Inhaled Formaldehyde: Exogenous and Endogenous DNA Adducts and Epigenetic Alterations of microRNAs

James Swenberg, D.V.M., Ph.D. jswenber@email.unc.edu 919-966-6139

Introduction
More than 20 million tons/year of formaldehyde is produced worldwide 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

FEMA trailers used after Hurricane Katrina

Limited evidence exists 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 convincing mechanism for the induction of leukemia has been identified

15ppm 12-month formaldehyde induced nasal tumor

Tumor Incidence and Cell Proliferation in Rats Exposed to Formaldehyde

70 60 50
Tumor Incidence (%)
Tumor Incidence 24-month St udy (Kerns, 1983)

14
Cell Proliferation (mean unit length labeling index) at Nasal Level II (fold increase over control)

12 10 8 6 4 2 0

Tumor Incidence 24-month St udy (M onticello, 1996)

40 30 20 10 0 0 2

Cell Proliferat ion Study 6-mont h (M onticello, 1990)

Cell Proliferat ion Study 12-month (M onticello, 1990)

Cell Proliferat ion Study 18-month (M onticello, 1990)

10

12

14

16

HCHO Concentration (ppm)

 Formaldehyde is a ubiquitous environment pollutant, but it is also an essential metabolite in all living cells. Therefore, both endogenous and exogenous formaldehyde need to be considered in risk assessment. Formaldehyde is very reactive with proteins and DNA, leading to diverse protein adducts and DNA damage.
Fate and metabolism of formaldehyde endogenous sources exogenous sources adduct formation

glutathione

S-hydroxymethylglutathione
ALDH1A1 ALDH2

ADH3 S-formylglutathione

one carbon pool

glutathione

S-formylglutathione hydrolase

formate CO2+H2O
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, mononuclear WBC 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. 20-40 g of DNA was used for nasal tissue, bone marrow and WBC, while 200 g was analyzed for other tissues. Thus, 5-10-fold more DNA was analyzed from Tissues distal to the site of contact. Capillary MS/MS methods were developed for N2-methyl-dG (detection limit 200 amol) and N6-CH3-dA (detection limit 50 amol) monoadducts. Nano-UPLC-MS/MS methods were developed for dG-dG cross-links (detection limit 60 amol). Endogenous and [13CD2]-adducts were measured.

Scheme 1. The formation of N2-hydroxymethyl-dG originating from both endogenous and exogenous formaldehyde.

100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 6.0 6.5 7.0 Time (min) 7.5 m/z 297.2 m/z 176.1 RT: 7.53 MA: 2266960 m/z 285.2 m/z 169.1 m/z 282.2 m/z 166.1 6.0 6.5 7.0 Time (min) 7.5 m/z 297.2 m/z 176.1 RT: 7.54 AA: 3130922 m/z 285.2 m/z 169.1 RT: 7.52 AA: 283694

A.
m/z 282.2 m/z 166.1

RT: 7.55 MA: 426271

100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 6.0 6.5 7.0 Time (min) 7.5 RT: 7.55 MA: 952352 m/z 282.2 m/z 166.1 m/z 297.2 m/z 176.1 RT: 7.54 AA: 3159370 m/z 285.2 m/z 169.1 RT: 7.54 AA: 623964

B. m/z 282.2 m/z 166.1

RT: 7.56 MA: 386661

C.

RT: 7.53 MA: 450110

80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0

D.

m/z 285.2 m/z 169.1

RT: 7.56 MA: 3205157

m/z 297.2 m/z 176.1

6.0

6.5

7.0 Time (min)

7.5

LC-ESI-MS/MS SRM chromatograms of N2-Me-dG in typical tissues: 1 day-exposed nasal epithelium (A), 5 day-exposed nasal epithelium (B), bone marrow (C) and spleen (D).

