Using Population-based Mouse Models to Explore Individual Variability and Toxicity

John E. (Jef) French, Ph.D.Host Susceptibility Group

N ti l T i l P NIEHSNational Toxicology Program, NIEHSResearch Triangle Park, NC 27709 USA

(french@niehs.nih.gov)A il 18 2012April 18, 2012

Hazard Identification & CharacterizationE id i l• Epidemiology– Exposure assessment– variability between intensity (peak

exposure), inter- and intra-individual exposures and lti l t i t it d i di itmultiple exposure routes; intensity and periodicity

– Disease endpoints are categorical– Population differences based upon individual variability

through candidate gene and genome wide association analysis (CGAS and GWAS)

• Animal Models of Human Disease– Historical – Use of 1 or 2 inbred strains or outbred stocks

to test specific agents or mixtures representing occupational exposures

– Routes and lengths of exposure (partial or lifetime)– Mechanism of action studies for causality (support Hill’s

postulates)p )• Type 1 and Type 2 errors based upon exposure

misclassification and population architecture

Mouse models for human diseases•Most diseases are heritable quantitative or continuous traits

Mouse models for human diseasesq

•Use population-based models to identify highly variable genotypes-phenotypes associated with a toxicity or disease phenotype ( i t & tibilit )(resistance & susceptibility)

•Functionally validate SNP/SV associations for causal relationships using reverse genetics and in vitro/in vivo targeted testing methodsg g g g

•Anchor extrapolation between species for exposure and disease risk to highly conserved orthologous loci and networks or path a s shared bet een the animal model and h manspathways shared between the animal model and humans

Benzene Metabolism and Toxicity

Rappaport et al. EHP 117, 946 (2009)

P l ti B d M d l f I b d M St i

• Research community resource

Population Based Model of Inbred Mouse Strains

• Proposed for population based model for environmental exposures, toxicity, and diseaseB d NTP/NIEHS P l DNA• Based upon NTP/NIEHS-Perlegen DNA sequencing of 15 inbred strains for comparison to the mouse reference strain – C57BL/6J. B6C3F1 and PWK/LacJ) were included for additional comparisons –

• Total 18 inbred strains• Total 18 inbred strains• Major impact on understanding the mouse

genomeg

Rank order blood [14C] benzene equivalents in males following a single oral exposure (100 g/kg; n=5; μ SE)

B6C3F1DBA/2J

MOLF/EiJNZW/LacJ

A/JBALB/cByJ

BTBR T+tf/JNOD/ShiLtJ

B6C3F1

C3H/HeJAKR/JKK/HlJ

A/J

CAST/EiJPWK/PhJ

129S1/SvImJC57BL/6J

• 10X difference AUC• B6C3F1 > B6 or C3• PWD/PhJ ≤ CAST/EiJ ≤ PWK/PhJ

FVB/NJWSB/EiJ

PWD/PhJPWD/PhJ ≤ CAST/EiJ ≤ PWK/PhJ

0 50000 100000 150000 200000 250000 300000

Blood AUC Males (min*µmol-Eq/ml)

Blood

CopaSox2;Atp11b Rybp

Wdr17Plod2

Bcap29;Tmx1

GmdsEdnrb

Best1

Cdh2;Dsc3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Rank order blood [14C] benzene equivalents CL_F in males following a single oral exposure (100 g/kg; n=5; μ SE)

BTBR T+ tf/JCAST/EiJ

FVB/NJPWD/PhJ

129S1/S I JKK/HlJAKR/J

C57BL/6JBTBR T+ tf/J

•10X differencesBALB/ B J

C3H/HeJNOD/ShiLtJ

WSB/EiJ129S1/SvImJ

•B6C3F1 < B6 or C3•PWK/PhJ < PWD/PhJ & CAST/EiJ

CDBA/2J

PWK/EiJA/J

BALB/cByJ

PWK/PhJ

0 00 0 50 1 00 1 50

NZW/LacJMOLF/EiJ

B6C3F1

0.00 0.50 1.00 1.50Male Blood CL_F ml/min

Blood

Elf2

Gstm6

Xpa(Mitf;Gm765)

(Otog;Myod1)Tmem45b

Entpd1Slc16a4

Nell1

Tlr5Gstm6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 171 8 19 20

Blood AUC-CL_F Correlation R2 = 0.59 and p = 0.0003

P di t d Y [ l/ i ]

