The Process of TransformationThe Process of Transformation in Toxicity Testing 

NAS Systems Biology-Informed Risk Assessment Workshop

Kim Boekelheide, M.D., Ph.D.Professor Department of Pathology & Lab Medicine Brown UniversityProfessor, Department of Pathology & Lab Medicine, Brown University Director, Superfund Research Program & Center for the Evaluation of

Environmental Impacts on Fetal DevelopmentJune 14 2012June 14, 2012

Disclosures: Funding from NIEHS, USEPA, and the American Chemistry Council. O i l t lt t f h i l d h ti l i d t kOccasional expert consultant for chemical and pharmaceutical companies, and stock owned in CytoSolv, an early stage biotechnology company developing a wound healing therapeutic.

My Charge

1) Gi i i t lk1) Give a visionary talk• Be inspiring about the new approaches

2) Define “systems biology”

3) Go beyond a nice science review• Give guidance on breaking the bonds g g

of the traditional framework

The Big PictureThe Big PictureHow Endocrinology and Toxicologygy gyhave differing perceptions of endocrine disruption and both areendocrine disruption, and both are right

Bisphenol A

Example: Bisphenol ABisphenol A is an endocrine disruptor that

Example: Bisphenol ABisphenol A is an endocrine disruptor that highlights differences between endocrinologicaland toxicological perceptions of effectand toxicological perceptions of effect– binds ERα with Kd of 10‐5 – 10‐4 Min vitro effects in the nM range– in vitro effects in the nM range

– non‐GLP studies report animal effects at ng/kgGLP st dies report animal effects at mg/kg– GLP studies report animal effects at mg/kg

– controversy over [bisphenol A] at target tissue  

Example: Bisphenol AExample: Bisphenol A

E d i l T i l

e

Endocrinology Toxicology

physiologic

spon

se

molecular

physiologicadversity?

Res

molecularadversity?

10-12 10-9 10-6 10-3

Dose

Diet and BPA effects on oogenesisDiet and BPA effects on oogenesis

“Exogenous estrogens are a serious confounding variable in rodent studies designedExogenous estrogens are a serious confounding variable in rodent studies designed to assess the effects of endocrine-disrupting chemicals, and, because estrogenic contaminants can be present in food, bedding materials, water, and caging materials, controlling for their presence is a daunting task.” Muhlhauser et al. Biol Reprod 2009.

Can we all agree on this?Can we all agree on this?

Can we all agree on this?Can we all agree on this?

Yes…..except:• My normal is different than your normal• My normal is different than your normal

– In fact, how do we define normal?

• My adverse is different from your adverse– In fact, how do we define adverse?

• And what about everything we don’t know……– Epigenetic effects are a good example of an p g g padaptive response that, when extreme, is adverse

“The conceptual creations of science becomeThe conceptual creations of science….become accepted as fact through a complex process of social consolidation These thoughtsocial consolidation. These thought products….are never finalized but can undergo transformation The older way of looking attransformation….The older way of looking at things may become incomprehensible under the new thought style and the process ofthe new thought style, and the process of transformation….may be a rapid gestalt switch or a slow process of differentiation….”or a slow process of differentiation….

From the Preface to Genesis and Development of a Scientific Fact Ludwik Fleck (1935)a Scientific Fact, Ludwik Fleck (1935)

The Ideal Toxicity Test SystemThe Ideal Toxicity Test System

Chemical X

Safe

fNot Safe

The Ideal Toxicity Test SystemThe Ideal Toxicity Test System

NO!NO!Ignores biologyg gy

LinearSimplisticSimplistic

Goal is to find one numberone number

The Ideal Toxicity Test SystemThe Ideal Toxicity Test System

NO! YES!NO!Ignores biology

YES!Biology-basedg gy

LinearSimplistic

gyInteractiveComplexSimplistic

Goal is to find one number

Complex

Probabilisticone number

Ignores Biology→ Biology‐BasedIgnores Biology → Biology Based

THE FUTURENOW

Too Linear→ Interac veToo Linear → Interac ve

Simplis c→ ComplexSimplis c → Complex

Simplicity“Simplicity often lies on the other side of complexity”p y(of ignorance)