Improved Methodology
LOD: 20 attomoles LOQ: 40 attomoles Instrumentation
Waters NanoAcquity UPLC
Waters C18 T3 Nano Flow Rate: 0.6 L/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

Dosimetry of N2-hydroxymethyl-dG Adducts in Nasal Epithelium of Rats

3000000 2500000 2000000 1500000 1000000 500000 0 3000000 2500000 2000000 Intensity 1500000 1000000 500000 0 3000000 RT: 10.31 2500000 2000000 1500000 1000000 500000 0 8 9 10 Time (min) 11 12 RT: 10.30

Endogenous 282.2 166.1 m/z

4.9 adducts/ 107 dG

Exposure (ppm) 0.70.2

Exogenous adducts/107 dG 0.0390.019 0.190.08 1.040.24 2.030.43 11.153.01

Endogenous adducts/107 n dG 3.621.33 6.093.03 5.511.06 3.410.46 4.240.92 3* 4** 4 5 5

Exogenous 285.2 169.1 m/z

RT: 10.30

9.0 adducts/ 107 dG

2.00.1 5.80.5 9.12.2 15.22.1

Internal Standard 297.2 176.1 m/z

20 fmol

15 ppm Rat NE

*4-6 rats combined ** 2 rats combined

Ratio of Exogenous to Endogenous Adducts

Exogenous

3 Ratio of Exogenous Versus Endogenous Adducts 2.5 2 1.5 1 0.5 0

Endogenous

5 10 15 20 Formaldehyde Exposure Dose(ppm)

N2-hydroxymethyl-dG Adduct Half-life Study

1

ln (Exogenous Adducts/10 dG)

t1/2 = 63 hours
0

-1

-2

Y= -0.011x 0.46 R2 = 0.771

20

40

60

80
n=5 per time point Mean SD

Hours Days

Non-Human Primate Study

13CD O 2

Exposure for 2 days (6 hours/day) at 2 or 6 ppm (n=4)

Cynomolgus Macaque Tissues (to date) Nasal turbinates Femoral Bone Marrow Brain Lung

Adduct Numbers in Primate Nasal Maxilloturinbates

Exposure concentrati on 1.9 ppm 6.1 ppm Exogenous adducts/107 dG 0.25 0.04 0.41 0.05 Endogenous adducts/107 dG 2.49 0.39 2.05 0.53
n = 3 or 4

Primate Femoral Bone Marrow Endogenous and Exogenous Adducts

RT: 10.52 20000000

Endogenous 282.2 166.1 m/z

2E7

RT: 10.62 6000000 5000000

15000000 Intensity

10000000

5000000

312 g DNA
Exogenous 285.2 169.1 m/z 4E4

Intensity

4000000 3000000 2000000 1000000 0

Endogenous 282.2 166.1 m/z

7E6

178 g DNA
6E4

0 40000 35000 30000 Intensity

Intensity

50000 40000 30000 20000 10000

Exogenous 285.2 169.1 m/z

25000 20000 15000 10000 5000 0 3000000 2500000 2000000 RT: 10.52

0 1800000

RT: 10.62

No Exogenous Adducts Detected with 5-10 fold >DNA Note: We used ~2030 ug for nasal tissue

Internal Standard 297.2 176.1 m/z

3E6

1600000 1400000 1200000 Intensity 1000000 800000 600000 400000

Intensity

Internal Standard 297.2 176.1 m/z

2E6

1500000 1000000 500000 0 8 9 10 Time (min) 11 12

200000 0 8 9 10 Time (min) 11 12

1.9 ppm 13CD2O

6.1 ppm 13CD2O

Adduct Numbers in Primate Bone Marrow

Exposure concentrati on 1.9 ppm 6.1 ppm Exogenous adducts/107 dG nd nd Endogenous adducts/107 dG 17.48 2.61 12.45 3.63
n=4

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 (rats and primates).

