issues related to uncertainty in risk assessment high to low dose extrapolation species to species...

HumanEPI

Data

Where the Question of

Health Risk is Raised

Resp

on

se Log Dose

Paustenbach (1995)

AnimalData

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

• Mode of action information– Role of cell proliferation– Mutagenicity

• 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

• Role of endogenous DNA damage

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

Internal Exposure

Metabolic Activation

Macromolecular Binding Detoxication

DNA RNA Protein

Biologically Effective Dose

X

Efficiency of Mispairing

X

Cell Proliferation

Biomarkers of Exposure

Mutation/Initiation

Progression Cancer

Biomarkers of Effect

ca

b

Increasing External Exposure

Inc

rea

sin

g A

dd

uc

t C

on

ce

ntr

ati

on

Sublinear

Supralinear

Role of Increased Cell Proliferation in Carcinogenesis

• Decreases time available for DNA repair

• Converts repairable DNA damage into nonrepairable mutations

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

• Clonally expands existing cell populations

Initiating

Event

Cell Proliferation

(clonal expansion)

Progression

Cell Proliferation

Cell Proliferation

Malignancy

Second Mutating Event

Third Mutating Event

SOURCES OF MUTATIONS

ENDOGENOUS DNA DAMAGE EXOGENOUS DNA DAMAGE

Depurination

DNA REPAIR

MUTATION

LifeStyles

EnvironmentalAgents

FreeRadicals

PolymeraseErrors

CELL REPLICATION

Sources of DNA Damage

Spontaneous DNA Chemistry

Depurination

TA

G C

T A

TA

C

T A

Deamination

Endogenous AP Sites in Rat Tissues and Human Liver

0

5

10

15

20

25

30

35

Rat Lung RatKidney

Rat Liver HumanLiver

RatTestis

Rat Heart Rat Colon Rat Brain

AP

sites

/10

6 nuc

leot

ides

[Nakamura and Swenberg, 1999]

Oxidative Stress Induced-DNA Damage

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 20

H2O 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

DL

s/H

2O2

conc

entr

atio

n

Efficiency of Low Doses of H2O2

Pentachlorophenol

Used as a Pesticide and Wood Preservatives

Introduction to Humans: Air, Food and Drinking water

Mutagen, Rodent Carcinogen

OH

Cl

Cl

Cl

Cl

Cl

OH

Cl

Cl

OH

Cl

Cl

O

Cl

Cl

O

Cl

Cl

O

Cl

Cl

OH

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

/dG

(10

e-6)

Steady-state Amounts of Endogenous DNA Damage

Endogenous DNA Lesions Number per Cell

Abasic sites 10,000-50,000

OHEtG 3,000

7-(2-Oxoethyl)guanine 3,000

8-oxodG 2,400

Formaldehyde 1,000-4,000

Acetaldehyde 1,000 - 4000

7-Methylguanine 1,200

AcrdG 120

M1dG 60

N2,3-Ethenoguanine 36

1N2-Etheno dG 30

1N6-Etheno dA 12

Total 20,000-60,000

Chemical-Specfic DNA Alkylation

N7AlkylGuanine

O6 Akyl Guanine

O4 Alkyl Thymine

O2 Alkyl Thymine

MMS 85% 0.3% - -

MNU

DMN

70% 7% 0.1% 0.4%

ENU

DEN

14% 7% 2% 7%

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)

Alkylation

s/10 gu

anin

es

Alkylation

s/10 gu

anin

es

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

Mo

lar

rati

o 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)/

dT

(M

)

0 5 10 15 200

5

10

15

0

5

10

15

O2-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, 197 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

Exposure-Response for Vinyl Chloride Metabolism and Carcinogenicity

0

5000

10000

0 1000 2000 3000 4000

VC Exposure (ppm)

n (

g / 6

hr)

(Gehring et al, 1978)

0.0

0.1

0.2

0 2000 4000 6000VC Exposure (ppm)

AS

L In

cid

ence

(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

dRib

O

NH

NH2NN

NH

O

dRib

O

NH

NNN

N

O

dRib

CYP450 2E1

vinyl chloride chloroethylene oxide

DNA

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

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

* ** *

* *

*

*

*

* *

*

**

Miscoding Properties of Vinyl Chloride DNA Adducts

7-(2-Oxoethyl)guanine(7OEG) None

N2,3-Ethenoguanine (εG) G → A

3,N4-Etheno-2’-deoxycytidine (εdC)

C → T

C → A

C → G

1,N6-Etheno-2’-deoxyadenosine (εdA)

A → T

A → C

A → G

Mutations in VC-induced Neoplasms in Humans and Rats

• Marion et al., found G:C→A:T mutations in codon 13 of the c-Ki-ras-2 gene in 5/6 human hepatic angiosarcomas.

• Hollstein et al., found A:T →T:A mutations in codon 249 and 255 of the p53 gene in 2/4 human hepatic angiosarcomas.

• Bolvin-Angele did not find ras gene mutations in hepatic angiosarcomas induced by VC or vinyl fluoride.

