Mechanisms of acute toxicity and using - PowerPoint Presentation

1. Mechanisms of acute toxicity and using ToxCast data to predict acute toxicityDan Wilson, PhD, DABTScience Leader – CheminformaticsThe Dow Chemical Companyddwilson@dow.com (989)636-0712EPA’s Computational Toxicology Communities of PracticeOct 22, 2015

2. OutlineHow did we come to focus on this research?Acute toxicity regulatory landscapePotential mechanisms of high acute toxicityOur researchFuture opportunities

3. AcknowledgementsWere AOPs 1st mentioned??…Back to the Future II ?

4. Dow ChemicalTyler AuernhammerMike BartelsBarun BhhataraiGreg BondEd CarneyShubhra ChaudhuriBhaskar GollapudiAmanda ParksPaul PricePam SpencerEPA/NTP/NIEHSLinda BirnbaumWarren CaseyRichard JudsonBob KavlockNCATSMenghang XiaSimulations PlusJohn DiBellaPETA/PCRMAmy ClippingerKristie SullivanAcknowledgements

5. AcknowledgementsDow ChemicalTyler AuernhammerMike BartelsBarun BhhataraiGreg BondEd CarneyShubhra ChaudhuriBhaskar GollapudiAmanda ParksPaul PricePam SpencerEPA/NTP/NIEHSLinda BirnbaumWarren CaseyRichard JudsonBob KavlockNCATSMenghang XiaSimulations PlusJohn DiBellaPETA/PCRMAmy ClippingerKristie Sullivan

6. AcknowledgementsDow ChemicalTyler AuernhammerMike BartelsBarun BhhataraiGreg BondEd CarneyShubhra ChaudhuriBhaskar GollapudiAmanda ParksPaul PricePam SpencerEPA/NTP/NIEHSLinda BirnbaumWarren CaseyRichard JudsonBob KavlockNCATSMenghang XiaSimulations PlusJohn DiBellaPETA/PCRMAmy ClippingerKristie Sullivan

7. Toxicity Study TypesAcute ToxOral ToxicityDermal ToxicityInhalation ToxicityDermal CorrosionEye CorrosionSkin SensitizationRepeated Dose ToxRat, mouse, dog28-day, 90-day, chronicOral, dermal, inhalationGenetic ToxicityCancer BioassayLifetime study of rat/mouseDevelopmental teratologyPotential for birth defectsReproductive ToxEffects on M/F fertilityEffects on the offspringMultigenerational effectsNeuro ToxImmunologyADME (Absorption, Distribution, Metabolism, Excretion)

8. Background: Focus on acute tox2007: NAS report Toxicity Testing in 21st Century2013: Linda Birnbaum publishes EHP editorial redirecting ICCVAM to focus on HTS and computational approaches2013: ICCVAM establishes acute toxicity as 1 of 3 strategic near-term priority areas for enhanced, immediate resource investment with expectation of short-term successAcute toxicity should be a simpler and easier endpoint

9. Acute Toxicity EndpointsAcute systemic toxicityOralInhalationDermalIrritation/CorrosionSkinOcularSkin sensitization

10. Where is acute toxicity testing required?USAEPA - PesticidesFDA - Medical devicesEU-REACHNew registered substances >1 MTChinaNew registered substances >1 MTKoreaNew registered substances >1 MTTaiwanNew registered substances >1 MT

11. Acute Oral Systemic Tox GuidelinesTest Method InternationalAcceptance Acute Toxicity GuidelineDeleted Dec 2002OECD 401Up-Down Procedure2001, Revised 2008OECD TG 425Fixed Dose Procedure2002OECD TG 420 Acute Toxic Class Method2002OECD TG 423In Vitro Cytotoxicity 3T3 Cells(For choosing starting dose)2010OECD GD 129 In Vitro Cytotoxicity NHK Cells(For choosing starting dose)2010OECD GD 129

12. Acute Inhalation Tox GuidelinesTest Method InternationalAcceptance Acute Inhalation ToxicityOECD TG 403Acute Toxic Class Method2009OECD TG 436Fixed Concentration ProcedureAnticipated 2016OECD TG 433