MicroRNA Study
Acquired nasal maxilloturbinate samples (stored in RNAlater) from cynomolgus macaques from the Moeller et. al. study Isolated small RNA molecules Generated Agilent miRNA Microarray using 2 controls 3 2ppm formaldehyde tissue samples 3 6ppm formaldehyde tissue samples Statistical analysis revealed 3 unique miRNAs with significantly different expression in monkeys exposed to 2 ppm formaldehyde (Fold Change >= +/- 1.5, ANOVA p < 0.05, FDR q < 0.10) Statistical analysis revealed 13 unique miRNAs significantly differentially expressed in monkeys exposed to 6 ppm formaldehyde

Significance of Findings
All 3 of the significantly differentially expressed miRNAs in the 2 ppm group were also significant in the 6 ppm group, where fold change magnitudes were larger (dose-response) Many of the significant miRNAs have known associations to cancer (based on literature searches): 4 of the 13 significant miRNAs were measured as significantly differentially expressed after 1 ppm formaldehyde exposure using human lung cancer cells in the Rager et al., 2011 study published in EHP.

Fold Change in Cancer-related miRNA

* Significant in exposed group compared to controls

Down-regulated by formaldehyde in Rager et al. 2011

RT-PCR of selected miRNAs with altered expression upon exposure to formaldehyde.

Predicted targets of formaldehyde-altered miR-125b are involved in apoptosis signaling

Apoptosis-related genes predicted to be targeted by miR-125b were confirmed using RT-PCR.

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 may arise from intracellular formation of formaldehyde secondary to intracellular metabolism or DPC. Endogenous DNA monoadducts (dG and dA) are present in all cells and tissues. Endogenous adducts are present in 2.5-3-fold greater amounts than exogenous adducts following 10 ppm exposures to [13CD2]-formaldehyde for 5 days, but 100-fold greater at ~1 ppm exposures for 1 day.

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.

Future Studies
We have just completed exposing rats to 2 ppm for up to 28 days.
DNA adducts to establish the time to steady-state and half-life at noncytotoxic exposures DNA protein cross-links DNA methylation MicroRNAs in nasal tissue and distant tissues

Human CD 34+ cells to establish endogenous adduct amounts. Human bone marrow to compare with monkey data. Human nasal turbinates to establish endogenous adduct amounts. A primate study to examine stem cells in CD 34+ primed monkeys exposed to [13CD2]-formaldehyde.

Repair of Aldehyde DNA Lesions

1000 RKO (parental) FANCG ko FANCC ko 100 Survival (% control)

10

0 0 20 40 Formaldehyde (M) 60 80

28 Ridpath, JR et al (2007) Cancer Res

DNA adduct analysis

Endogenous 296.1 180.1 m/z

us no ge do en
ex og en

ou

Exogenous 298.1 182.1 m/z

N2-ethylidene-dG reduced to N2-ethyl-dG for stability and LC-MS/MS analysis

DNA Adduct Analysis

DAD1 B, Sig=254,4 Ref=360,100 (BEN\10192011_AA_CALCURVE 2011-10-20 11-10-08\101911_AA_002.D)

Nucleosides

1.

Reduce DNA with NaCNBH3 at 37C, 6 hours with phosphate buffer, pH 7.2

2.

Digest DNA
500

Fraction Collection Chromatogram

3. 4. 5. 6.

Filter with MW cutoff (Pall 3Kd Nanosep) HPLC Fraction collection, ~60 min/sample
A 0.1 % acetic acid B - 0.1 % acetic acid in ACN

Dry in speedvac Re-dissolve in 10 L water, inject 5 L

Waters nanoUPLC and Thermo Quantum Ultra

nim

1. 2. 3.

Enyzmes: AP, PDE, DNAse Tris/MgCl2, pH 7.2, IS - 5 fmol 60 min with 10 min pre-incubation w/ DNAse at 37C

UAm
2000 1500 1000 0

1.

Isolate DNA using Nucleobond anion exchange columns

3.685

13.146

14.769

16.598

N2-ethyl-dG

10

15

20

25

30

35

40

45

UPLC-MS/MS Chromatograms
Control TK6 Cells
100 Relative Abundance 80 60 40 20 0 100 Relative Abundance 80 60 40 20 0 100 Relative Abundance 80 60 40 20 0 14 15 16 Time (min) 17 18
80 60 40 20 0 100 Relative Abundance 80 60 40 20 0 RT: 16.58 AA: 36539 RT: 15.70 AA: 2064135 Relative Abundance Relative Abundance