Sample Spectrum of 7-OEG

2.5 3.0 3.5 4.0 4.5Time (min)

010

20

3040

50

60

70

80

90

100

Rel

ativ

e A

bu

ndan

ce

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bu

ndan

ce

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bu

ndan

ce RT: 3.36MA: 163809

2.593.98 4.09 4.384.56

RT: 3.35MA: 26517530

3.06 3.67 3.884.042.72 4.442.54 4.29 4.61RT: 3.35MA: 1981473

2.772.68 3.06 3.68 4.543.90 4.29

m/z 265152 AST

m/z 267152 13C2-OEG

m/z 270157 IST

2.5 3.0 3.5 4.0 4.5Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bu

ndan

ce

010

20

30

40

50

6070

80

90

100

Rel

ativ

e A

bu

ndan

ce

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bu

ndan

ce RT: 3.35MA: 298897

2.493.74 4.13 4.37 4.60

RT: 3.35MA: 16059051

3.06 3.683.872.74 3.992.62 4.39

4.58RT: 3.35MA: 2216725

2.77 3.072.56 3.59 3.83 4.444.07 4.664.27

A B

A. Adult rat liver(1100 ppm [13C2]-VC, 5days)B. Weanling rat liver (1100 ppm [13C2]-VC, 5days)

Relative Amounts of Endogenous and Exogenous DNA Adducts in Liver DNA From Rats Exposed to [13C2]-VC

(1100 ppm, 6 hr/day, 5 days)

[12C2]-

7OEG/

105 Gua

[13C2]-

7OEG/

105 Gua

[12C2]-

N2,3-εG/

108 Gua

[13C2]-

N2,3-εG/

108 Gua

[12C2]-

1N6- εdA/

108 dA

[13C2]-

1N6- εdA/

108 dA

Adult Rats at End of Exposure

0.2± 0.1 10.4± 2.3 4.1 ± 2.8 18.9 ± 4.9 4.9 ± 0.6 5.1 ± 0.6

2 Weeks Post Exposure

0.1 ± 0.03 0.4± 0.3 3.7 ± 3.1 14.2 ± 4.2 8.6 ± 0.9 ND


0.2 ± 0.04 0.1± 0.06 3.1 ± 1.0 16.9 ± 1.6 6.2 ± 1.3 ND


0.2 ± 0.07 ND 3.7 ± 1.5 13.2 ± 2.5 4.1 ± 0.5 ND

None

1100 ppm VC; 4 wk

1100 ppm VC; 4 wk10 ppm VC; 4 wk

None

None known

None known

Exposure

110 ± 40

15 ± 2

3.0 ± 0.8SD rat hepatocytes

16 ± 5Human colon

17 ± 2Human liver

10 ± 11

7.8 ± 2.1

SD rat brain

N2,3-G / 108 GSample

N2,3-G in Control and VC-exposed Samples

T1/2 and Repair Pathways For VC-Induced DNA Adducts

Adduct T1/2 Repair Pathways

7OEG 4 Days Chemical depurination

N2,3-εG 150 Days Unknown

1,N6- εdA ~1 Day

MPG/Aag

AlkB

3,N4- εdC ~1 Day DNA glycosylases

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 g/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 is One of the Oldest Chemicals in the World

• One-carbon pool• Methanol metabolism• Amino Acid metabolism• Lipid Peroxidation• P450 dependent demethylation (O-, N-, S-methyl)

Sources of Endogenous

Formaldehyde

Formaldehyde was Part of the Origin of Life

Ubiquitous Environmental Chemical • Global production is >20

million tons/yr

• Wide use in industrial and consumer products

• Carcinogenic in rodent bioassays

• Listed as a human carcinogen NTP 2011, IARC 2006

• Mode of Action is complex Cytotoxic/cell proliferation

Mutagenic Site of contact vs distant

sites Endogenously formed in all

cells

Carcinogenesis Bioassays• CIIT/Battelle studies in rats and mice

– 12 month sacrifice/interim report – 18 month data published in Cancer

Research (Swenberg ,et al 1980)– Final report and Cancer Research

paper on the study (Kerns, et al. 1983)• CIIT expanded the exposure range and

mechanistic designs in a second bioassay published in Cancer Research (Monticello, et al, 1996)

• Subsequent cancer bioassays– Inhalation studies– Oral studies

0

10

20

30

40

50

60

70

0 2 4 6 8 10 12 14 16

HCHO Concentration (ppm)

Tu

mor

In

cid

ence

(%

)

0

2

4

6

8

10

12

14

Cel

l Pro

lifer

atio

n (m

ean

unit

leng

th la

belin

g in

dex)

at

Nas

al L

evel

II

(fol

d in

crea

se o

ver

cont

rol)

Tumor Incidence 24-month Study(Kerns, 1983)

Tumor Incidence 24-month Study(Monticello, 1996)

Cell Proliferation Study 6-month(Monticello, 1990)



Tumor Incidence and Cell Proliferation in Rats Exposed to Formaldehyde

Epidemiology of Formaldehydeand Cancer

• Nasopharyngeal Cancer– The NCI cohort found an increase in NPC, while other

studies have been negative.• Only 1 plant out of 10 had an increased incidence of NPC

• The same plant was in a region known for silversmithing and metal working, two known causes of NPC.

• The extent of formaldehyde exposure was not associated with the increase in NPC.

• While biologic plausibility is clearly present, the lack of consistency between studies and the lack of an exposure relationship in positive studies weakens the conclusion.

• Confounding cannot be eliminated.

Epidemiology of Formaldehydeand Cancer (Cont.)