13. Acute Dermal Systemic Tox GuidelinesTest Method InternationalAcceptance Acute Dermal ToxicityOECD TG 402

14. Acute toxicity study designsStudySpeciesSexNLengthDoseObservationsORAL TOXUP/DOWNOECD 425RatsF5: 2000 limit3: 5000 limit5 reversals in 6 rats dosed14 Days Limit: 2000 mg/kg Dose one at a time 1st dose slightly below est LD50Wait 48 hr, adjust dose depending on mortality Default doses spaced 3.2 x/ previous doseDaily clinical,Weekly BW; Gross necropsyACUTE DERMAL TOXOECD 402RatM/F5/sex14 DaysLimit: 2000 mg/kgDaily clinical,Weekly BW; Gross necropsyACUTE INHALATIONOECD 403 RatM/F5/sex14 DaysLimit: 5 mg/L for 4 hrLimit: 2 mg/L for 4 hr, USADaily clinical,Weekly BW; Gross necropsy

15. Mammalian vs. aquatic GHS bracketsGHS ClassAcute OralLD50 (mg/kg)Acute Aquatic LC50 (mg/l)I≤ 5≤ 1II5 - 501 - 10III50 - 30010 - 100IV300 - 2000>100Not ClassifiedV> 2000--

16. Oral rat GHS classes not evenly distributed

17. Regulations require to classify by LD/LC50This can be done using guideline animal studiesDo in silico alternative methods offer a replacement?Do in vitro alternative methods offer a replacement?

18. In silico approaches - QSAR

19. In Vitro ApproachesBasal cytotoxicity in 3T3 or NHK cellsSet starting dose for acute animal toxicity studiesMiss compounds acting via some mechanismsBasal cytotoxicity in differentiated neuronal cell modelsAxonopathy; Neurite outgrowth; Ach receptor signaling; Noradrenalin uptake; Cell membrane potential; Voltage-operated Ca++ channelsSmall organismsZebra fish embryoC. ElegansHigh-throughput-screening dataToxCastTox21

20. Why study acute mechanisms?May direct in vitro HTS assaysEnable building of QSARsThe ‘future’ is mechanism- (AOP-) basedBetter enable read-acrossFocus on identifying compounds of high inherent toxicityImportant in poisoning casesAcute mechanisms in scope for repeat-dose studiesUnderstand if animal data relevant to humansUnderstanding mechanisms makes us better toxicologists

21. Challenges of identifying acute MOAsUntil recently, acute toxicity not a priority for studyRisk assessors didn’t consider acute toxicity ‘sexy’ – rare focusUnderstanding MOAs not a guideline study requirementAcute studies not include organ weights, histopath or clin pathDBs of LD/LC50 values don’t contain other mechanistic infoStudies often conducted at CROs blinded to TM identitySpecific mechanisms rarely examined on a compound-basisRelationship of mechanistic effect to apical effect not clearMechanistic in vitro HTS assays may only look above cytotoxicity noise level yet the MOA may drive cytotoxicity

22. Ways to identify potential mechanismsDetermine whether ‘reactive’ or ‘pharmacologic’3-D crystalline protein structure mappingHT Gene expression data-miningIdentify protein targets using wet-lab binding interactionsExamine pathology and clinical pathology dataConsider Time-to-deathExamine relationship of acute toxicity to HTS dataRead-across to compounds with known mechanismsYears of experience resulting in a logical ‘hunch’Use systems biology approach

23. Some mechanisms of acute toxicityAntimetabolitesAnticoagulantsChelantsInhibit signal transductionIon channel blockersInhibit Na+/K+ ATPaseProtein synthesis inhibitorsInhibit energy productionNon-specific chemical reactivity

24. Attention on acute mechanismsBy oral route, most highly acutely toxic compounds are “pharmacologic”Chemically reactive compounds easier to model in silicoPharmacologic compounds harder to model by any meansTargets often unknownSubtle structural differences can be importantBy inhalation route, highly acutely toxic compounds may be pharmacologic or reactiveBy intravenous route, compounds that pass limit-dose orally can be lethal at low doses within secondsCompounds average 40x more toxic by iv than oral routeHighlights need to address bioavailability and metabolism

25. Non-specific chemical reactivityElectrophilicityHardness (HOMO/LUMO)AcylationSchiff base formationMichael acceptorsSN1 mechanismSN2 mechanismSNAr mechanismPolarizabilityMolecular weightProtein/DNA bindingSubstructure scaffoldsSolubilitypKa, pKbLog KoWSurfactant properties

