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