• Myeloid Leukemia– No evidence has been provided that demonstrates that

formaldehyde gets to sites distant to the portal of entry.

– While several studies have shown associations, equal numbers of studies have not.

– No mechanisms have been identified for the induction of leukemia by formaldehyde.

– Thus, the biologic plausibility of inhaled formaldehyde causing leukemia is weak.

• Formaldehyde is a ubiquitous environment pollutant, but it is also an essential metabolite in all living cells. Therefore, both endogenous and exogenous formaldehyde is present.

• Formaldehyde is very reactive with DNA and proteins, leading to diverse protein adducts and DNA damage.

ProteinN CH2

ProteinCH2

CH2OH

H2C

O

H H

glutathione

S-hydroxymethyl-glutathione

ADH3

S-formylglutathione

S-formylglutathionehydrolase

formate

CO2+H2O

glutathione

endogenous sources

exogenous sources

ALDH1A1ALDH2

one carbon

pool

adduct formation

Fate and metabolism of formaldehyde

Adapted for IARC monograph 88

Experimental Design

• Rats were exposed to 10 ppm [13CD2]-formaldehyde for 6 hrs/day for 1 or 5 days and sacrificed within 2 hr.

• Nasal mucosa, lung, liver, spleen, thymus and bone marrow were collected for DNA adduct analysis.

• DNA was reduced with NaCNBH3, hydrolyzed to nucleosides and adducts were separated by HPLC and fraction collection. ~40 µg DNA was used for nasal tissue and 200 µg for all others. Thus, 5-fold more DNA was analyzed from distal sites.

• Ultrasensitive Capillary-MS/MS and UPLC-MS/MS methods were developed for N2-methyl-dG (detection limit 240 and 20 amol) and N6-CH3-dA (detection limit 50 and 5 amol) monoadducts.

• Endogenous and [13CD2]-adducts were measured.

NH

N

N

O

NH2N

O

HOH

HHHH

HO

NH

N

N

O

NH

N

O

HOH

HHHH

HOCH3

NH

N

N

O

NH

N

O

HOH

HHHH

HOCH2OH

NaCNBH3

HH

O

NH

N

N

O

NH

N

O

HOH

HHHH

HO

13CD2H

NH

N

N

O

NH

N

O

HOH

HHHH

HO

13CD2OH

NaCNBH3D13C

D

O

m/z=282.2

m/z=285.2

endogenous

exogenous

The formation of N2-hydroxymethyl-dG originating from both endogenous and exogenous formaldehyde.

6.0 6.5 7.0 7.5

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

RT: 7.55

RT: 7.52

RT: 7.54

6.0 6.5 7.0 7.5

RT: 7.56

RT: 7.54

RT: 7.54

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

6.0 6.5 7.0 7.5

Time (min)

RT: 7.53

RT: 7.53

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100A. B. C.

Endogenous

Exogenous

Internal standard

Endogenous

Exogenous

Internal standard

Endogenous

Exogenous

Internal standard

100

100

6.0 6.5 7.0 7.5

RT: 7.55

RT: 7.56

0

20

40

60

80

0

20

40

60

80

0

20

40

60

80

100 D.

Endogenous

Exogenous

Internal standard

Time (min) Time (min) Time (min)

1 day-exposed nasal epithelium (A), 5 day-exposed nasal epithelium (B), bone marrow (C) and spleen (D).

LC-ESI-MS/MS SRM chromatograms of N2-Me-dG in typical tissues

Exposure period Tissues

N2-HOCH2-dG (adducts/107 dG)

N6-HOCH2-dA (adducts/107 dA)

exogenous endogenous exogenous endogenous

1 day

Nose 1.28±0.49* 2.63±0.73 n.d. 3.95±0.26

Lung n.d.+ 2.39±0.16‡ n.d. 2.62±0.24

Liver n.d. 2.66±0.53 n.d. 2.62±0.46 #

Spleen n.d. 2.35±0.31 n.d. 1.85±0.19

Bone Marrow n.d. 1.05±0.14 n.d. 2.95±1.32

Thymus n.d. 2.19±0.36 n.d. 2.98±1.11

5 day

Nose 2.43±0.78 2.84±1.13 n.d. 3.61±0.95

Lung n.d. 2.61±0.35 n.d. 2.47±0.55

Liver n.d. 3.24±0.42 n.d. 2.87±0.65

Spleen n.d. 2.35±0.59 n.d. 2.23±0.89

Bone Marrow n.d. 1.17±0.35 n.d. 2.99±0.08

Thymus n.d. 1.99±0.30 n.d. 2.48±0.11

Formaldehyde-induced monoadducts in tissues of rats exposed to 10 ppm [13CD2]-formaldehyde for 1 day or 5 days

# Endogenous N6-HOCH2-dA was present in control rat liver at 1.96±1.86 adducts/107 dA

From Cheng et al., Chem. Res. Toxicol. 21, 746-751,2008.

