1. Endogenous DNA Adducts: Key Events in the MOA for Low Dose Risk Assessment
James A. Swenberg, D.V.M., Ph.D.
University of North Carolina
Chapel Hill, NC 27599

2. DNA Adducts: Biomarkers of Exposure
DNA adducts are biomarkers of exposure, not effect.
DNA adducts are not mutations, are repairable and have vastly different abilities to cause mutations
The molecular dose of DNA or protein adducts integrates our knowledge of metabolism, detoxication and DNA repair. Thus, it provides important information on species differences.
DNA adducts are expected to be linear at low doses.
An exception to this is when identical adducts are formed endogenously and methods used cannot discriminate between endogenous and exogenous adducts.
Many forms of endogenous DNA adducts have been quantified. These include direct oxidative adducts, exocyclic adducts, AP sites, ethylene oxide, formaldehyde, deamination products, etc. totaling >40,000 per cell.

3. 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.
Issues related to low dose extrapolation are important, as vinyl chloride is present in many Superfund sites and some public drinking water in ppb amounts.
Identical endogenous adducts are present in rodent and human tissues.

4. Formation of DNA Adducts by Vinyl ChlorideCl O
CYP450 2E1
vinyl chloride
chloroethylene oxide O
DNA O O N H N H N H N N N N N H N N 2
7-(2-oxoethyl)guanine N 2
,3-ethenoguanine N d R i b O
3,N4-ethenodeoxycytidine N d R i b
1,N6-ethenodeoxyadenosine

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

6. Sample Chromatogram of 7-OEG
2.5
3.0
3.5
4.0
4.5
Time (min) 10 20 30 40 50 60 70 80 90
100
Relative Abundance
RT: 3.35
MA:
2.49
3.74
4.13
4.37
4.60
MA:
3.06
3.68
3.87
2.74
3.99
2.62
4.39
4.58
MA:
2.77
3.07
2.56
3.59
3.83
4.44
4.07
4.66
4.27 A
2.5
3.0
3.5
4.0
4.5
Time (min) 10 20 30 40 50 60 70 80 90
100
Relative Abundance
RT: 3.36
MA:
2.59
3.98
4.09
4.38
4.56
RT: 3.35
MA:
3.06
3.67
3.88
4.04
2.72
4.44
2.54
4.29
4.61
MA:
2.77
2.68
3.68
4.54
3.90 B
m/z 265152 AST
m/z 267152 13C2-OEG
m/z 270157 IST
A. Adult rat liver(1100 ppm [13C2]-VC, 5days)
B. Weanling rat liver (1100 ppm [13C2]-VC, 5days)

7. 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]-
N2,3-εG/
108 Gua
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
4 Weeks Post Exposure
0.2 ± 0.04
0.1± 0.06
3.1 ± 1.0
16.9 ± 1.6
6.2 ± 1.3
8 Weeks Post Exposure
0.2 ± 0.07
3.7 ± 1.5
13.2 ± 2.5
4.1 ± 0.5

8. T1/2 and Repair Pathways For VC-Induced DNA Adducts
7OEG
4 Days
Chemical depurination
N2,3-εG
150 Days
Unknown
1,N6- εdA
~1 Day
MPG/Aag
AlkB
3,N4- εdC
DNA glycosylases

9. N2,3-eG in Control and VC-exposed Samples
None
1100 ppm VC; 4 wk
1100 ppm VC; 4 wk
10 ppm VC; 4 wk
None known
Exposure
110 ± 20
15 ± 1.0
3.0 ± 0.3
SD rat hepatocytes
16 ± 5
Human colon
17 ± 2
Human liver
5.6 ± 1.1
6.7 ± 0.6
SD rat brain
N2,3-eG / 108 G
Sample

10. Introduction
Formaldehyde is produced worldwide more than 20 million tons/year and used in a wide spectrum of applications. Therefore, formaldehyde exposures from environmental and occupational sources are quite common.
Formaldehyde is a known animal and human carcinogen, causing nasal cancer.
1.rats: 15ppm formaldehyde induced 50% incidence of nasal carcinomas after 2 year-exposure (10ppm formaldehyde caused 22% incidence).
2.humans: “sufficient epidemiological evidence that formaldehyde causes nasopharyngeal cancer in humans” according to IARC
Limited evidence to support formaldehyde inducing leukemia.
1.“strong but not sufficient evidence for a causal association between leukemia and occupational exposure to formaldehyde” based on IARC ( in 2006)
2.no mechanism of induction of leukemia in human has been identified
FEMA trailers used after Hurricane
Katrina
15ppm 12-month formaldehyde
induced nasal tumor

11. Formaldehyde-induced cell proliferation
Therefore, significant cell proliferation could convert both endogenous and exogenous DNA adducts into mutations during enhanced cell replication.