↓ity

complexity = many interactions and detailscomplication = obscuring of ↓

Complexity↓C

ompl

exi

the detailsWith toxicity pathways (and biology), the details may at fi t h l i d↓

Simplicity

C first seem overwhelming and unconnected, but ultimately it will be possible to identify those details that matter(of knowledge)Time →

Eric Berlow, an ecologist and network theorist, in a recent TED Talk(see www.ericlberlow.net)

those details that matter

Goal Is to Find One Number → Probabilistic

“….a complete and accessible….a complete and accessible account of the theoretical foundations and computationalfoundations and computational methods that underlie plausible reasoning under uncertainty….”reasoning under uncertainty….Probabilistic Reasoning in Intelligent Systems: Networks of Plausible Inference Judea Pearl 1988

The Ideal Toxicity Test SystemThe Ideal Toxicity Test System

Chemical X

PlausiblyPlausiblySafe

PlausiblyPlausiblyNot Safe

Systems Biology  ̶  Definitionan interconnected network of eventsan interconnected network of eventspredictive of emergent properties

ligand receptortranscriptional

activationmetabolic

ti tiphysiologic 

e

ligand-receptoractivation

nuclear t

kinase

activation activation endpoint

spon

se receptor translocation

activation

Res

seconds minutes hours daysTime

NAS Report, 2007

Challenges……. 

• Assay Design/Development for Toxicity 

g

Pathways• Improved methods to identify (predict) and test 

metabolites• Co‐ordinated development of ‘functional 

genomic tools’ to map and model pathways and use results to establish safe levels of exposures

• Train toxicologists and regulators about need for new approach and then in the tools and methods that will be involved in the transformation

Toxicity Testing

Chemical Characterization

Toxicity Testing Dose‐Response Assessment

Exposure Standard

Implementation Plan for Toxicity Testing in the 21st Century

Characterization Assessment Standard

Prototype A hApproach

es

MetabolismQSARb

PlatformsAssay 

O i i i

ComputationalSystems Biology 

h d l

Safe Dose Estimation

H

hallenge Databases OptimizationAssay Integration

& DisplayHuman Genetic 

Pathway ModelsIn Vitro – to – In 

Vivo Extrapolations

Human ExposureAssessmentHuman

Biomarker

chnicalC Variability

Human SusceptibilityAdversity

p& ReverseDosimetry

DevelopmentHuman

Surveillance

Tec Adversity

MixturesEpigenetics

g Cross‐cutting

Capabilities

Validation

EducationalInitiatives

Representativetoxicitypathwayswithmoleculartargets,prototypicalactivator/agonist,andcelllinesforinvestigation

Molecular Target Activator/Agonist Human Cell Line

AHR TCDD HepG2, HepaRG

ER estradiol tamoxifen MCF-7 T47D IshikawaER estradiol, tamoxifen MCF 7, T47D, Ishikawa

CAR phenytoin, CITCO HepG2, HepaRG

PPARα WY-14643, fenofibric acid, GW7647

HepG2, HepaRG

HIF1α hypoxia, CoCl2 A549, Hep3B

NRF2 tBHQ, H2O2, AsHepG2, MDA-MB-231, H h 7

Q, 2 2, Huh-7

p53 x- or -irradiation, etoposide HepG2, A549

Systems biology‐based toxicity pathway model 

Risk EvaluationConsideration ofvarability and sensitivityIVIVE

In Vitro Based Risk Evaluation ApproachRisk Evaluationvarability and sensitivity

of subpopulationsIn vitro – in vivo extrapolation

Exposure assessmentExposure assessmentDetermination of appropriatePOD (point of departure)

Determination of appropriatePOD (point of departure)