RT: 8.00 - 12.00 SM: 9G

8 9 10 11 12Time (min)

0

500000

1000000

1500000

2000000

2500000

30000000

500000

1000000

1500000

2000000

2500000

3000000

Inte

nsi

ty

0

500000

1000000

1500000

2000000

2500000

3000000

RT: 10.30

RT: 10.30

RT: 10.31

NL: 3.00E6TIC F: + p NSI SRM ms2 282.200 [165.600-166.600] MS ICIS Me_dG_09272010_05



Endogenous282.2 → 166.1 m/z

Exogenous285.2 → 169.1 m/z

Internal Standard297.2 → 176.1 m/z

4.9 adducts/107 dG

9.0 adducts/107 dG

20 fmol

Exposure (ppm)

Exogenous adducts/107

dG

Endogenous adducts/107

dGn

0.7±0.2 0.039±0.019 3.62±1.33 3*

2.0±0.1 0.19±0.08 6.09±3.03 4**

5.8±0.5 1.04±0.24 5.51±1.06 4

9.1±2.2 2.03±0.43 3.41±0.46 5

15.2±2.1 11.15±3.01 4.24±0.92 5

*4-6 rats combined** 2 rats combined

Dosimetry of N2-hydroxymethyl-dG Adducts

15 ppm Rat NE

Non-Linear Dose Response for Formaldehyde DNA Monoadducts

Non-Human Primate Study

• 13CD2O Exposure for 2 days (6 hours/day)

• Cynomolgus Macaque

• Tissues (to date)

– Nasal Maxilloturbinate

– Femoral Bone Marrow

• Exposure Levels

– 2 ppm (n=4)

– 6 ppm (n=4)

Nasal Maxilloturbinate N2-hydroxymethyl-dG Adducts

RT: 8.04 - 12.00 SM: 13G

9 10 11 12Time (min)

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

Inte

nsi

ty

0

20000

40000

60000

80000

100000

120000

Inte

nsity

0

500000

1000000

1500000

2000000

2500000

3000000

Inte

nsity

RT: 10.44

RT: 10.46

11.77

RT: 10.43

NL: 3.00E6TIC F: + p NSI SRM ms2 282.200 [165.600-166.600] MS ICIS Monkey_Me_dG_092910_06

NL: 1.39E5TIC F: + p NSI SRM ms2 285.200 [168.600-169.600] MS Monkey_Me_dG_092910_06





RT: 8.04 - 12.00 SM: 11G

9 10 11 12Time (min)

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

Inte

nsi

ty

0

20000

40000

60000

80000

100000

120000

140000

160000

Inte

nsi

ty

0

100000

200000

300000

400000

500000

600000

Inte

nsi

ty

RT: 10.40

RT: 10.38

RT: 10.42







1.9 ppm 13CD2O 6.1 ppm 13CD2O

Adduct Numbers in Primate Nasal Maxilloturinbates

Exposure concentrati

on


dG


dG

1.9 ppm 0.25 ± 0.04 2.49 ± 0.39

6.1 ppm 0.41 ± 0.05 2.05 ± 0.53

n = 3 or 4

Species Comparisons

1 day 1 day 2 day2 day

Primate Femoral Bone Marrow Endogenous and Exogenous Adducts

1.9 ppm 13CD2O 6.1 ppm 13CD2O

312 µg DNA

RT: 8.00 - 12.00 SM: 7G

8 9 10 11 12Time (min)

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

Inte

nsity

0

10000

20000

30000

40000

50000

Inte

nsity

0

1000000

2000000

3000000

4000000

5000000

6000000

Inte

nsity

RT: 10.62

RT: 10.62




7E6

6E4

2E6

Endogenous282.2 → 166.1

m/z

Exogenous285.2 → 169.1

m/z

Internal Standard

297.2 → 176.1 m/z

178 µg DNA

No Exogenous Adducts

Detected with 5-10 fold >DNA

RT: 8.00 - 12.00 SM: 7G

8 9 10 11 12Time (min)

0

500000

1000000

1500000

2000000

2500000

3000000

Inte

nsity

0

5000

10000

15000

20000

25000

30000

35000

40000

Inte

nsity

0

5000000

10000000

15000000

20000000

Inte

nsity

RT: 10.52

RT: 10.52




2E7

4E4

3E6




Note: We used ~20-30 ug for

nasal tissue

Adduct Numbers in Primate Bone Marrow

Exposure concentrati

on


dG


dG

1.9 ppm nd 17.48 ± 2.61

6.1 ppm nd 12.45 ± 3.63

n = 4

Conclusions• Both cytotoxicity and genotoxicity are key events for the

induction of nasal carcinoma.

• The sustained increase in cell proliferation that results from formaldehyde cytotoxicity “fixes” both endogenous and exogenous DNA adducts into heritable mutations.

• If a rat was placed in a FEMA trailer for 6 hours, only 91/100,000 formaldehyde adducts would come from the exposure. The rest are endogenous.

• A 6 hr exposure of a rat to the USEPA proposed safe level of formaldehyde (0.07 ppt) would induce 83/100,000,000 adducts.

• The lack of exogenous formaldehyde adduct formation in bone marrow and other distant sites does not support the biologic plausibility of leukemia.

Application to Risk Assessment

• Because no [13CD2]-N2-MedG adducts were detectable in primate bone marrow, we can state that they must be below the LOD.

• Therefore, the LOD represents a worst case upper bound for the amount of DNA analyzed.

• We have assumed that the relationship between airborne formaldehyde concentration and exogenous dG adducts is linear through zero.

• We calculated steady state concentrations based on the adduct half life and a 24/7 exposure.

• Risk estimates were calculated for all data sets.

