HumanEPI

Data

Where the Question of

Health Risk is Raised

Res

pons

e

Log DosePaustenbach (1995)

AnimalData

General Approaches To Risk Assessment

• Qualitative approach using scientific judgment

• Quantitative approach using safety factors

• Quantitative approach using mathematical models

• Quantitative approach using linear extrapolation

What Drives Most Types of Environmental Health

Risk Assessment?

• There are many types of risk assessment – Cancer– Developmental toxicity– Neurotoxicity

Cancer Risk Assessment

• Population risks for environmental carcinogens are usually set at one additional cancer per 100,000 or 1,000,000 individuals

• Occupational risks are frequently much higher, with one additional cancer per 1,000 workers being not uncommon

Hazard Identification

• A qualitative risk assessment

• Does an agent have the potential to increase the incidence of cancer under any conditions

Dose-Response Assessment

• The relationship between dose and response (cancer incidence)

• Two sets of data are usually available– Data in the observable range– Extrapolation to responses below the

observable range

Exposure Assessment

• EPA uses the cumulative dose received over a lifetime

• This is expressed as the average daily exposure

• Occupational exposures are usually based on exposure during the work week

Risk Characterization

• Provides an overall conclusion and confidence of risk for the risk manager

• Gives the assumptions made

• Explains the uncertainties

• Outlines the data gaps

Issues Related to Uncertainty in Risk Assessment

• High to low dose extrapolation

• Species to species extrapolation

• Mechanism of carcinogenesis

• Interindividual differences

• Chemicals that are carcinogenic in animals are expected to be carcinogenic in humans

• Humans are assumed to be as sensitive as the most sensitive animal

• The dose-response is assumed to be linear

Major Default Assumptions in Cancer Risk Assessment

Emerging Issues in Biologically-based Risk Assessment

• Incorporation of PBPK models• Use of molecular dosimetry as a

surrogate of exposure• Role of cell proliferation• Mode of action information• Life stage differences in susceptibility

Potential of Molecular Dosimetry in Risk Assessment

• High to low dose extrapolation– Saturation of metabolic activation– Saturation of detoxication– Saturation of DNA repair

• Route to route differences• Species to species differences

How is Causality Determined?

Bradford Hill Criteriafor Cancer Causation

• Consistency• Strength• Specificity• Temporality• Coherence

• Dose Response• Biological

Plausibility• Experimental

Support• Analogy

IPCS/EPA Framework for Evaluating Mechanistic Data

• Introduction• Postulated mode of

action• Key events• Dose-response

relationship• Temporal association• Strength, consistency

and specificity of association with key events

• Biological plausibility and coherence

• Other modes of action• Assessment of mode of

action• Uncertainties,

inconsistencies and data gaps

Chemical Exposure (air, water, food, etc.)

Internal Exposure

Metabolic Activation

Macromolecular Binding Detoxication

DNA RNA Protein

Biologically Effective Dose

Efficiency of Mispairing

Cell Proliferation

X

XInitiation

k

k

k k

k

k

1

2

3 4

6

5(Biomarker)

c a

b

Increasing External Exposure

Incr

easi

ng A

dduc

t C

once

ntra

tion

Sublinear

Supralinear

SOURCES OF MUTATIONS

ENDOGENOUS DNA DAMAGE EXOGENOUS DNA DAMAGE

Depurination

DNA REPAIR

MUTATION

LifeStyles

EnvironmentalAgents

FreeRadicals

PolymeraseErrors

CELL REPLICATION

Initiating

Event

Cell Proliferation

(clonal expansion)

Progression

Cell Proliferation

Cell Proliferation

Malignancy

Second Mutating Event

Third Mutating Event

D D

S

S

SS

I

I I

I

Mµ1 µ2

β2

First Event Second Event

A Moolgavkar Representation of Multistage Carcinogenesis

Role of Increased Cell Proliferation in Carcinogenesis

• Decreases time available for DNA repair

• Converts repairable DNA damage intononrepairable mutations

• Necessary for chromosomal aberrations, insertions, deletions and gene amplification