26. Systems biology approach

27. Consideration of bioavailabilityPhysicalMucousChewingMixing/churningAcidEmulsificationHormonesEnzymes

28. Protein DigestionStomachHCl denaturesPepsinogen → pepsinSmall IntestineHormonesCholecystokininSecretinPancreatic enzymesTrypsin, peptidases, elastase Amino acids ↑ insulin, ↓ glucagonNo storage form for proteinamino acids → protein; carbons → carbohydrate/lipid; amino “N” as urea

29. Carbohydrate DigestionStarch: glucose polymer α(1→4) glycosidic bondsAmyloselinear, 100’s glucosesAmylopectinbranched, 1000’s unitslinear α(1→4)branch α(1→6) each 24-30 unitsGlycogenbranch each 8-12 unitsPancreatic amylase breaks α-1,4-bonds

30. Lipid DigestionStomachLingual and Gastric LipaseSmall intestineCystokinin → gallbladder↓ gastric motilitySecretin → pancreasbicarbonate neutralizes pHEmulsification → Bile saltssPancreatic Lipase → FA at C1 and C3Colipase → stabilizes LipaseCholesteryl ester hydrolasePhospholipase A2 → FA at C2Lysophospholipase → C2

31. Potential chemically labile fragments

32. GlucoseDihydroxyacetone PGlycolysis

33. Kreb’s TCACycleLactateDihydroxyacetone-PGlucoseCO2TCA CycleGluconeogenesis

34. NADPHNADPH Ribose 5-PRibulose 5-P6-P Gluconate6-P GluconolactoneXyulose 5-PSedoheptulose 7-PErythrose 4-P Glyceraldehyde 3-PKreb’s TCACycle LactateDihydroxyacetone-PGlucoseCO2UDP GlucosePentose-Phosphate Pathway

35. NADPHNADPH Ribose 5-PRibulose 5-P6-P Gluconate6-P GluconolactoneXyulose 5-PSedoheptulose 7-PErythrose 4-P Glyceraldehyde 3-PKreb’s TCACycleLactateAcetoacetateβ-HydroxybutarateMethylmalonyl CoAPropionyl Co AFatty acyl CoA (odd-number carbons)FructoseFructose 1PGlyceraldehydeDihydroxyacetone-PGlycerol-P TriacylglycerolFatty acyl CoAMalonyl CoAGlycerolFatty acidsGlucoseCO2UDP GlucoseKetonesFructoseLipids

36. NADPHNADPH Ribose 5-PRibulose 5-P6-P Gluconate6-P GluconolactoneXyulose 5-PSedoheptulose 7-PErythrose 4-P Glyceraldehyde 3-PKreb’s Urea CycleKreb’s TCACycleAsnAspAlaCysGlySerThr NH3 CO2Carbamoyl-PLactateAcetoacetateβ-HydroxybutarateMethylmalonyl CoAPropionyl Co AFatty acyl CoA (odd-number carbons)IleMetValThrHomocysteineFructoseFructose 1PGlyceraldehydeDihydroxyacetone-PGlycerol-P TriacylglycerolFatty acyl CoAMalonyl CoAUreaGlycerolFatty acidsGlucoseLeuPheTyrTrpLysCO2ArgHisProGlnGluPheTyrIleFumarateUDP GlucoseAmino Acids

37. NADPHNADPH Ribose 5-PRibulose 5-P6-P Gluconate6-P GluconolactoneXyulose 5-PSedoheptulose 7-PErythrose 4-P Glyceraldehyde 3-PKreb’s Urea CycleAsnAspAlaCysGlySerThr NH3 CO2Carbamoyl-PLactateAcetoacetateβ-HydroxybutarateMethylmalonyl CoAPropionyl Co AFatty acyl CoA (odd-number carbons)IleMetValThrHomocysteineFructoseFructose 1PGlyceraldehydeDihydroxyacetone-PGlycerol-P TriacylglycerolFatty acyl CoAMalonyl CoAUreaGlycerolFatty acidsGlucoseLeuPheTyrTrpLysCO2ArgHisProGlnGluPheTyrIleFumarateUDP GlucoseNiacinThiaminNiacinNiacinNiacinNiacinRiboflavinNiacinVit B12PyridoxineBiotinNiacinRiboflavinPyridoxineNiacinRiboflavinNiacinThiaminNiacinLipoic AcidThiaminNiacinLipoic AcidRiboflavinBiotinVit B12PyridoxineBiotinVitamins