12. Formaldehyde is a ubiquitous environment pollutant, but it is also an essential metabolite in all living cells. Therefore, both endogenous and exogenous formaldehyde is always present.
Formaldehyde is very reactive with DNA and proteins, leading to diverse protein adducts and DNA damage.
Fate and metabolism of formaldehyde
glutathione
S-hydroxymethyl-
ADH3
S-formylglutathione
hydrolase
formate
CO2+H2O
endogenous
sources
exogenous
ALDH1A1
ALDH2
one
carbon
pool
adduct
formation
Adapted for IARC monograph 88

13. in vivo data in rats exposed to [13CD2]-formaldehyde
Rats were exposed to 10 ppm [13CD2]-FA for 1 day (6h/day) and 5 days.
To determine whether or not formaldehyde has distant genotoxic effects.
Both DNA monoadducts and DNA-DNA cross-links were measured.
30-50µg DNA was used for nasal epithelium, while 200µg DNA was used for distant tissues.
Digested DNA was separated by HPLC. All the adducts from DNA digestion was loaded on the column. Therefore, we have 5 times higher detecting capability for distant tissues.
Lu, K., Collins, L.B, Ru, H.Y., Bermudez,E., Swenberg, J.A. DNA Adducts Caused by Inhaled Formaldehyde Are Found
in Nasal Epithelium, but not Bone Marrow. Toxicological Sciences 116: 441–451 (2010).

14. LC-ESI-MS/MS SRM chromatograms of N2-Me-dG in typical tissues
6.0
6.5
7.0
7.5 20 40 60 80
100
RT: 7.55
RT: 7.52
RT: 7.54
RT: 7.56
Time (min)
RT: 7.53 A. B. C.
Endogenous
Exogenous
Internal standard D.
1 day-exposed nasal epithelium (A), 5 day-exposed nasal epithelium (B), bone marrow (C) and spleen (D).

15. LC-ESI-MS/MS SRM chromatograms of N6-Me-dA of typical tissues of rats:
8.0
8.5
9.0
9.5
10.0
Time (min) 20 40 60 80
100
RT: 8.46
MA:
RT: 8.43
MA:
RT: 8.51
MA:
RT: 8.49
MA:
RT: 8.45
MA:
RT: 8.44
MA:
RT: 8.57
MA:
RT: 8.55
AA:
m/z → m/z 150.1
m/z → m/z 153.1
m/z → m/z 155.1 A. B. C. D.
LC-ESI-MS/MS SRM chromatograms of N6-Me-dA of typical tissues of rats:
nasal epithelium of a 1 day-exposed rat (A); nasal epithelium of a 5 day-exposed rat (B)
bone marrow of a 5 day-exposed rat (C); spleen of a 5 day-exposed rat (D).

16. From Cheng et al., Chem. Res. Toxicol. 21, 746-751,2008.
Formaldehyde-induced monoadducts in tissues of rats exposed to 10 ppm [13CD2]-formaldehyde for 1 day or 5 days
Exposure period
Tissues
N2-HOCH2-dG
(adducts/107 dG)
N6-HOCH2-dA
(adducts/107 dA)
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‡
2.62±0.24
Liver
2.66±0.53
2.62±0.46 #
Spleen
2.35±0.31
1.85±0.19
Bone Marrow
1.05±0.14
2.95±1.32
Thymus
2.19±0.36
2.98±1.11
5 day
2.43±0.78
2.84±1.13
3.61±0.95
2.61±0.35
2.47±0.55
3.24±0.42
2.87±0.65
2.35±0.59
2.23±0.89
1.17±0.35
2.99±0.08
1.99±0.30
2.48±0.11
# 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, ,2008.