• Clonally expands existing cell populations

8-oxo-G, FapyGua

Lipid Peroxidation

Lipid PeroxideMDA, 4-HNE AP Sites

DNA Base Modification DNA Base

adductROS

SugarDamage

DNA Base adduct

M1G, edG, edA

Oxidative Stress Induced-DNA Damage

GlycosylasehOGG1

GlycosylaseMPG

Base Propenal

Control Rat Liver Tissues

0. 0

1. 0

2. 0

3. 0

4. 0

5. 0

6. 0

4- wk 12- wk 2- yr

Rat Ages

8OH

dG/d

G (1

0e-6

)

Non-smoker Lymphocytes

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

1 2 3 4 5 6 7 8 9 10

8OH

dG/d

G (1

0e-6

)

PentachlorophenolUsed as a Pesticide and Wood Preservatives

Introduction to Humans: Air, Food and Drinking water

Mutagen, Rodent Carcinogen

OHCl

ClCl

Cl

ClOH

Cl

ClOH

Cl

Cl

OCl

ClO

Cl

Cl

OCl

ClOH

Cl

Cl

O2

O2-

H2O2

OH

Induced Oxidative StressCalf Thymus DNA Exposed to TCHQCalf Thymus DNA Exposed to TCHQ

0

10

20

30

40

50

0.1 1 10 100 1000

TCHQ (uM)

8OH

dG/d

G (1

0e-6

)

M1G formation from 1,4-TCBQ treatment

0

5

10

15

20

25

30

35

1,4-TCBQ (µM) 0 1 10

M1G

in

108

Nt.

0.1mM NADPH + + +0.1mM CuCl2 + + +

Aldehydic DNA lesions (ADL) in HeLa cells exposed to H2O2 (0.06-20 mM) for 15 min

0

5

10

15

20

25

0 5 10 15 20H2O 2 (mM)

AD

L/1,

000,

000

ntd

0

10

20

30

40

50

0.01 0.1 1 10 100H2O2 (mM)

Incr

ease

d A

DLs

/H2O

2

conc

entr

atio

n

Efficiency of Low Doses of H2O2

DNA Alkylation

7%2%7%14%ENUDEN

0.4%0.1%7%70%MNUDMN

--0.3%85%MMS

O2 Alkyl Thymine

O4 Alkyl Thymine

O6 AkylGuanine

N7AlkylGuanine

N-7-Methylguanine

O -Methylguanine

10000

1000

100

10

1

0.1

0.01

0.001 0.001 0.01 0.1 1 10 100

40

30

20

10

03210

6

DMN (mg/kg)

Alky

latio

ns/1

0 g

uani

nes

Alk y

latio

ns/1

0 g

uani

nes

6 6

A B

0.00E+00

2.00E-06

4.00E-06

6.00E-06

8.00E-06

1.00E-05

1.20E-05

0 10 20 30 40 50 60 70 80

Duration of DEN exposure, days

Mol

ar ra

tio in

DN

A

O4-EtdThd

O6-EtdGuo

0 20 40 60 80 100

0

20

40

60

80

100

0

20

40

60

80

100

Dose (ppm DEN)

ET (p

M)/d

T(µ

M)

0 5 10 15 200

5

10

15

0

5

10

15O2-ET O4-ET

Molecular Dosimetry of DEN

Vinyl Chloride

• Vinyl chloride is a known human and animal carcinogen that induces hepatic angiosarcomas

• Carcinogenic response is associated with high exposure (>50 ppm)

• To date, 178 VC workers have developed hepatic angiosarcomas. All of them started work prior to lowering the occupational exposure 1 ppm

• Vinyl chloride is present in many Superfund sites and some public drinking water in ppb amounts

Vinyl Chloride MetabolismCl

O Cl

Cl

O

P-450

Epoxide Hydrolase

HO

O

Cl

OH

Alcohol dehydrogenaseGlutathioneDetoxication

DNA Adducts

Exposure-Response for Vinyl Chloride Metabolism and Carcinogenicity

0

5000

10000

0 1000 2000 3000 4000

VC Exposure (ppm)

ν ( µ

g / 6

hr)

(Gehring et al, 1978)

0.0

0.1

0.2

0 2000 4000 6000VC Exposure (ppm)