Table 1. Endogenous and exogenous DNA adduct concentrations (per 107dG) in nasal epithelial tissue and bone marrow of cynomolgus macaques exposed via inhalation for 6 hours on two consecutive days to 2 ppm [13CD2]-formaldehyde (data taken from Moeller et al. (2011)). Also shown are the 8.85-fold higher steady-state exogenous adduct concentrations that are expected to result from lifetime continuous inhalation exposure to 2 ppm [13CD2]-formaldehyde (see text for details).

Tissue Endogenous Adducts at 30 hrs

Exogenous Adducts at 30 hrs

Exogenous Adducts at Steady-State

Nasal Epithelium Mean ± se

Lower 95% Bound

2.49 ± 0.23

2.11

0.250 ± 0.020

2.21 ± 0.18

Bone Marrow Mean ± se

Lower 95% Bound

17.5 ± 1.31

15.34

< 0.00103a

<0.00912a

a: no exogenous adducts were detected in bone marrow; upper limits estimate based on the detection limit reported in Moeller et al. (2011).

Table 2. Comparison of estimated lifetime risks of developing nasopharyngeal cancer (NPC) and leukemia (LEU) from continuous lifetime inhalation exposure to 1 ppm formaldehyde, as estimated with the bottom-up approach and, alternatively, by USEPA using top-down linear extrapolation from epidemiologic data (as taken from Table 6-3, pp 6-41-6-42 of the Agency’s 2 June 2010 draft assessment).

Cancer

Background Risk, P0

Bottom-Up Slope, P0/C0L

a Bottom-Up

Risk at 1 ppmb USEPA

Risk at 1 ppm

NPC 7.25 x 10-4 3.44 x 10-4 0.038 x 10-2 1.1 x 10-2

LEU 1.30 x 10-2 8.50 x 10-4 < 3.9 x 10-6 5.7 x 10-2

a: for NPC, 3.44 x 10-4 = 7.25 x 10-4 / 2.11 for LEU, 8.50 x 10-4 = 1.30 x 10-2 / 15.3 b: for NPC, 0.038 x 10-2 = 3.44 x 10-4 x (2.21/2) for LEU, < 3.9 x 10-6 = 8.50 x 10-4 x (< 0.00912/2)

Risk Assessment Model is Conservative

• Attributes all background risk to dG adducts.

• Only utilizes endogenous dG adducts, even though endogenous dA adducts are also present.

• Risk model is linear.

• Used lower 95% confidence bounds of measured endogenous adducts to generate upper 95% bounds on slopes.

• Made conservative assumptions on kinetics and dG adduct half-life data.

• Used same scaling methods used by the USEPA.

Epigenetic Mode of Action Studies

• Adduction to histone lysines• Altered expression of miRNAs

– Primate maxilloturbinates, bone marrow, WBC and CD34+ cells

– Rat tissues following 28 days of inhalation exposure to 2 ppm (6 hr/day, 7 days/week)

• DNA methylation in tissues from inhalation exposed rats and primates

• Discovered in early 1990s• Recognized as important biological regulators in early 2000s

(Filipowicz, 2008)

DNA

miRNA

mRNA

Protein

Transcription

Translation

Transcription

1. Decay of target mRNA

2. Translational repression

3. Cleavage of newly translated polypeptides

miRNAs regulate gene expression in three ways:

MicroRNAs (miRNAs) are Important Epigenetic Regulators of Gene Expression

Nonhuman Primate Project

• Cynomolgus macaques were exposed to 0, 2, or 6 ppm 13CD2 formaldehyde for 6 h/day for 2 days

• Time-matched control macaques received clean air under the same conditions

• RNA samples were collected from the nose

• RNA samples were hybridized to miRNA microarrays to compare genome-wide miRNA expression profiles of formaldehyde-exposed versus unexposed samples

Formaldehyde Exposure Changes the Expression Levels of 13 miRNAs in the Nonhuman Primate Nasal Epithelium

Plot displaying the fold changes in miRNA expression induced by formaldehyde exposure in the nasal epithelium of nonhuman primates. 3 and 13 miRNAs were significantly (FC ≥ ±1.5, p<0.05, q<0.10) differentially expressed upon exposure to 2 ppm and 6 ppm formaldehyde, respectively.

Computationally predicted mRNA

targets for miR-125b

RT-PCR of selected miRNAs with altered

expression upon exposure to formaldehyde.

Systems Biology-Based Analysis of the Transcriptional Targets Predicted to be Regulated by miR-125b Revealed an Enrichment for

Apoptosis Signaling

Apoptosis Signalingp = 0.003

Predicted targets of formaldehyde-responsive miR-125b are shown in BLUE

All Apoptosis-Related Targets of miR-125b Showed Decreased Expression in Formaldehyde-Exposed Samples

FormaldehydeExposure

↑ miR-125b Expression

↓ Predicted Targets of miR-125b

* *

* Significantly altered at the expression level by formaldehyde exposure in the rat nasal epithelium (Andersen et al. 2010)

Rodent Project Design

Nasal Epithelium

Bone Marrow

White Blood Cells

Rats were exposed to 2 ppm 13CD2 formaldehyde for 6 h/day for 28 days

Time-matched control rats received clean air under the same conditions RNA samples were collected from the nose, circulating white blood cells, and bone marrow RNA samples were hybridized to the Agilent Rat miRNA Microarray to compare genome-wide miRNA expression profiles of formaldehyde-exposed versus unexposed samples

Genome-wide miRNA expression profiles were assessed throughout three regions: (1) nose, (2) circulating white blood cells, and (3) bone marrow

Plot displaying the fold changes in miRNA expression induced by formaldehyde exposure in the rat nasal epithelium. 59 miRNAs were significantly (FC ≥ ±1.5, p<0.05, q<0.10) differentially expressed after 28 days of exposure to 2 ppm formaldehyde.