87000

97000

107000

ol/m

l)

Predicted Y [ml/min]

47000

57000

67000

77000

(min

*nm

17000

27000

37000

0.1 0.3 0.5 0.7 0.9 1.1

AU

C

Blood CL F (ml/min)_ ( )

68000

78000

88000

mol

/ml

Predicted Y [Systolic]

R2 = 0.55p = 0.004

18000

28000

38000

48000

58000

UC

min

*nm p 0.004

84 94 104 114 124 134 144 154 164AU

Systolic BP

Predicted Y [Systolic]

0.8

0.9

1

1.1

1.2

ml/m

inPredicted Y [Systolic]

R2 = 0.16p = 0.16

0.3

0.4

0.5

0.6

0.7

CL_

F m

0.284 104 124 144 164

Systolic BP

Rank order bone marrow AUC [14C] benzene equivalents in males following a single oral exposure (100 g/kg; n = 5; μ SE)

B6C3F1MOLF/EiJPWK/EiJ

following a single oral exposure (100 g/kg; n 5; μ SE)

NOD/ShiLtJPWD/PhJ

KK/HlJNZW/LacJ

B6C3F1

AKR/JDBA/2J

BALB/cByJ129S1/SvImJNOD/ShiLtJ

• 19 fold difference• B6C3F1 > B6 or C3

WSB/EiJA/J

CAST/EiJC57BL/6J

AKR/J • B6C3F1 > B6 or C3• CAST/EiJ - low AUC but > WSB or C3

0 2 4 6 8 10

FVB/NJBTBR T+ tf/J

C3H/HeJWSB/EiJ

0 2 4 6 8 10Male Bone Marrow AUC (min*micromoles/mg protein x103)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 8 19 20

R k d b CL F [14C] b i l t l

FVB/NJBTBR_T+_tf/J

Rank order bone marrow CL_F [14C] benzene equivalents clearance rate in males following a single oral exposure (100 g/kg; n=5; μSE)

C57BL/6JA/J

AKR/JC3H/HeJFVB/NJ

CAST/EiJNOD/LtJ

BALB/cByJDBA/2J

C57BL/6J

• 36 fold differences

WSB/EiJPWD/PhJ

KK/HlJ129S1/SvImJ

CAST/EiJ • 36 fold differences• B6C3F1 > B6 or C3• CAST intermediate CL_F phenotype

PWK/PhJMOLF/EiJ

B6C3F1NZW/LacJ

WSB/EiJ

0 10 20 30 40 50 60 70 80 90Male Bone Marrow CL_F (mg protein/min)

All i t i d 5’ t did t

C3: Ints12C15: Tbc1d22a

All intergenic and 5’ to candidate

C9: Vps13c

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 8 19 20

Conclusions• ADME/TK studies in a limited panel of 18 inbred strains

demonstrated a 10-36X difference across strains for AUC and CL Fdemonstrated a 10 36X difference across strains for AUC and CL_F in blood and bone marrow

• Model lacked power to achieve genome wide significance and minimize potential false positives candidate genes using haplotype association mapping tools

• The phenome panels of inbred strains of mice (35-40 strains) are difficult to use for genetic/epigenetic analysis due to lack of genetic power and efficiency required to load studies due to breeding andpower and efficiency required to load studies due to breeding and reproductive differences

• Laboratory derived inbred strains are largely identical by descent with limited linkage disequilibrium (LD) networks with pervasive high inter-limited linkage disequilibrium (LD) networks with pervasive high interchromosomal linkage, leading to low accuracy in genetic analysis

• Consequently, new models are required for genetic analysis in order to to demonstrate population based differences for individual variability

The J:DO diversity outbred mouse • A population based model for environmental

exposures, toxicity, and disease• Complex traits community resource• Complex traits community resource• DO mice derived from the Collaborative Cross

(CC) advanced recombinant intercross lines( )

The Collaborative Cross*

Strain Letter Color

The Collaborative Cross(8-way advanced recombinant inbred lines)

Strain Letter ColorA/J A

C57BL/6J B129S1/SvImJ CNOD/ShiLtJ D

NZO/LtJ ECAST/EiJ FPWK/PhJ GWSB/EiJ HWSB/EiJ H

*See suggested references provided for supplementary reading

Collaborative Cross (CC) & Diversity Outbred (J:DO) Models≈45 million segregating SNPs