ASL

Inci

denc

e(Maltoni et al, 1981)

Formation of [13C2]-DNA Adducts by Vinyl Chloride

CH2Cl

O

Cl

N

NH

NN

N

OOH

dRib

N

NN

N

dRib

N

N

N

N

dRibO

NH

NH2NN

NH

O

dRib

O

NH

N NN

N

O

dRib

CYP450 2E1

vinyl chloride chloroethylene oxide

DNA

HO-ethanodeoxyguanosine1,N6-ethenodeoxyadenosine 3,N4-ethenodeoxycytidine

7-(2-oxoethyl)-deoxyguanosine N2,3-ethenodeoxyguanosine

* ** *

* *

**

** *

*

**

Immunoaffinity/GC-HRMS Method for N2,3-Ethenoguanine

0

20

40

60

80

100

9.5 9.6 9.7 9.8 9.9 10 10.1 10.2Retention Time (min)

Rel

ativ

e R

espo

nse

(%)

4.6 fmol N2,3-εGua (m/z=354.0413)

204 fmol Internal Standard (m/z=360.0498)

Molecular Dosimetry of N2,3-Ethenoguanine in Adult Rats Exposed to Vinyl Chloride

HEP

0

4

8

12

0 25 50 75 100

0

5

10

15

20

0 200 400 600 800 1000

VC Concentration (ppm)

mol

N2 ,3

G /

107 m

ol G

0

4

8

12

16

0 200 400 600 800 1000

VC Concentration (ppm)

mol

N2 ,3

-G

/ 10

7 mol

G

0

2

4

6

8

0 25 50 75 100

NPC

Vinyl Chloride Exposure-Response Relationship Between Endogenous and

Exogenous N2,3-Ethenoguanine

130 ± 50

99 ± 25

16.2 ± 0.5

3.5 ± 1.0

εG/108 G

4 wk

371671 ± 101100

298.238 ± 4100

4.72.29.9 ± 6.510

----4.6 ± 3.50

Fold-Increase

Fold-IncreaseεG/108 Gppm VC

1 wk

* Whole-body inhalation (6 hr/d; 5 d/wk)

Formation of Endogenous εGua Adducts from Lipid Peroxidation

HN

N

N

N

O

H2N

OHO

OH

HN

N

N

N

O

N

OR

OHHO

H

OHO

OH

H

N

N

N

N

O

N

OHO

OH

H

HN

N

N

N

O

N

OHO

OH

+

+CH3(CH2)4CH CH CH CHOOOH

Formation of [13C2]-EG in Hepatocyte DNA by Vinyl Chloride

0.0

0.5

1.0

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention Tim e (m in)

Rel

ativ

e In

ten

sity

0.0

0.5

1.0

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention T im e (m in)

Rel

ativ

e In

ten

sity

0.0

0.5

1.0

9.4 9.5 9.6 9.7 9.8 9.9 10.0

Retention T im e (m in)

Rel

ativ

e In

ten

sity

A

B

C

m/z=354N2,3-εG(Endogenous)

m/z=356[13C2]N2,3-εG(VC-Derived)

m/z=360[13C4,15N2]N2,3-εG(Internal Standard)

0.0

0.5

1.0

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention Tim e (m in)

Rel

ativ

e In

ten

sity

0.0

0.5

1.0

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention T im e (m in)

Rel

ativ

e In

ten

sity

0.0

0.5

1.0

9.4 9.5 9.6 9.7 9.8 9.9 10.0

Retention T im e (m in)

Rel

ativ

e In

ten

sity

A

B

C

0.0

0.5

1.0

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention Tim e (m in)

Rel

ativ

e In

ten

sity

0.0

0.5

1.0

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention T im e (m in)

Rel

ativ

e In

ten

sity

0.0

0.5

1.0

9.4 9.5 9.6 9.7 9.8 9.9 10.0

Retention T im e (m in)

Rel

ativ

e In

ten

sity

0.0

0.5

1.0

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention Tim e (m in)

Rel

ativ

e In

ten

sity

0.0

0.5

1.0

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention T im e (m in)

