Traditional Testing Strategies for Traditional Testing Strategies for Detecting Later Life Effects Detecting Later Life Effects

Following Early Life Following Early Life Stressors: Animal ModelsStressors: Animal Models

John M. RogersJohn M. Rogers

United States Environmental United States Environmental Protection AgencyProtection Agency

NAS 10-14-10

OutlineOutline

In vivo regulatory tests for developmental toxicity

Study designs for detecting DOHaD effects

Animal models of IUGR

Effects and mechanisms: Blood pressure

Effects and mechanisms: Glucose intolerance/insulin resistance

Effects on body composition

Transgenerational effects

Conclusions

Animal Tests for Developmental ToxicityAnimal Tests for Developmental Toxicity

Prenatal developmental toxicityPrenatal developmental toxicity

TwoTwo--generation reproduction studygeneration reproduction study

Developmental neurotoxicityDevelopmental neurotoxicity

Developmental immunotoxicityDevelopmental immunotoxicity

Extended oneExtended one--generation study generation study

NTP toxicity and carcinogenicity studyNTP toxicity and carcinogenicity study

Lifetime exposure beginning GD6 (rats)Lifetime exposure beginning GD6 (rats)

Prenatal Developmental Toxicity StudyPrenatal Developmental Toxicity Study

Fetal viability, weight, structure

Two Generation Reproduction StudyTwo Generation Reproduction Study

Survival, growth, puberty, reproduction, necropsy

Developmental Neurotoxicity StudyDevelopmental Neurotoxicity Study

Developmental Immunotoxicity StudyDevelopmental Immunotoxicity Study

P♂

&

♀

dosing Selected subsets F1♂

& ♀

dosing

Pre X: 4W

Pre X: 2W

X: 2WPost X: up to 6W

Gestation Lactation

Post wean: up to PND 70Set 1 : clinical path/ neurotox

Post wean: up to PND 70Set 2 : estrous cycles / immunotox

Post wean: up to PND 70Set 3 :

TK /

triggered reprotox - extend to F2

Surplus F1 pup necropsy

P♂

& P♀necropsyP♂

P♀

LifeLife--Stages F1Stages F1--’’extendedextended’’

: 1: 1--Gen StudyGen Studymodified from Cooper et al., Critical Reviews in Toxicology, 2006

Other Endpoints?-Blood pressure-Glucose/insulin-Body composition-GR

DOHaD Animal Study DesignsDOHaD Animal Study Designs

Stages of development

“Critical Periods”

not well defined

Preimplantation to postnatal “exposures”

Treatments

Under-, over-, mal-nutrition, dexamethasone, toxicants, maternal stress, obesity, diabetes

Apical endpoints examined

Blood pressure, glucose tolerance, obesity, cancer, behavior

Underlying mechanistic endpoints

Species

Rodents, sheep, nonhuman primates, pigs

Animal Models of IUGRAnimal Models of IUGR

Caloric restrictionCaloric restriction

3030--50% of 50% of ad libitumad libitum food intake (rats)food intake (rats)

Protein restrictionProtein restriction

88--9% protein vs 189% protein vs 18--20% in controls20% in controls

Southampton dietSouthampton diet

Micronutrient deficiency (e.g. , Fe, Zn)Micronutrient deficiency (e.g. , Fe, Zn)

Glucocorticoid (dexamethasone) exposureGlucocorticoid (dexamethasone) exposure

Uterine artery ligation/placental insufficiencyUterine artery ligation/placental insufficiency

Pig Pig ––

natural variationnatural variation

Crowded Uterine Horn mouse modelCrowded Uterine Horn mouse model

Consistent findings across species and methods

Maternalpheno-, geno-, epigeno-type

In Utero Environment

Nutrition(over-, under-, mal-)

Toxicants Stress

Postnatal Environment

placenta

Offspring pheno-, epigeno-type

NutritionNurture

ToxicantsStress

Disease

hypertension, insulin resistance, diabetes, obesity, behavior

obesity diabetes

“Predictive Adaptive

Responses”

Blood Pressure TestingBlood Pressure Testing Tail Cuff or TelemetryTail Cuff or Telemetry

Armitage et al, 2004

Offspring Blood PressureOffspring Blood Pressure Dietary ModelsDietary Models

Systolic BP at 11 Weeks Systolic BP at 11 Weeks Maternal LPD on GD 0Maternal LPD on GD 0--4.254.25

100

110

120

130

140

150

Male Female

18% Casein9% Casein*

ratsratsKwong et al. Development 127:4195 ( 2000)