Endogenous 296.1 180.1 m/z

RT: 15.76 AA: 5557894

4.7E5

0.01 mM [13C2]Acetaldehyde
100

RT: 15.69 AA: 10457675

8.1E5

100

2.0 mM [13C2]Acetaldehyde
80 60 40 20 0

RT: 15.67 AA: 7391301

5.5E5

Relative Abundance

Exogenous 298.1 182.1 m/z

6.0E3

1.0E5

100 80 60 40 20 0 16.66

RT: 15.68 AA: 471225815

3.4E7

80 60 40 20 0 13 14 15

Relative Abundance

Relative Abundance

IS 301.1 185.1 m/z

16.55 RT: 15.75 AA: 1707053

1.9E5

100

2.9E5

RT: 15.69 AA: 2462242

100 80 60 40 20 0 13 14

RT: 15.67 AA: 3544716

2.9E5

16 Time (min)

17

18

15

16 Time (min)

17

18

Presence of a signal in exogenous chromatogram (298.1 182.1 m/z) is from the natural isotopic abundance (0.7%) of endogenous N2-ethyl-dG (296.1 180.1 m/z).

Exogenous and Endogenous DNA Adducts of Acetaldehyde in AHH-1 Cells

Sum of Adducts
y = 2.2 + 1.31 x + 0.30 x2 + 0.02 x3
500

Sum Adducts/107 dG

100 50

10 5

Endogenous Mean

1 5e-05 1e-04 0.001 0.005 0.01 0.05 [13C2-Acetaldehyde] mM 0.25 0.5 1.0 2.0

Polynomial Model of the Sum of Adducts. The mean of the endogenous adducts was 2.98 (green line) and the 95% prediction interval (dotted lines) of the log10(Total DNA adducts) are shown. Thus, we have 95% confidence that if the acetaldehyde concentration is above 0.02 mM, the sum of the adducts will be higher than the endogenous background.

34

95% Prediction Interval for Micronucleus

20

%MN = 0.53 + 1.38 X -1.02 X2 + 2.92 X3 Spearman rank correlation 0.698 (p-value 3.7e-05)

10 5

% MN

35
2 1 0.5

Vehicle Control

0.2 0.1

5e-05

5e-04

0.001

0.005

0.01

0.05

0.25

0.5

1.0

2.0

[13C2-Acetaldehyde] mM

% Micronuclei Formation versus [Acetaldehyde]. The mean % MN of the vehicle control was 0.61% (red dash) and the 95% prediction interval (dotted lines) are shown. Thus, we have 95% confidence that if the acetaldehyde concentration is above 0.35 mM, the future observation of % MN will be larger than the background of 0.61%.
n = 3 per except for 2.0 mM with n = 1

35

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.

Steady-state Amounts of Endogenous DNA Damage

Endogenous DNA Lesions Abasic sites OHEtG 7-(2-Oxoethyl)guanine 8-oxodG Formaldehyde Acetaldehyde 7-Methylguanine AcrdG M1dG N2,3-Ethenoguanine 1N2-Etheno dG 1N6-Etheno dA Total Number per Cell 50,000 3,000 3,000 2,400 1,000-4,000 1,000 1,200 120 60 36 30 12 60,000 +

Mutations Are Biomarkers of Effect, but They 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.

Historical Control Data for HPRT and TK Mutations in vitro

7 6

Mutant Fraction (x10 )

-6

4 tk locus n=87

q1 min median max q3

hprt locus n=34

0 TK6 AHH-1

Cell Line

95th

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

Collaborators and Sponsors

Kun Lu Ben Moeller Natalie Herr Tom Starr Leonard Collins Patricia Upton Betsy Bermudez Ed Bermudez Jacob McDonald Melanie Doyle-Eisele Andrew Gigliotti Julia Rager Rebecca Fry Formaldehyde Council FormaCare-CEFIC American Chemistry Council Hamner Institutes for Health Sciences Lovelace Respiratory Research Institute NIEHS Superfund Basic Research Program (P42-ES 5948) NIEHS Center for Environmental Health and Susceptibility (P30 ES 10126)