Rel

ativ

e In

ten

sity

0.0

0.5

1.0

9.4 9.5 9.6 9.7 9.8 9.9 10.0

Retention T im e (m in)

Rel

ativ

e In

ten

sity

A

B

C

m/z=354N2,3-εG(Endogenous)

m/z=356[13C2]N2,3-εG(VC-Derived)

m/z=360[13C4,15N2]N2,3-εG(Internal Standard)

m/z=354N2,3-εG(Endogenous)

m/z=356[13C2]N2,3-εG(VC-Derived)

m/z=360[13C4,15N2]N2,3-εG(Internal Standard)

Formation of [13C2]-EG in Brain DNA by Vinyl Chloride

0.1

0.6

1.1

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention Tim e (m in)

Rel

ativ

e In

ten

sity

0.4

0.9

1.4

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention Tim e (m in)

Rel

ativ

e In

ten

sity

0.0

0.5

1.0

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention Tim e (m in)

Rel

ativ

e In

ten

sity

A

B

C

m/z=354N2,3-εG(Endogenous)

m/z=356[13C2]N2,3-εG(VC-Derived)

m/z=360[13C4,15N2]N2,3-εG(Internal Standard)

0.1

0.6

1.1

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention Tim e (m in)

Rel

ativ

e In

ten

sity

0.4

0.9

1.4

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention Tim e (m in)

Rel

ativ

e In

ten

sity

0.0

0.5

1.0

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention Tim e (m in)

Rel

ativ

e In

ten

sity

0.1

0.6

1.1

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention Tim e (m in)

Rel

ativ

e In

ten

sity

0.4

0.9

1.4

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention Tim e (m in)

Rel

ativ

e In

ten

sity

0.0

0.5

1.0

9.4 9.5 9.6 9.7 9.8 9.9 10

Retention Tim e (m in)

Rel

ativ

e In

ten

sity

A

B

C

m/z=354N2,3-εG(Endogenous)

m/z=356[13C2]N2,3-εG(VC-Derived)

m/z=360[13C4,15N2]N2,3-εG(Internal Standard)

A

B

C

A

B

C

m/z=354N2,3-εG(Endogenous)

m/z=356[13C2]N2,3-εG(VC-Derived)

m/z=360[13C4,15N2]N2,3-εG(Internal Standard)

m/z=354N2,3-εG(Endogenous)

m/z=356[13C2]N2,3-εG(VC-Derived)

m/z=360[13C4,15N2]N2,3-εG(Internal Standard)

10.0

Vinyl Chloride Cancer Risk Estimates

1.6-3.7Rat

PBPK/LMS1995Clewell et al

2000

1996

1989

1994

Year

1.4EpiChen & Blancato

4.4Rat (f)

1.0-2.3Mouse

0.3-2.8Epi

PBPK/LMSEPA

0.6RatPBPK/LMSReitz et al

0.7-1.4RatPBPK/LMS

84RatLMSEPA

Inhalation Risk(per µg/m3 x 10-6)

DataModelAuthor(s)

Use of Mechanistic Evidence in Vinyl Chloride Risk Assessment

• PBPK Modeling– Conversion of animal exposures to human

equivalent concentrations– Route-to-route extrapolation

• DNA Adducts– Selection of low dose extrapolation model– Inclusion of 2-fold protection factor for young– Increased confidence in risk assessment

Uncertainties in Vinyl Chloride Risk Assessments

• Relationship between low exposure and cancer has large uncertainty.

• High quality human exposure data are not available for individuals with angiosarcoma.

• There has not been any utilization of new data on endogenous DNA adducts.

Formaldehyde

• Chronic exposure to high concentrations (10-15 ppm) causes nasal cancer in rats

• Listed as a Probable Carcinogen for humans

• Formaldehyde causes DNA-protein cross-links

• Highly nonlinear response due to increased cell proliferation

0

0.2

0.4

0.6

0 2 4 6 8 10 12 14 16

Parts per million Formaldehyde

Tum

or R

ate

Nasal Carcinogenesis in Rats Exposed to Formaldehyde

DNA-Protein Cross-links versus Formaldehyde Exposure

0.00

0.02

0.04

0.06

0 2 4 6 8 10 12 14 16

ppm Formaldehyde

Cov

alen

tly B

ound

HC

HO

per

ppm

0

10

20

30

40

50

60

70

0 2 4 6 8 10 12 14 16

HCHO Concentration (ppm)