Preconceptional LPD and Offspring BP

Watkins et al. J Physiol 586:2231 (2008)MF-1 Mice

Systolic Blood PressureSystolic Blood Pressure Toxicants, Toxicants, Block IBlock I

week 7 week 52

mm

Hg

0

105

120

135

150

165

180Control DexPFOSAtrazine *

** * * *

Rat

Systolic Blood PressureSystolic Blood Pressure Toxicants, Toxicants, Block IIBlock II

week 10 week 270

105

120

135

150

165

180Control PFNAArsenic Nicotine **

* *

Rat

# of Nephrons at 14 Weeks# of Nephrons at 14 Weeks Low Protein Diet GD 0Low Protein Diet GD 0--4.54.5

6000

7000

8000

9000

10000

11000

12000

13000

14000

Male Female

ADUN

**

Kwong et al. Development 127:4195 ( 2000)ratsrats

Nephron EndowmentNephron Endowment Whole Kidney EstimateWhole Kidney Estimate

0

7000

8000

9000

10000

11000

12000

Control Dex. PFOS PFNA Atrazine Arsenic Nicotine

** * *

Toxicants

Rat

Kidney Glucocorticoid Receptor Kidney Glucocorticoid Receptor Expression Expression --

NewbornNewborn

0

5000

10000

15000

20000

25000Control Dex. PFOS PFNA Atrazine Arsenic Nicotine

*

**

Toxicants

Rat

Augustyniak, R. A. et al. Am J Physiol Regul Integr Comp Physiol

298: R1375 (2010)

Baseline BP Baseline BP ––

Effects of Low ProteinEffects of Low Protein By TelemetryBy Telemetry

Rats

Augustyniak, R. A. et al. Am J Physiol Regul Integr Comp Physiol

298: R1375 (2010)

Stressed BP Stressed BP ––

Effects of Low ProteinEffects of Low Protein By TelemetryBy Telemetry

1 Hour

Rats

O’Regan and Seckl, J Endocrinol. 196:343 (2008)

Vascular Response to Pressors Vascular Response to Pressors Effects of DEXEffects of DEX

Isolated mesenteric vasculature

Rats

Torrens C et al. Exp Physiol 2009;94:1024-1033

Arterial Response to VasodilatorsArterial Response to Vasodilators (sheep)(sheep)

Acetylcholine Isoprenaline Na nitroprusside

Sheep pre/periconceptual LPD

Mechanisms of Programmed Mechanisms of Programmed HypertensionHypertension

Reduced nephron endowmentReduced nephron endowment

→→ sclerosissclerosis

Increased glucocorticoid responsivenessIncreased glucocorticoid responsiveness

↑↑

GR, GR, ↓↓

1111ββ--HSD2 in kidneyHSD2 in kidney

Increased vascular reactivityIncreased vascular reactivity

Decreased reninDecreased renin--angiotensin systemangiotensin system

↑↑

BW (=BW (=↑↑BW/nephron)BW/nephron)

IUGR and Glucose ToleranceIUGR and Glucose Tolerance

Simmons et al., Diabetes 50:2279 (2001)Rat -

Uterine artery ligation

15 weeks of age

Glucose Tolerance Glucose Tolerance ––

Effects of DEXEffects of DEX (non(non--human primate)human primate)

De Vries et al, J Clin Invest 117:1058 (2007)

Offspring at 8 months of age

Chlorocebus aethiops(African vervet)

Armitage et al, 2004

Offspring Insulin/GlucoseOffspring Insulin/Glucose Dietary ModelsDietary Models

IUGR and Pancreas IUGR and Pancreas ββ--Cell MassCell Mass

Simmons et al., Diabetes 50:2279 (2001)Rat -

Uterine artery ligation

Effects of DEX on the PancreasEffects of DEX on the Pancreas

Nonhuman primate De Vries et al, J Clin Invest 117:1058 (2007)

Oral Glucose Tolerance Testing Oral Glucose Tolerance Testing –– Week 10 Insulin ResponseWeek 10 Insulin Response

Time (min.)

0 30 60 90 120

Insu

lin (

g/L)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5Control UN1-15 UN10-21 Control High Fat UN1-15 High Fat UN10-21 High Fat

* = Postnatal high fat diet increased significantly from normal diet postnatal.