17. dG-CH2-dG (adducts/107 dG)
Formaldehyde-induced dG-dG cross-links in tissues of rats exposed to10 ppm [13CD2]-formaldehyde for 1 day or 5 days
Exposure period
Tissues
dG-CH2-dG (adducts/107 dG)
exogenous
endogenous
1 day
Nose
0.14±0.06§
0.17±0.05
Lung
n.d.
0.20±0.04¶
Liver
0.18±0.05
Spleen
0.15±0.06
Bone Marrow
0.09±0.01
Thymus
0.10±0.03
5 day
0.26±0.07
0.18±0.06
0.20±0.03
0.21±0.08
0.16±0.08
0.11±0.03
0.19±0.03

18. Improved Methodology LOD: 20 attomoles LOQ: 40 attomoles
Instrumentation
Waters NanoAcquity UPLC
Waters C18 T3 Nano
Flow Rate: 0.6 µL/min
24 minute reverse phase gradient
Mobile Phases:
A) Water with 0.1% Acetic Acid
B) ACN with 0.1 % Acetic Acid
Thermo Quantum Ultra Triple Quadrupole MS
Scan Speed: 0.1 seconds per transition
Collision Energy: 17 eV
Peak Width
Q1: 0.3 dalton
Q3: 0.5 dalton
Scan Width: 1 dalton
ESI nano source – positive mode

19. Dosimetry of N2-hydroxymethyl-dG Adducts
Exposure
(ppm)
Exogenous adducts/107 dG
Endogenous adducts/107 dG n
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
Endogenous
282.2 → m/z
Exogenous
285.2 → m/z
Internal Standard
297.2 → m/z
4.9 adducts/
107 dG
9.0 adducts/
20 fmol
15 ppm Rat NE
*4-6 rats combined
** 2 rats combined

20. Ratio of Exogenous to Endogenous Adducts

21. N2-hydroxymethyl-dG Adduct Half-life Study
t1/2 = 63 hours
Y= x – 0.46
R2 = 0.771
n=5 per time point
Mean ± SD
Hours

22. Adduct Numbers in Primate Nasal Maxilloturinbates
Exposure concentration
Exogenous adducts/107 dG
Endogenous adducts/107 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

23. Primate Femoral Bone Marrow Endogenous and Exogenous Adducts
282.2 → m/z
Exogenous
285.2 → m/z
Internal Standard
297.2 → m/z
7E6
6E4
2E6
Endogenous
282.2 → m/z
Exogenous
285.2 → m/z
Internal Standard
297.2 → m/z
312 µg DNA
178 µg DNA
No Exogenous Adducts Detected with 5-10 fold >DNA
On average we used between ug of DNA for the Nasal tissues
Note: We used ~20-30 ug for nasal tissue
1.9 ppm 13CD2O
6.1 ppm 13CD2O 23

24. 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.

25. Risk Assessment Model is Public Health 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.

27. Conclusions to date
Exposure-induced DNA monoadducts and cross-links only occur in nasal epithelial DNA in rats and primates.
Only dG monoadducts and cross-links are formed following inhalation and in vitro exposures to formaldehyde.
dA monoadducts arise from intracellular formation of formaldehyde secondary to intracellular metabolism.
Endogenous DNA monoadducts (dG and dA) are present in all cells and tissues.
Endogenous adducts are present in fold greater amounts than exogenous adducts following 10 ppm exposures to [13CD2]-formaldehyde, but 100-fold greater at 1 ppm.
These data strongly challenge the biological plausibility that formaldehyde causes leukemia.

28. Conclusions to date
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 would be 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.

29. Ethylene Oxide
Ethylene oxide (EO) is a classified as a known human and animal carcinogen.
EO is genotoxic and mutagenic.
DNA and hemoglobin adducts of EO are present in all individuals and animals.
These arise from metabolism of ethylene to EO and from oxidative stress.
Everyone in this room is breathing off ppm 24/7.
The draft IRIS risk assessment gives a virtually safe exposure as <1 ppt. This is ~1000 times less than our endogenous daily exposure.