Literature reviewLiterature reviewConcentration-responsemodeling

Concentration-responsemodeling

Structural physicochemicalreactivity characterizationStructural physicochemicalreactivity characterizationIn vitro concentration

responseIn vitro concentration

response

In vitro risk assessment

Determination ofbiokinetic behaviour

Determination ofbiokinetic behaviourIn vitro effects batteryIn vitro effects battery

Identification of appropriatetests and conditions

Identification of appropriatetests and conditions

Go beyond a nice science review“Gi id b ki th b d f th“Give guidance on breaking the bonds of the

traditional framework”

1) Screening/ranking of chemicals – determining h th h i l l lik l t bwhether chemicals are more or less likely to be

hazardous to human health2) Hazard identification – determining whether a2) Hazard identification determining whether a

compound may cause a particular type of effect3) Mode of action analysis – testing a hypothesis ) y g yp

about how a compound causes a particular effect4) Adversity – the third rail of the new toxicity

itesting

Screening/ranking of chemicals –d t i i h th h i ldetermining whether chemicals are more or less likely to be hazardous to human health

Of course this already happens based upon:public concern politics economicsQSAR TOXCASTQSAR exposure TOXCASTAmes tests animal tests production volumeetcetera…….

The BIG question: Do we create an official screening/ranking/prioritization protocol and is it distinctscreening/ranking/prioritization protocol, and is it distinct from our “real” testing protocol?

Is our screen in vitro/in silico testing?I “ l” t ti i l t ti ? I hIs our “real” testing animal testing? I hope we are beyond this idea….

Screening/ranking of chemicals –T M d lTwo Models

SScr Real Test Systemee

TestTest System

Screeningn Elements

Same conceptual and technical approachDifferent conceptual and technical approaches

Systems Biology  ̶ an interconnected network of eventsan interconnected network of eventspredictive of emergent properties

ligand receptortranscriptional

activationmetabolic

ti tiphysiologic 

e

ligand-receptoractivation

nuclear t

kinase

activation activation endpoint

spon

se receptor translocation

activation

Res

seconds minutes hours daysTime

Screening/ranking of chemicals –R f ifi d hReasons for a unified approach

• Parsimonious• Continued test system

Test System

development improves the screening process Test System

Screening• More likely that the

screen and test will Elementshave the same order

• A common system is Same conceptual and technical approach

easier to defend

Hazard identification –determining whether a compound may cause a particular type of effect

What is meant by “a particular type of effect?”Does our in vitro test system need to predict inDoes our in vitro test system need to predict in

vivo endpoints? (or just give us a probability estimate of safe or not safe)estimate of safe or not-safe)

Put another way, do we have to have models (actual or conceptual) for each tissue and its(actual or conceptual) for each tissue and its responses? Or……do we just have to be sure that the full range of potential biologicalsure that the full range of potential biological responses is covered?

•Targeted testingg g•Tissue-specific

testing

Blaauboer BJ (2003) Tox Letters

g

Development of the Toxicity Pathway Portfolio

Start Finish

AgnosticApproach

Di t d

Repro/DevNeuroI

+Directed

ApproachImmunoCancerCardiacRenalHepaticHepatic

ParallelApproach

Systems Biology  ̶ an interconnected network of eventsan interconnected network of eventspredictive of emergent properties

physiologic

For risk assessment, the physiologic endpoints of concern are cellular perturbations

For risk assessment, the physiologic endpoints of concern are cellular perturbations

For risk assessment, the physiologic endpoints of concern are cellular perturbations

For risk assessment, the physiologic endpoints of concern are cellular perturbations physiologic 

endpointperturbations.perturbations.We should regulate on these cellular endpoints.

perturbations.We should regulate on these cellular endpoints.E trapolation of these cell lar endpoints

perturbations.We should regulate on these cellular endpoints.E trapolation of these cell lar endpointsExtrapolation of these cellular endpoints to effects at the organ/tissue level is important and interesting, but is not

Extrapolation of these cellular endpoints to effects at the organ/tissue level is important and interesting, but is not necessary for risk assessment.necessary for risk assessment.We should avoid this unnecessary complication.complication.