Tum

or In

cide

nce

(%)

0

2

4

6

8

10

12

14

Cel

l Pro

lifer

atio

n (m

ean

unit

leng

th

labe

ling

inde

x) a

t Nas

al L

evel

II (f

old

incr

ease

ove

r co

ntro

l)

Tumor Incidence 24-monthStudy (Kerns, 1983; EPA,1987)

Tumor Incidence 18-monthStudy (Monticello, 1990)

Cell Proliferation Study 6-month (Monticello, 1990)

Cell Proliferation 12-month(Monticello, 1990)

Cell Proliferation 18-month(Monticello, 1990)

Tumor Incidence and Cell Proliferation in Rats Exposed to Formaldehyde

Propylene Oxide (PO)

• Workplace exposure: inhalation

• Limit of exposure: 20 ppm (8hr time weighted average)

• Mutagenic in bacterial and mammalian systems in vitro

• In vivo tests (rat and mouse dominant lethal tests and mouse sperm-head morphology test) were negative

• No difference in chromosome aberrations or significant increase in sister chromatid exchanges in lymphocytes of monkeys exposed to 100 or 300 ppm PO for 104 weeks

Detoxication (glutathione S-transferase, epoxide hydrolase))

Intracellular ExposurePO

Proposed mode of action: Events and pathways involved in PO-induced nasal tumor formation

Macromolecular binding

DNA adductscellular injury

Cellhyperplasia

Papillary adenomas

?

Comparison of Carcinogenicity, DNA and Hemoglobin Adducts, and Cell Proliferation

0

2

4

6

0 100 200 300 400 500

Tum

or In

cide

nce

0

50

100

150

200

250

300

0 100 200 300 400 500

pmol

HPG

/mg

DN

A

0

50

100

150

200

0 100 200 300 400 500

pmol

HPV

al/m

g gl

obin

0

2

4

6

8

10

0 100 200 300 400 500

fold

incr

ease

ove

r co

ntro

l

Carcinogenicity DNA Adducts

Hemoglobin Adducts Cell Proliferation

Butadiene• Carcinogenicity

– Mouse - highly carcinogenic– Rat - weakly carcinogenic– Human – considerable uncertainty

• Major metabolites differ by species and include: – Epoxybutene (EB)– Diepoxybutane (DEB): 100x more mutagenic than EB

and 200x more mutagenic than EBD– Epoxybutane diol (EBD): Major cause of

trihydroxybutyl adducts– Butene diol (BD-diol)– Hydroxymethylvinylketone (HMVK): Precursor of M1,

the major urinary metabolite of humans

HO

SG

HO

OH

SG

HO

OH

HO

OH

OH

OH

HO

OH

SG

OH

1,3-Butadiene

O

1,2-Epoxy-3-butene

O

HO

OH

1,2-Dihydroxy-3,4-epoxybutane

O

O

1,2,3,4-Diepoxybutane

Erythritol

EH

M3

EHGSHEH

3-Butene-1,2-diol

CYP2E1

CYP2E1

GSH

M1GSH

M2

EH

GSH

DNAProtein

CYP2E1

Comparison of Mutagenicity at HPRT

20

TK6 cells

0 200 400 600 800 1,0000

5

10

15

Dose (µM)EB EBDDEB

-6In

duce

d M

utan

t Fre

quen

cy X

10

Molecular Dosimetry of Butadiene Epoxide DNA Adducts

0

10

20

30

40

50

60

70

80

0 100 200 300 400 500 600Butadiene Exposure (ppm)

Add

ucts

/mill

ion

Gua

Mouse THB-GRat THB-GMouse EB-GRat EB-G

What is the Source of THB-Guanine?