**

*

Oral Glucose Tolerance TestOral Glucose Tolerance Test Week 10 Insulin AUCWeek 10 Insulin AUC

Insu

lin A

UC

0

50

100

150

200

250

300

Control Diet High Fat Diet

a

b

Mechanisms of Glucose IntoleranceMechanisms of Glucose Intolerance

PancreasPancreas

↓↓

ββ--cell proliferationcell proliferation

↑↑

ββ--cell apoptosiscell apoptosis

↓↓

Insulin secretionInsulin secretion

MuscleMuscle

↓↓

Muscle massMuscle mass

↓↓

Insulin sensitivityInsulin sensitivity

Adipose tissueAdipose tissue

↓↓

InsulinInsulin--stimulated glucose uptakestimulated glucose uptake

LiverLiver

↑↑

GluconeogenesisGluconeogenesis

Effects of Dex on Cell SignalingEffects of Dex on Cell Signaling Adenylyl Cyclase ActivityAdenylyl Cyclase Activity

Adigun et al, Neurotox Teratol 32:193 (2010)

Heart Liver

Doses below BW effects

Organophosphates, Obesity and Organophosphates, Obesity and DiabetesDiabetes

Slotkin, TA, Reprod Toxicol in press (2010)

Body Composition ApparatusBody Composition Apparatus

Gestational PFOA and Body Wt 20-29 Weeks-

Females

35

37

39

41

43

45

47

49B

ody

Wei

ght

(g)

0 0.01 0.1 0.3 1

PFOA Dose (mg/kg/d)

* **

Hines et al., Mol Cell Endocrinol 304:97 (2009)CD-1 Mouse

Females

mg/kg/day PFOA

0 0.01 0.1 0.3 1 5

Bod

y W

eigh

t (gr

ams)

0

20

25

30

35

Males

0 0.01 0.1 0.3 1 50

20

25

30

35

40

45

PFOA PFOA --

Body Weight at 12 WeeksBody Weight at 12 Weeks

N=5 N=11 N=6 N=10 N=11 N=9 N=5 N=10 N=6 N=10 N=12 N=8

PFOA PFOA --

% Body Fat at 12 Weeks% Body Fat at 12 Weeks

Females

mg/kg/day PFOA

0 0.01 0.1 0.3 1 5

% B

ody

Fat

0

6

8

10

12

14

16

18

Males

0 0.01 0.1 0.3 1 50

6

8

10

12

14

16

N=5 N=11 N=6 N=10 N=11 N=9 N=5 N=10 N=6 N=10 N=12 N=8

Maternalpheno-, geno-, epigeno-type

In Utero Environment

Nutrition(over-, under-, mal-)

Toxicants Stress

Postnatal Environment

placenta

Offspring pheno-, epigeno-type

NutritionNurture

ToxicantsStress

Disease

hypertension, insulin resistance, diabetes, obesity, behavior

obesity diabetes

Maternalpheno-, geno-, epigeno-type

In Utero Environment

Nutrition(over-, under-, mal-)

Toxicants Stress

Postnatal Environment

placenta

Offspring pheno-, epigeno-type

NutritionNurture

ToxicantsStress

Disease

hypertension, insulin resistance, diabetes, obesity, behavior

obesity diabetes

↑

Maternal corticosteroid ↓

11-β-HSD 2

early tissuematuration↓tissue mass

Altered physiology, metabolism

↑tissue GR

Altered Offspring HPA AxisAltered Offspring HPA Axis

Hippocampus

CRH

ACTH

CORTISOL

GR

GR GR

GR

Adrenal

Pituitary

Hypothalamus

Hippocampus

↑CRH

↑ACTH

↑CORTISOL

GR

NORMAL DEX, UN, LPD

Liver, Kidney:↑GR↓11βHSD2

Plasma Cortisol* Plasma Cortisol* ––

Effects of DEXEffects of DEX nonnon--human primatehuman primate

Offspring at 8 months of age

*Following brief restraint stressDe Vries et al, J Clin Invest 117:1058 (2007)

Youngson and Whitelaw Annu. Rev. Genomics Hum. Genet. 9:233, 2008

Transgenerational EffectsTransgenerational Effects

Exposing a pregnantfemale potentially exposes

three generations

Effects on F1 physiology can affect F2

Torrens, Poston and Hanson, 2008

Transmission of Raised Blood Transmission of Raised Blood Pressure to the FPressure to the F

2 2 GenerationGeneration

Phenylephrine response

PRCon

Copyright ©2006 The Endocrine SocietyAnway, M. D. et al. Endocrinology147:5515 (2006)

Intergenerational Effect of VinclozolinIntergenerational Effect of Vinclozolin

McMullen and Mostyn, 2009

Agents, Timing and MechanismsAgents, Timing and MechanismsOthertoxicants

ConclusionsConclusions

Consistent results across species and strains Consistent results across species and strains for effects on glucose/insulin, blood for effects on glucose/insulin, blood pressure, HPA activity.pressure, HPA activity.

Need to challenge the system, Need to challenge the system, e.ge.g., glucose ., glucose challenge, stress, high fat diet (mismatch)challenge, stress, high fat diet (mismatch)

HPA and glucocorticoid signaling pathway HPA and glucocorticoid signaling pathway may represent common mode of actionmay represent common mode of action

Most effects evident early in lifeMost effects evident early in life

Tissue GR as early marker?Tissue GR as early marker?

AcknowledgmentsAcknowledgments