Let’s do our risk assessment here

systemstoxicology

epidemiology& medicine

systemsrisk

t

molecularepidemiology& di iassessment & medicine

systems level d t di funderstanding of 

health and disease

Mode of action analysis –testing a hypothesis about how atesting a hypothesis about how a compound causes a particular effect

chemicalproperties

molecularevent

cellular response

tissueresponse

organismresponse

Mechanism of action

Toxicity pathway

Mode of action

Adverse outcome pathway

Adversity– the third rail of the new toxicity t titesting

The industry fear: that every little molecular blip in a cell y y pwill be considered adverse, and nothing will be considered safe

The endocrinologist’s fear: that if we ignore low dose phenomena, we are missing biologically important effects

The toxicologist’s fear: that by failing to consider the integrated physiological response (like with a whole g p y g p (animal), that we are underestimating the capacity for compensation (missing the forest for the trees)

Everyone’s fear: that what we don’t know might kill us

Autumn 1944 – Western Holland was blockaded by the Nazis, who turned away all 

Fetal Origins

shipments.  This led to a food shortage during one of Europe’s coldest winters.

The Dutch Hunger WinterThe Dutch Hunger Winter• The Dutch survived on < 500 calories per 

day (¼ of pre‐war consumption)• Food restriction during pregnancy had 

dramatic consequences for the offspring• Higher rates of obesit diabetes heartobesit diabetes heart• Higher rates of obesity, diabetes, heart obesity, diabetes, heart 

disease, higher blood pressure, disease, higher blood pressure, hypercholesterolemia, and glucose hypercholesterolemia, and glucose intoleranceintolerance

St di b T R b d ll t bli h d f th l ti hiSt di b T R b d ll t bli h d f th l ti hiStudies by Tessa Roseboom and colleagues established many of these relationships during the 1990’s when the children of the Hunger Winter were in their 50’s.

Studies by Tessa Roseboom and colleagues established many of these relationships during the 1990’s when the children of the Hunger Winter were in their 50’s.

Chemical Characterization

PhysicalProperties

ChemicalProperties Metabolites Environmental

Concentrations Uses

Toxicity Pathways

DeleteriousPathways

ProtectivePathways

ActivationIntensity

ActivationSpread Reversibility

Dose Response & Extrapolation Modeling

Pathways Pathways Intensity Spread

EffectiveConcentration

TissueDoses

PBPKModels

ResponseCharacteristics

HostSusceptibilityConcentration DosesModelsCharacteristics Susceptibility

Diet and BPA effects on oogenesisDiet and BPA effects on oogenesis

“Exogenous estrogens are a serious confounding variable in rodent studies designedExogenous estrogens are a serious confounding variable in rodent studies designed to assess the effects of endocrine-disrupting chemicals, and, because estrogenic contaminants can be present in food, bedding materials, water, and caging materials, controlling for their presence is a daunting task.” Muhlhauser et al. Biol Reprod 2009.

Estimate acceptable concentration in vitro (ug/ml)(ug/ml)

Implementation challenges…..

•It is important not to over-promise and fail to deliver, because this is a long-term effortis a long-term effort

•Experience is important and cumulative... this will take time, patience, and accepting wrong turnspatience, and accepting wrong turns

•A centralized-enough effort is very important

•More focus on pathways and commonality of responses

•Taking a ‘systems’ approach to human biology that relies on mechanisms and pathways has inherent value, contributing both to improved toxicity testing and to the fundamental molecular elucidation of human disease

Lessons Learned ̶ Human Genome Project

• Build the best teams

• Process must be science-driven

• Meet managerial challenges Meet managerial challenges

• International participation important

• Explicit milestones and quality assessment are valuablevaluable

• Technology matters

Borrowed from Chris Austin and Collins et al., Science 300:286, 2003