38. NADPHNADPH Ribose 5-PRibulose 5-P6-P Gluconate6-P GluconolactoneXyulose 5-PSedoheptulose 7-PErythrose 4-P Glyceraldehyde 3-PKreb’s Urea CycleAsnAspAlaCysGlySerThr NH3 CO2Carbamoyl-PLactateAcetoacetateβ-HydroxybutarateMethylmalonyl CoAPropionyl Co AFatty acyl CoA (odd-number carbons)IleMetValThrHomocysteineFructoseFructose 1PGlyceraldehydeDihydroxyacetone-PGlycerol-P TriacylglycerolFatty acyl CoAMalonyl CoAUreaGlycerolFatty acidsGlucoseLeuPheTyrTrpLysCO2ArgHisProGlnGluPheTyrIleFumarateUDP GlucoseNiacinThiaminNiacinNiacinNiacinNiacinRiboflavinNiacinVit B12PyridoxineBiotinNiacinRiboflavinPyridoxineNiacinRiboflavinNiacinThiaminNiacinLipoic AcidThiaminNiacinLipoic AcidRiboflavinBiotinVit B12PyridoxineBiotinMitochondrialElectron Transport

39. Pyruvate Dehydrogenase ComplexMitochondriaArsenicinhibits

40. Lipoic Acid: Cofactor for 2nd EnzymeDihydrolipoyl transacetylaseArsenicinhibits

41. Niacin – Vitamin B3

42. Riboflavin – Vit B2FMN and FAD tightly bound to enzymes that catalyze oxidation or reduction

43. Purine synthesisFOLIC ACID ANALOGSMethotrexate etc inhibit reduction of dihydrofolate to THF; ↓ DNA replication in cancer and normal cellsPurines built on existing ribose sugar supplied by Pentose Phosphate Path

44. Pyrimidine BiosynthesisBase synthesized then added to preformed ribose

45. AntimetabolitesTetrahydrofolate (THF)N10-Formyl-THFN5N10-Methenyl-THFN5N10-Methylene-THFN5-Methyl-THFNADPH + H+NADP+NADH + H+NAD+MethionineTMPPurinesDihydrofolate reductase2-steps requiring 2 NADPHMethotrexateLD50 135 mg/kg

46. AnticoagulantsCofactor of enzyme that carboxylates γ-glutamyls in Prothrombin and Factors VII, IX and XWithout carboxylation, don’t bind membrane phospholipidsDeficiency in infants - hemorrhagic disease of the newbornWarfarin is structural analog of Vit KLD50 8.7 mg/kg

47. Chelators104mg/kg rat iv280 ug/kg rat iv

48. Signal transduction

49. TetrodotoxinInhibits voltage-gated sodium channelsOral LD50 334ug/kg

50. Cardiac glycosides (Inh Na+/K+ ATPase) 10.8 mg/kg rat LD50 iv28.3 mg/kg rat LD50 oral

51. Michael acceptorsAcroleinLD50 26 mg/kgMethyl-methacrylateLD50 7872 mg/kg

52. AcidsTrifluoromethanesulfonic acidpH of 10% solution = 0.1Acute oral LD50: 1605.3 mg/kg bwGHS Cat 4; H302: Harmful if swallowedAcute dermal LD50: > 50 mg/kg bwtest results inconclusive because of severe local effects on skin at 2000 mg/kg bwAcute inhalation LC50: ????study scientifically unjustified

53. Our Acute Toxicity ResearchCatalogue and assess alternative modelsIn VivoIn VitroIn SilicoCompile toxicity databasesAcute rat oral, intravenous, inhalationAcute daphnia, fishIn vitro high-throughput screening dataEye and skin irritation/corrosionDerive plausible AOPs/mechanismsAddress bioavailability and metabolismSystems Biology approach to read-across

54. Alternative approaches to acute mammalian toxicity.Use of ToxCast™ mitochondrial inhibition data.PROOF-OF-CONCEPT

55. Current HypothesisMechanisms of acute toxicity likely conserved across invertebrate, aquatic and mammalian speciesSuggests dose-response concordance would be high and in vitro mechanistic data could predict responses in multiple species under conditions of similar bioavailability

56. Fish acute toxicity vs. ToxCast HTS

57. Produces 90% of cell’s energyCell uses 10 million ATP molecules/sec and recycles every 30 secKnown mechanism for acute lethalityFocused on mitochondria