Formaldehyde Exposure Changes the Expression Levels of 59 miRNAs in the Rat Nasal Epithelium

miR-203 shows decreased expression levels by formaldehyde exposure within the nonhuman primate and rodent nose

Evaluated miR-203 further by predicting its transcriptional targets

Systems Biology-Based Analysis of the Transcriptional Targets Predicted to be Regulated by miR-203 Revealed an Enrichment for

Carcinogenesis-Related Signaling

*

** Involved in carcinogenesis

Altered at the expression level by formaldehyde in the rodent nasal epithelium (Andersen et al. 2010)

*

*

*

**

**

*p < 10-51

** Member of the RAS oncogene family Altered at the expression level in nasopharyngeal carcinoma (Zheng et al. 2007)

Most significant network associated with miR-203 targets

Plot displaying the fold changes in miRNA expression induced by formaldehyde exposure in the rat leukocytes. 8 miRNAs were significantly (FC ≥ ±1.5, p<0.05, q<0.10) differentially expressed after 28 days of exposure to 2 ppm formaldehyde.

Formaldehyde Exposure Changes the Expression Levels of 8 miRNAs in Rat White Blood Cells

miR-31 shows formaldehyde-induced increased expression in the rat nose and circulating white blood cells

Summary of the Number of miRNAs with Altered Expression by Formaldehyde Exposure throughout Multiple Regions of the Body

Nasal Epithelium

Bone Marrow

White Blood Cells

59 miRNAs59 miRNAs

8 miRNAs8 miRNAs

0 miRNAs0 miRNAs

1,3-Butadiene• An important industrial chemical

• Classified by IARC, the NTP and EPA as a “Known Human Carcinogen”

• Epidemiologic data: SBR process is associated with increase leukemia in workers; monomer workers have increased lymphoma, but not leukemia

• Significant species differences in carcinogenicity: mice are much more sensitive than rats, the sites of tumors also differ.

• Formation of diepoxybutane is much greater in mice than rats. The diepoxide is 100-200 times more mutagenic than the monoepoxide or diolepoxide. Numerous DNA adducts are formed, with N7-guanine adducts being most prevalent.

Goggin, M. et al. Cancer Res 2009;69:2479-2486

Metabolic activation of BD to reactive electrophiles and the formation of DNA adducts


Dose-dependent formation of bis-N7G-BD in liver DNA of female B6C3F1 mice and female F344 rats exposed to BD by inhalation

BD-induced N-7 Guanine Adductsin Female and Male Mice (Adducts per 108 Guanine)

Exposure

(ppm)N7-HBG Bis-N7G-BD N7-THBG

Male Female Male Female Male Female

0.5 ND ND N/A 0.9 ± 0.6 9.5 ±1.5 8.3 ± 1.5

1.0 N/A ND N/A 1.4 ± 1.1 N/A 17.1 ± 1.6

1.5 ND ND N/A 2.2 ± 1.1 35.3± 6.7 31.6 ±3.6

6.25 N/A 8.3 ± 4.9 N/A 16.4 ± 4.62 N/A 70.1 ± 8.7

62.5 N/A 29 ± 11 N/A 41 N/A 732 ± 150

200 122 ± 20 138 ± 12 N/A 102 3914 ± 682 3635 ± 353

625 659 ± 85 728 ± 824 N/A 202 ± 46 6205 ± 5 6103 ± 1140

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

Lack of Gender Differences in Pyr-Val in Mice Exposed to Butadiene

0

500

1000

1500

2000

0 10 20 30 40 50 60 70

Am

ou

nts

of

pyr

-Val

[p

mo

l/g

]

BD Exposure evels [ppm]

males

females

Lack of Gender Differences in Pyr-Val in Rats Exposed to Butadiene

0

20

40

60

80

100

120

140

160

0 100 200 300 400 500 600 700

Am

ounts

of pyr-

Val [p

mol/

g]

BD exposure [ppm]

males

females

0

2

4

6

8

10

0 2 4 6

Hprt Mutation Induction and pyr-Val Adduct Formation in Female Mice Exposed to BD for 10 Days

3 6.25 20 62.5 625 1250

BD exposure [ppm]

0

5

10

15

20

25pyr-Val adducts/ppm BD Induced Hprt MF x 10(-8)/ppm BD

Georgieva et al. Tox. Sci. 2010

Copyright ©2009 American Association for Cancer Research


Figure 4. Gender differences in the formation of racemic bis-N7G-BD in B6C3F1 mice and F344 rats exposed to 625 ppm BD, and mice exposed to 200 ppm BD by inhalation for 2

wk

Male Female

BD

-in

du

ced

Mu

tan

t F

req

ue

nci

es

(x 1

0-6)

0

2

4

6

8

10

12

14

16

18

20

Black bars = mice; Gray bars = rats

Gender and species differences in BD-induced Hprt MFs in mice and rats exposed for 2 weeks to 1250 ppm BD

DNA Damage Response to Butadiene Diepoxide in DT-40 Cells

HR

1

10

100

0.00 0.25 0.50 0.75

DEB (M)

Su

rviv

al

(%)

wt

fancd2

xrcc2

xrcc3

BER/NER

1

10

100

0.00 0.25 0.50 0.75

DEB (M)

Su

rviv

al

(%)

wt

polb

fen1

parp1

xpa

Czech BD Gender Study

• A second molecular epidemiology study was conducted in the same plant to investigate gender differences in biomarkers using 26 female controls, 23 female BD-exposed workers, 25 male controls and 30 male BD-exposed workers (Albertini RJ, et al., Chem Biol Interact. 166:63-77, 2007.)