• DEB and EB are equally reactive with DNA• DEB and EB have been measured in tissues of rats

and mice• DEB-Guanine can be converted to THB-Guanine• DEB-Guanine/[DEB] = EB-Guanine/[EB]• DEB-Guanine = EB-Guanine x [DEB]/[EB]• THB-Guanine = DEB-Guanine +EBD-Guanine• ~95% of THB-Guanine came from EBD

02468

101214161820

0 10 20 30 40 50 60Butadiene Exposure (ppm)

Add

ucts

/mill

ion

Gua

Mouse THB-GRat THB-GMouse EB-GRat EB-G

Dose-response Relationship of DNA Adducts in Female Rats and Mice Exposed to Butadiene for 4 Weeks

0

500

1000

1500

2000

2500

3000

3500

4000

4500

0 10 20 30 40 50 60 70BD Exposure (ppm)

pmol

val

ine

addu

ct/g

glo

bin

Mouse THBV

Rat THBVMouse HBV

Rat HBV

Dose-response Relationship of Hemoglobin Adducts in Female Rats and Mice Exposed to Butadiene for 4 Weeks

0

2000

4000

6000

8000

10000

12000

14000

16000

0 5 10 15 20 25 30

EB Exposure (ppm)

pmol

val

ine

addu

ct/g

glo

bin

Mouse THBV

Rat THBV

Mouse HBV

Rat HBV

Dose-response Relationship of Hemoglobin Adducts in Female Rats and Mice Exposed to Epoxybutene for 4 Weeks

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0 1 2 3 4 5 6

DEB exposure (ppm)

pmol

val

ine

addu

ct/g

glo

bin

Mouse THBV

Rat THBV

Mouse HBV

Rat HBV

Dose-response Relationship of Hemoglobin Adducts in Female Rats and Mice Exposed to Diepoxybutane for 4 Weeks

Hemoglobin Adducts in CYP2E1 Knock-out Mice Exposed to 62.5 ppm 1,3-butadiene for 6 h by Inhalation

5.6%16%Percent of Wild-Type

73 ± 219.4 ± 3.2CYP2E1 Knock-out Mouse

1300 ± 32058.1 ± 18.5Wild-Type Mouse

THBVal(pmol /g globin)

HBVal(pmol /g globin)Genotype

Efficiency of THBVal formation and Hprt Mutant Induction in Female B6C3F1 Mice Exposed to BD

3 20 62.5 625 1250

Butadiene Exposure (ppm)

0

5

10

15

20

THB

Val

(10

m

ol/g

glo

bin)

/BD

Con

cent

ratio

n (p

pm)

0

5

10

15

20

Indu

ced

Hpr

t MF

X 1

0 /B

D C

once

ntra

tion

(ppm

)THBVal formation in erythrocytesMutant induction in T-lymphocytes

-13

-9

Biomarkers Facility Core 67

Molecular Dosimetry and Epidemiology Studies on Butadiene

Trihydroxybutyl-valineAdduct vs. 1,3-ButadieneExposure by GSTT1 Genotype

10

100

1000

10000

0.001 0.01 0.1 1 10

Average Butadiene Exposure (ppm)

Trih

ydro

xy a

dduc

t (pm

ol/g

Hb) GSTT1 + GSTT1 -

R2=0.737

Control Monomer Polymer

Butadiene Exposure Group

0

100

200

300

400

500

600

700

800

THB

Val (

pmol

/g g

lobi

n)

0.0

1.0

2.0

3.0

4.0

HB

Val (

pmol

/g g

lobi

n)

THBValHBVal

Monoepoxide and Triol Hemoglobin Adducts in Butadiene Workers from the Czech Republic

Effect of Butadiene Exposure on Hemoglobin Adducts, Urinary Metabolites, and Indicators of

Genotoxicity in Humans

Endpoint Control Monomer Polymer Significance (Kruskal-Wallis)

Butadiene Exposure (mg/m3)

0.02 0.64 1.79 p<0.05

HB-Val (pmol/g Hb) THB-Valine (pmol/g Hb)

0.2 95

0.5 180

2.2 715

p<0.05 p<0.05

Net M1 (ug/L) Net M2 (ug/L)

-219 -0.05

213 5.3

2700 84

p<0.05 p<0.05

Hprt VF (×106) MF (×106)

10.75 13.00

5.73 10.69

6.48 18.83

p<0.05 --

SCE (SCE/cell) CA (%)