58. Mitochondrial Electron Transport I II III Intermembrane spaceMatrixATP SynthaseH+TCACycleSuccinateFADH2QH+H+ III IVH2OO2ADPATPH+NADH H+H+ H+

59. Mitochondria membrane potential assay Sakamuru S et al. Physiol. Genomics 2012;44:495-503

60. Data AcquisitionRat Oral and Intravenous LD50 DataPublic literatureEuropean Chemicals Agency (ECHA)e-ChemPortalChemID PlusFish 96h LC50 and daphnia 48h LC50 dataEU Centre for Ecotox and Tox of Chemicals (ECETOC)Aquatic JapanUS-EPA ecotoxicology DB from OECD QSAR Toolbox ECHAToxCast Mitochondrial toxicity dataUS-EPA ToxCast-II data download (v 12/13/2013)Name: Tox21 Mitochondrial Toxicity RatioAssessed in HepG2 cells using Mito-MPS (BD Biosciences)AC50 (in µM) was provided

61. Amount of DataRat Oral LD5013,156Rat Intravenous LD50931Fish 96h LC502605Daphnia 48h LC502406ToxCast Mitochondrial toxicity1800

62. Mitochondrial toxicity predicts upper boundary to acute fish toxicityAC50 Inhibition of Mitochondrial Membrane Potential (Fish acute LC50 (mg/l)AC50 Inhibition of Mitochondrial Membrane Potential (uM) AC50 Inhibition of Mitochondrial Membrane Potential (

63. AC50 Inhibition of Mitochondrial Membrane Potential (Daphnia acute LC50 (mg/l)AC50 Inhibition of Mitochondrial Membrane Potential (uM) AC50 Inhibition of Mitochondrial Membrane Potential (Mitochondrial toxicity predicts upper boundary to Daphnia toxicity

64. Mitochondrial toxicity predicts upper boundary to acute rat intravenous toxicityMethylene BlueAC50 Inhibition of Mitochondrial Membrane Potential (uM) Rat Intravenous LD50 (mg/kg)AC50 Inhibition of Mitochondrial Membrane Potential (AC50 Inhibition of Mitochondrial Membrane Potential (

65. Mitochondrial toxicity does not predict upper boundary to acute oral rat toxicityAC50 Inhibition of Mitochondrial Membrane Potential (uM) Rat Oral LD50 (mg/kg)AC50 Inhibition of Mitochondrial Membrane Potential (AC50 Inhibition of Mitochondrial Membrane Potential (

66. Simulation of BioavailabilityGastroPlus (v8.5, Simulations Plus Inc, Lancaster, CA, USA) Predicted fraction (F) of chemical absorbed from GI tractOne compartment PK model set; single oral 300 mg/kg dose/250gm ratIncluded CYP P450 2C, 2D, 3A rat liver metabolismIdentified potential UGT substratesPrediction of metabolic clearance (ADMET Predictor, v7.0 Simulations Plus)

67. Rat oral LD50 values adjusted downward by % systemic bioavailabilityAC50 Inhibition of Mitochondrial Membrane Potential (uM) Rat Oral LD50 (mg/kg)AC50 Inhibition of Mitochondrial Membrane Potential (AC50 Inhibition of Mitochondrial Membrane Potential (

68. Rat oral LD50 adjusted down by % F, removal of esters amides ester groupAC50 Inhibition of Mitochondrial Membrane Potential (uM) Rat Oral LD50 (mg/kg)AC50 Inhibition of Mitochondrial Membrane Potential (AC50 Inhibition of Mitochondrial Membrane Potential (

69. Rat oral LD50 further adjusted by removal of substrates for UGTs Rat Oral LD50 (mg/kg)AC50 Inhibition of Mitochondrial Membrane Potential (uM) AC50 Inhibition of Mitochondrial Membrane Potential (

70. ConclusionsHTS assays of mitochondrial toxicity can be used to predict high acute systemic toxicity in multiple speciesPredictions of oral toxicity from HTS assays routes would often be confounded by chemical-specific differences in uptake and metabolismWith proper in silico estimations of absorption and first pass metabolism, HTS assays can eventually be used to predict acute oral toxicity in mammalsThere is a need for in silico models that address hydrolysisOther mechanistic endpoints need to be covered in vitro

71. Future opportunities: acute inhalation

72. Thank You!Questions?