• The design was similar to the 2003 study, but BD exposures were lower (Exposed Males - 0.81 mg/m3 [0.37 ppm], Exposed Females – 0.397 mg/m3[0.18 ppm]).

• Biomarkers of exposure and effect were measured including: urinary metabolites, hemoglobin adducts, chromosomal aberrations and HPRT mutations, as well as metabolic genotypes.

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)

0.295

0.5180

2.2715

p<0.05p<0.05

Net M1 (ug/L)

Net M2 (ug/L)

-219-0.05

2135.3

270084

p<0.05p<0.05

Hprt VF (106)

MF (106)

10.7513.00

5.7310.69

6.4818.83

p<0.05--

SCE (SCE/cell)

CA (%)

6.321.56

6.141.52

6.471.54

----

Albertini et al., Res. Rep. Health Eff. Inst., 1-141 (2003).

Calculation for the EB-dose Calculation for the EB-dose Equivalent in Humans, Rats and MiceEquivalent in Humans, Rats and Mice

Dose-equivalent = EB × 1 + DEB × 32 + EBdiol × 0.21 Dose-equivalent = EB × 1 + DEB × 32 + EBdiol × 0.21

CONCLUSIONS• The collective data from nearly 20 years of research by many

investigators has provided an excellent understanding of the mode of action for butadiene carcinogenesis.

• It is clear that mice are much more sensitive than rats and humans as a result of more efficient metabolism of BD to BRIs.

• Potentially important gender differences have been demonstrated in rats and mice, where increases in DNA cross-links and mutagenesis were shown in female rats and mice, compared to males. This is most likely due to differences in DNA repair, as metabolism was not different.

• Using the EB-equivalents concept, we found that mice produce ~44 and 174 times greater numbers of EB dose equivalents than rats and humans, respectively.

• In three blinded molecular epidemiology studies, no increases in HPRT or chromosomal mutations have been demonstrated under current occupational exposures.

ED0.01 Carcinogenicity Study

• A carcinogenicity study has been conducted on Dibenzo[a,l]pyrene using 42,000 rainbow trout.

• This model is 50 times more sensitive than rodent bioassays due to the low background incidence of neoplasia.

• The EPA linear risk model over estimated the actual observed liver cancer incidence by three orders of magnitude.

Bailey et al, Chem. Res. Tox., 2009

2005 EPA Guidelines for Carcinogen Risk Assessment

• Linear extrapolation should be used when there are Mode Of Action data to indicate that the dose-response curve is expected to have a linear component below the POD.

– Agents that are DNA-reactive and have direct mutagenic activity.

• The EPA Guidelines also suggest using a Framework Analysis approach to support or not support a proposed Mode Of Action.

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

MOA Key Events

Genotoxicity

DNA Adducts

Mutations in reporter genes

Mutations in cancer genes

Cancer

Genotoxicity• A chemical is defined as genotoxic if the weight

of evidence is positive in a battery of genetic toxicology assays.

• This is not a quantitative data set.

• Such data represents Hazard Identification, not Risk Assessment.

MOA Key Events

Genotoxic

DNA Adducts



Cancer

Molecular Dosimetry of DNA Adducts

• DNA adducts are expected to be linear at low doses.

• An exception to this is when identical adducts are formed endogenously.

• Many forms of endogenous DNA adducts have been identified and measured. These include direct oxidative adducts, exocyclic adducts, AP sites and deamination products.

MMS 1 hr exposure (HeLa cells)(912-06 to 927-06, combined)

y = 0.2757x + 1.642

R2 = 0.9922

y = 0.2832x - 4.0077R2 = 0.9977

0

50

100

150

200

250

300

350

400

450

0 200 400 600 800 1000 1200

MMS (uM)

N7-m

e(d

)G(R

O)

(fm

ol/u

g D

NA

)

0

50

100

150

200

250

300

0 100 200 300 400 500

pmol

HP

G/m

g D

NA

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)

Alkylations/10 guanines

Alkylations/10 guanines

6 6

A B

Linear DNA Adducts at Low Doses

MMS DBP

PO DMN

MOA Key Events

Genotoxic

DNA Adducts



Cancer

Mutations Do Not Go Through Zero

• In contrast to most DNA adducts, mutations do not go through zero.

• Rather, they reach a spontaneous level that reflects the summation of endogenous DNA damage and repair that occurs in cells.

• The point in the dose response curve where the number of mutations significantly increase above the spontaneous level represents the point at which the exogenous DNA damage starts driving the biology that results in additional mutations.