J:DO mice(CC G4 G5)(CC G4:G5)

4-8 Mb

≥10% minor allele frequency

Benzene 28 Day Inhalation Study at NIEHS/NTP

• 0 1 10 or 100 ppm benzene exposure groups• 0, 1, 10 or 100 ppm benzene exposure groups• 28 days - 6 hr TWA daily at target for each exposure• Diversity outbred (J:DO) male mice (7 & 8th randomlyDiversity outbred (J:DO) male mice (7 & 8 randomly

outbred generation; selected from 175 breeding pairs)• Randomly assigned to exposure group by weight• 75 mice/exposure group × 4 exposure concentrations• 300 male mice /study (600 total with 2 cohorts)

NIH31 di t h d i• NIH31 diet; housed on wire • NTP Specifications (with cited exceptions)

Peripheral Blood %MN-RET

Each mouse is genetically different…Peripheral Blood %MN-RET

DNA Damage

Significant

DNADNA DamageMinimal

Benzene, 100 ppm – Bone Marrow %MN-RET Chr 10 QTL

Whole Genome Plot Chr 10:27-35Mb

QTL segment

129S1/SvlmJ

Coefficient for CAST/EiJ

LOD

allele identity

LOD

Benzene, 100 ppm – Bone Marrow %MN-RET Chr 10 QTLChr 10:27-35Mb

14 43

Chr 10:27 35Mb

67

7

3

5

5

3

Susceptible

7

4

1

66

2 3 5 2 2Susceptible

10 12 26 17

7

Resistant

Resistant SusceptibleGenotypeGenotype

Benzene, 100 ppm – Bone Marrow %MN-RET Chr 10 QTL

Benzene, 100 ppm – Bone Marrow %MN-RET

Chr 2:136-144MB

QTL segment

C ff fCoefficient for allele identity

LOD

Benzene, 100 ppm – Bone Marrow %MN-RET Chr 2 QTL

Chr 2:136 -144 Mb

Benzene 100 ppm – Bone Marrow %MN-RETChr 10:27-35Mb Chr 2:136 -144 Mb

MGI Data set 11:UNC MUGA

Nkain2

Data set 11:UNC-MUGA

Tasp1_RPTPk_Flrt3_bCat_FGFR1_BZ100ppm_QTLsp53_Trdn_Tasp1_cRaf_AKT1_BZ100ppm QTLs

p = 2.2 x 10-07

Nkain2p = 7.1 x 10-05

Nkain2Sult3aMacrod2

p = 2.45 x 10-07

CyclinD1_C20orf7_ABTAP_UBL7_BZ_100ppm QTLs

Conclusions• Population based models required to determine inter• Population based models required to determine inter-

individual variability in response to toxic exposures (J:DO)• Benzene – ADME/TK (heritable quantitative trait)• Benzene – DNA damage (heritable quantitative trait)• Benzene – BMD/RfC and uncertainty factors/defaults• Discovery statistical analysis of genotype phenotype• Discovery - statistical analysis of genotype-phenotype

relationships and candidates for causal mechanisms• Validation – hypothesis based research for confirmation

f l l ti hi i CC AIL i i d i itof causal relationships using CC AILs in vivo and in vitro reverse genetics

AcknowledgementsUniversity of Arizona (ADME)• Gabe Knudsen - ADME

NTP/NIEHS (ADME & Toxicity)• Dan Morgan – Inhalation toxicology• Gabe Knudsen - ADME

• Glenn Sipes - ADME• Bob Kuester – ADME

• Dan Morgan – Inhalation toxicology• Grace Kissling - Biostatistics• Debra King - Hematology

ILS-Inc. (Genetic Toxicology)• Kim Shepard• Cheryl Hobbs

• Kristine Witt – Genetic Toxicology• Keith Shockley – Bioinformatics• Mike Cunningham – ADME

• Les RecioThe Jackson Laboratory (Genetics)• Dan Gatti (Systems Genetics)

Alion ( Animal Care & Exposures)• Herman Price• Bob O’Connor ( y )

• Gary Churchill (Systems Genetics)UNC-CH (Genetics)• David Threadgill (NCSU)

• Bob O Connor• Norm Gage

David Threadgill (NCSU)• Fernando Pardo-Manuel de Villena

Questions/DiscussionQuestions/Discussion