525.05651428572882.798354329371089.59536078652.176812072

085122627.0233311.03666.2206-0.3053575524

385120423.81266382.035327.64060.5975593102

385120623.62300208.226004.16440.6494769655

385120823.45335474.786709.49560.6977142628

385121024.11218335.954366.7190.5111796362

385122823.74277471.55549.430.615272768

385123024.22203943.784078.87560.4815648505

385123222.9480286.919605.73820.8535551363

385123424.55164779.333295.58660.3889571187

3085121224.2206563.24131.2640.4871073389

3085121423.68288951.635779.03260.6328795347

3085121623.04437150.068743.00120.8126849284

3085123624.2207233.784144.67560.4885149348

3085123824.33189326.223786.52440.4492651501

3085124022.83502370.1310047.40260.8730781952

3085124225.29102174.762043.49520.1813980123

F10NTC35.250

F11NTC35.150

F12NTC35.120

H10QA- 5110025.3101176.852023.537Mean2179.5973333333

H11QA- 5110025.11114742.842294.8568SD140.1860959888

H12QA- 5110025.16111019.912220.3982CV6.4317428658

G10QA-Gonad25.4791005.311820.1062Mean2032.3144

G11QA-Gonad25.21107627.592152.5518SD184.3203468945

G12QA-Gonad25.23106214.262124.2852CV9.0694799434

&A

&F

Fadrozole Phase 3 ovary, Arom-A

547.6822690753547.6822690753

980.5975400569980.5975400569

641.229579791641.229579791

24 h

Fadro

Systems Biology in Small Fish Models

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