6.32 1.56

6.14 1.52

6.47 1.54

-- --

Albertini et al., in press

Effect of Butadiene Exposure on Hemoglobin Adducts, Urinary Metabolites, and Indicators of Genotoxicity in Humans

Endpoint Unexposed Exposed Significance (Wilcoxin)

THB-Valine 37.6 74 P<0.0001 Hprt 17.2 16.8 0.76 SCE 10.3 9.7 0.20

Aneuploidy (%) 11 10 0.30

GPA:Nφ NN

6.0 7.2

3.9 6.0

0.12 0.11

Hayes et al. (2000)

Effect of Butadiene Exposure on Hemoglobin Adducts, Urinary Metabolites, and Indicators of Genotoxicity in

Humans with GSTT1 Polymorphisms

Endpoint Wild-Type Null (n=15)

Significance (Wilcoxin)

THB-Valine 72.8 74.0 0.89

hprt 16.7 18.9 0.92

SCE 9.6 9.8 0.78

In vitro SCE + BDO2 86.2 138.9 p<0.0001

Aneuploidy (%) 6 8 0.43

GPA:Nφ NN

4.0 7.0

3.9 5.3

0.90 0.31

Hayes et al. (2000)

O

O

OOH

OOH

OH

OH

OH

O

P450

P450

mEH

mEH

P450

GSTP450+ Several Steps

MI

GST + SeveralSteps

MII

BD EB BD-diol HMVK

DEB EB-diol

O

OHHBAL

ADHSpontaneous Rearrangement

GSTGlutathioneConjugate

Figure 1. 1,3-Butadiene Metabolism Scheme

0

10

20

30

40

0 10 20 30 40

BD-diol (ppm)

THB

-Val

-PFP

TH

(pm

ol/m

g gl

obin

)

Rat

Mouse

THB-Val adducts in mice and rat exposed to 3-butene-1,2-diol (BD-

diol)

O

O

O

OH

OH

OH

OHO

Globin

PeptideOH

NOH

OH

OHPeptide

ON

HO

HO

1,3-Butadiene (BD)

1,2-Epoxy-3-butene(EB)

3-Butene-1,2-diol(BD-diol)

3,4-Epoxy-1,2-butanediol(EBD)

1,2:3,4-Diepoxybutane(DEB)

EH

Globin

P450s

P450s

PeptideOH

NOH

EH

Globin

HB-Val THB-Valpyr-Val

P450s

Butadiene Hemoglobin Adducts

Total Ion Chromatogram (ESI+/MS, m/z 100-1100) Of A Tryptic Digest Of Globin From DEB-exposed

Human Red Blood Cells

15 20 25 30 35

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

37.06

36.34

26.23 34.84

27.1929.7021.1219.70

28.7732.46

25.2117.81 21.95 38.35

23.7116.92

N,N-(2,3-dihydroxybuta-1,4-diyl)VLSPADK

VLSPADK

Human: pyr-Val-Leu*-Ser- Pro-Ala-Asp-Lys/-Thr-Asn-Val-Lys(pyr-hV) V - L - S - P - A - D - K / -T - N - V - K

Mouse: pyr-Val-Leu*-Ser-Gly-Glu-Asp-Lys/-Ser-Asn-Ile-Lys(pyr-mV) V - L - S - G - E - D - K /- S - N - I - K

Rat: pyr-Val-Leu*-Ser-Ala-Asp-Asp-Lys/-Thr-Asn-Ile-Lys(pyr-rV) V - L - S - A - D - D - K / -T - N - I - K

* d3 in Internal standards/ hydrolyzed by trypsin

pyr-Val containing α-N-Terminal Peptides (1-11)

Globin↓

Add IST (1-11) peptide ↓

Trypsin hydrolysis (1-7 N-terminal peptide)

↓Centricon-3 filtration

↓Applying on IA column

↓Filtration (2 µm)

↓LC-ESI-MS/MS

Protocol for pyr-Val-Peptide Analysis

pyr-Val in Mice and Rats Treated with 3 ppm BD

0 2 4 6 8 10 12 14 16 18 20Time (min)