30. EO Low Dose Mutagenesis

31. Oxidative Stress Induced-DNA Damage

32. Endogenous AP Sites in Rat Tissues and Human Liver
[Nakamura and Swenberg, 1999]

33. Effects of PCB 126 and 153 on Oxidative DNA Adducts

34. Steady-state Amounts of Endogenous DNA Damage
Endogenous DNA Lesions
Number per Cell
Abasic sites
30,000
OHEtG
3,000
8-oxodG
2,400
7-(2-Oxoethyl)guanine
1,500
Formaldehyde
960
AcrdG
120
M1dG 60
N2,3-Ethenoguanine 36
1N2-Etheno dG 30
1N6-Etheno dA 12
Total
38,118

35. What are the Issues?
Linear extrapolations of cancer risks lead to highly conservative assessments of risk that represent science policy aimed at protecting the public’s health.
The accuracy of these estimates has a high degree of uncertainty
Genotoxicity issues drive a linear default
This presentation will examine our present understanding of the science involved in mutagenesis and carcinogenesis that should drive low dose risk assessment.

36. Mutations and Cancer
There is general consensus that multiple mutations are the primary mechanism involved in carcinogenesis (Hanahan and Weinberg, 2000).
The dose-response of this biomarker of effect has not received the same degree of consideration as have DNA adducts in cancer risk assessment, even though their use could be highly informative.

37. MOA Key Events for Mutations
Genotoxicity
DNA Adducts
Mutations in Reporter Genes
Mutations in Cancer Genes (Bioindicators)
Cancer

38. Mutations Do Not Go Through Zero
In contrast to most DNA adducts, mutations do not go through zero.
Rather, they reach a background level that reflects the summation of mutations arising from endogenous DNA damage and repair that occurs in cells.
The dose-response may be linear or nonlinear.
There may be an inflection point for a dose response curve where the number of mutations increases nonlinearly above the spontaneous level, or there may be a linear increase with data points that are not significantly different from controls at lower doses.
The point at which the mutations increase is where the exogenous DNA damage starts driving the biology that results in additional mutations.

39. MMS induced N7 and O6 adducts39

40. Relationships Between DNA Adducts and Micronucleus
Induction in Mice with Carcinogenic Doses of Acrylamide
Abramsson-Zetterberg, 2003
Zeiger, et al., 2007
Twaddle et al, 2004
Tareke et al, 2006
Young, et al, 2007

41. Comparison of Acute and 28-Day Treatments
350 mg/kg
12.5 mg/kg/day

42. 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

43. 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, reporter gene mutations can be used to examine dose response relationships in cells, animals and humans.

44. Conclusions (cont.)
New scientific approaches are needed to determine where in the dose response paradigm mutations are increased over background and where they are driven by endogenous biological processes.
The Bier Report (1980) Concluded “Use simple linear interpolation between the lowest reliable dose data and the spontaneous …rate.”
Such data represent a critical need to support a Point of Departure for setting acceptable exposures.
This could be accomplished by using a Margin of Exposure approach to protect susceptible individuals.

45. Conclusions (cont.)
All living cells have large amounts of endogenous DNA damage and this damage is responsible for background mutations.
Chemical exposures can cause DNA lesions that are unique or identical to endogenous DNA damage.
At high exposures, such as used in carcinogenicity bioassays, ROS-induced and exogenous DNA damage represent key factors driving mutagenesis.
At very low exposures, such as used in linear low dose extrapolations, the amount of endogenous DNA damage vastly exceeds chemical specific damage .
Focused research to increase our scientific knowledge related to the role of endogenous DNA adducts should play an important role in risk assessment.

46. Default The word default first came into use in the 1200’s.
A failure to meet one’s obligation
A sin
The above concept is certainly applicable to risk assessment.
We have failed to meet our obligation to use the best science when we resort to defaults.

47. Collaborators and Sponsors
Jun Nakamura
Paul Chastain
Eric Morinello
Esra Mutlu
Lina Gao
Kun Lu
Gunnar Boysen
Leonard Collins
Patricia Upton
Darrell Winsett
Gary Hatch
Ed Bermudez
Superfund Basic Research Program (P42-ES 5948)
NIEHS (P30 ES 10126)
American Chemistry Council
Formaldehyde Council
Environmental Protection Agency
Hamner Institutes for Health Sciences