0

50

100

150

200

250

300

350

0 100 200 300 400 500

DNA Adducts

Mut

ant F

requ

ency

Inflection point

Dose

Typical Mutation Dose Response

0

1

2

3

4

5

6

7

TK6 AHH-1

Cell Line

Mu

tan

t F

ract

ion

(x1

0-6)

q1

min

median

max

q3

tk locusn=87

hprt locusn=34

95th

Historical Control Data for HPRT and TK Mutations in vitro

Penman and Crespi, Environ Mol Mut 10:35-60, 1987

The Formation of Endogenous and Exogenous N2-Ethylidene-dG in TK6 Cells Using [13C2]-Acetaldehyde

The Sum of Endogenous and Exogenous N2-Ethylidene-dG in TK6 Cells Using [13C2]-Acetaldehyde

Micronucleus Formation in TK6 Cells exposed to [13C2]-Acetaldehyde

Survival of TK6 Cells exposed to [13C2]-Acetaldehyde

0

0.5

1

1.5

2

0 0.5 1 1.5 2 2.5 3

Acrylamide Dose (mg/kg)

fMP

CE

(m

ea

n/m

ile

)

0

50

100

150

200

250

300

DN

A A

dd

uc

ts (/1

08 n

td)

fMPCEDNA Adducts

0

0.05

0.1

0.15

0.2

0.25

0 0.5 1 1.5 2

Acrylamide Dose (mg/kg/d)

%M

N0

10000

20000

30000

40000

50000

GA

Gu

a (fmo

l/mg

DN

A)

%MN-NCE

GAGua

Abramsson-Zetterberg, 2003 Zeiger, et al., 2007

Relationships Between DNA Adducts and Micronucleus Induction in Mice with Carcinogenic Doses of Acrylamide

Twaddle et al, 2004Tareke et al, 2006Young, et al, 2007

0

5

10

15

20

0 50 100 150 200

Ethylene Oxide (or equivalent) Dose (ppm)

Hp

rt M

F (

X10

6 )

0

1

2

3

4

5

0 10 20 30 40 50

Ethylene Oxide (or equivalent) Dose (ppm)

Hp

rt M

F (

X10

6 )

DNA Repair Can Modulate Where Increased Mutations Occur

• If DNA repair is impaired or absent, the inflection point for mutations occurs at lower doses.

• This results from increased numbers of DNA adducts relative to a cell with normal DNA repair.

6

0.1

1

10

100

0.01 0.1 1 10 100

7-HPG/10 nucleotides

ind

uc

ed

RL

mu

tan

t fr

eq

ue

nc

y

PO-NER+PO-NER-

6

Exposure-response for Mutagenesis in Drosophila ExposedBy Inhalation To Propylene Oxide

Nivard et al, Mut. Res. 529: 95-107, 2003.

12,000 ppm hr PO

EMS and ENU in MutaMouse, GI tract 4-week treatment

acute treatment, sampling day 7acute treament, sampling day 30

0

50

100

150

200

250

300

350

400

450

0 50 100 150 200 250 300 350 400cumulated dose (mg/kg)

Mu

tan

t fr

equ

ency

(10

-6)

EMS 4-week

ENU acute day31

ENU acute day 7

ENU 4-week

EMS acute day 7

EMS acute day 31

Comparison of Acute and 28-Day Treatments

350 mg/kg

12.5 mg/kg/day

MOA Key Events

Genotoxic

DNA Adducts



Cancer

Gaps in Knowledge

• Most mutation assays are done at high doses to establish that a compound is or is not genotoxic.

• There is a real need to generate dose response data at low exposures to establish NOAELs for mutation in CA, MN and surrogate genes such as hprt.

• These data will further establish the exposures where the background number of mutations become significantly increased.

Conclusions

• As our knowledge of carcinogenesis has expanded, concepts of “one molecule → cancer “ have little to no scientific support.

• Mutations in genes controlling cell proliferation and cell death appear to play major roles in the induction of cancer.

• While these genes are difficult to monitor in noncancer tissues, surrogate mutations can be used to examine dose response in cells, animals and humans.

Conclusions (cont.)

• Such mutations do not have linear relationships with exposure. Rather, they reach a spontaneous incidence that is driven by endogenous biological processes.

• The exposures where mutagenesis becomes significantly increased over background represents a scientifically based Point of Departure for setting acceptable exposures.

• This could be accomplished by using a Margin of Exposure approach to protect susceptible individuals.

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

The Exposome• Chris Wild proposed that we should be considering the

“Exposome” for cancer etiology. Wild, C: CEBP 14: 1847-1850, 2005

– Under this view, the assessment of exposures should not be restricted to chemicals entering the body from air, water, food, smoking, etc., but should also include internally generated toxicants produced by the gut flora, inflammation, oxidative stress, lipid peroxidation, infections, and other natural biological processes. In other words, we must focus upon the ‘internal chemical environment’ arising from all exposures to bioactive chemicals inside the body

• More recently, Martyn Smith et. al. made similar statements. Smith, M: Chemico Biological Interactions 192: 155-159, 2011

– The question arises as to how to find the causes of the majority of de novo AMLs that remain unexplained. We propose that we should attempt to characterize the 'exposome' of human leukemia by using unbiased laboratory-based methods to find the unknown 'environmental' factors that contribute to leukemia etiology.

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

issues related to uncertainty in risk assessment high to low dose extrapolation species to species...

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