10.07

13.84

10.05

NL: 1.18E5TIC F: + p SRM ms2 833.5 → 158.2pyr-val_188

NL: 1.42E5TIC F: + p SRM ms2 836.5→158.2pyr-val_188

Rel

ativ

e A

bund

ance

50

100

50

100

0

0

A: Mouse 3 ppm 10 days~39.7 pmol/g

NL: 3.03E4

Rel

ativ

e A

bund

ance

50

100

50

100

0

0

10.44

10.44TIC F: + p SRM ms2 833.5 → 158.2 pyr-val_200

NL: 2.01E5TIC F: + p SRM ms2 836.5 → 158.2 pyr-val_200

B: Rat 3 ppm 10 days4.5 pmol/g

0 2 4 6 8 10 12 14 16 18 20Time (min)Boysen et al (2004) Cancer Research, 64:8517-8520

BD-derived globin adducts in mice and rats

Duration Species

Exposure [ppm BD]

HB-Val [pmol/g]

HB-Val ppm BD

pyr-Val [pmol/g]

pyr-Val ppm BD

THB-Val [pmol/g]

THB-Val ppm BD

10 days Mice (B6C3F1)

3 53 ± 7.6 17.7 48.7 ± 3.23 16.2 452 ± 38 150.0 62.5 137 ± 12.2a 1.6 130.4 ± 64 2.1 3410 ± 177 54.6 1250 7143 ± 537 5.7 2487.0 ± 426 2.0 13755 ± 1651 11.0

Rats (F344) 3 13 ± 2.4 4.3 3.9 ± 0.8 1.3 339 ± 41 113.0 62.5 87 ± 7.6 1.4 38.3 ± 1.2 0.6 3202 ± 302 51.2

90 days Rats (Crl:CD®)

Females 1000 8690 ± 930 b 8.6c 58.1 ± 17.3 0.058 c 24066 ± 9292 26.6c Males 1000 5480 ± 2880 b 5.4c 16.7 ± 6.6 0.017 c 12095 ± 3712 12.7c

a One data point was excluded with 95% CI according to Q-test (n=2). b Swenberg et al HEI Report 92, 2000, n=3. c Efficiencies were based on 90 day exposures and were not adjusted per day of exposure.

Boysen et al (2004) Cancer Research, 64:8517-8520

pyr-Val Adduct levels in Mice and Rats exposed to BD for 10 days

�y = 1.97x + 25.54

R2 = 1.00

0

500

1,000

1,500

2,000

2,500

3,000

3,500

0 200 400 600 800 1000 1200 1400

Exposure to BD [ppm]

pyrV

al a

dduc

t lev

el [p

mol

/g]

Rats

0

10

20

30

40

50

60

70

0 400 800 1,200

Boysen et al 2004, in preparation

Dose Response of pyr-V in Mice and Rats Exposed to BD for 4 Weeks

male rats

female rats

male mice

female mice

0

100

200

300

400

500

600

0 10 20 30 40 50 60 70 80

BD Exposure [ppm]

Am

ount

of

pyr

-Val

[pm

ol/g

]

0

4

8

12

16

20

0 25 50 75

male rats

female rats

Future Studies

• Extend the exposure-response data (HB-Val, THB-Val and pry-Val) for mice and rats.

• Compare molecular dose versus mutations in epoxide hydrolase null vs wild type mice.

• Compare molecular dose versus mutations in XPC null versus wild type mice.

• Examine exposure-response and gender effects in BD workers.

Biologically-Based Risk Assessment• Refine estimates of dose to relevant targets

through use of biomarkers of exposure and PBPK modeling

• Improve hazard characterization through a better understanding of the mode(s) of action for endpoints of concern

• Strengthen inferences regarding the shape of dose/response curves outside the range of traditional observations

• Identify/investigate opportunities for research in human populations, such as susceptibility factors

Systematic Characterization of ComprehensiveExposure-Dose-Response Continuum and the Evolution of Protective to Predictive Dose-Response Estimates

Conclusions

• Biomarkers of exposure and effect can identify key events that drive dose-response relationships.

• These responses can be the result of metabolism, cell proliferation and DNA repair.

• Understanding and utilizing these key events in risk assessment will reduce uncertainty and improve accuracy.