Inhaled Formaldehyde: Exogenous
and Endogenous DNA Adducts and
Epigenetic Alterations of
microRNAs
James Swenberg, D.V.M., Ph.D.
[email protected]
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.
FEMA trailers used after Hurricane
Katrina
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 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
14
Tumor Incidence 24-month St udy
(Kerns, 1983)
60
Tumor Incidence 24-month St udy
(M onticello, 1996)
50
Tumor Incidence (%)
12
10
Cell Proliferat ion Study 6-mont h
(M onticello, 1990)
40
8
Cell Proliferat ion Study 12-month
(M onticello, 1990)
30
6
Cell Proliferat ion Study 18-month
(M onticello, 1990)
20
4
10
2
0
0
0
2
4
6
8
10
HCHO Concentration (ppm)
12
14
16
Cell Proliferation (mean unit length labeling
index) at Nasal Level II (fold increase over control)
70
• 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
one
carbon
pool
S-formylglutathione
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 µg
of DNA was used for nasal tissue, bone marrow and WBC, while 200
µg 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
RT: 7.55
MA: 426271
A.
60
m/z 282.2 → m/z 166.1
100
60
40
40
20
20
0
0
100
m/z 285.2 → m/z 169.1
60
RT: 7.52
AA: 283694
80
40
20
20
RT: 7.54
AA: 3130922
100
0
RT: 7.54
AA: 3159370
100
80
80
m/z 297.2 → m/z 176.1
60
40
20
20
0
6.0
6.5
100
7.0
Time (min)
C.
80
7.5
m/z 282.2 → m/z 166.1
40
6.0
6.5
100
RT: 7.53
MA: 450110
60
m/z 297.2 → m/z 176.1
60
40
80
7.0
Time (min)
D.
7.5
RT: 7.55
MA: 952352
60
m/z 282.2 → m/z 166.1
40
20
20
0
0
100
100
80
80
60
60
m/z 285.2 → m/z 169.1
40
20
0
0
100
RT: 7.53
MA: 2266960
80
m/z 285.2 → m/z 169.1
40
20
RT: 7.56
MA: 3205157
100
80
60
60
m/z 297.2 → m/z 176.1
40
m/z 297.2 → m/z 176.1
40
20
20
0
m/z 285.2 → m/z 169.1
60
40
0
RT: 7.56
MA: 386661
RT: 7.54
AA: 623964
100
80
0
B. m/z 282.2 → m/z 166.1
80
0
6.0
6.5
7.0
Time (min)
7.5
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 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
Dosimetry of N2-hydroxymethyl-dG
Adducts in Nasal Epithelium of Rats
3000000
Endogenous
282.2 →
166.1 m/z
2500000
2000000
4.9 adducts/
107 dG
Exposure
(ppm)
Exogenous
adducts/107
dG
Endogenous
adducts/107 n
dG
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
1500000
RT: 10.30
1000000
500000
0
3000000
Exogenous
285.2 →
169.1 m/z
2500000
Intensity
2000000
9.0 adducts/
107 dG
RT: 10.30
1500000
1000000
500000
0
3000000
RT: 10.31
Internal
Standard
297.2 →
176.1 m/z
2500000
2000000
1500000
20
fmol
1000000
500000
0
8
9
10
Time (min)
11
12
15 ppm Rat NE
*4-6 rats combined
** 2 rats combined
Ratio of Exogenous to Endogenous
Adducts
Exogenous
Ratio of Exogenous Versus
Endogenous Adducts
3
2.5
2
1.5
1
0.5
0
Endogenous
0
5
10
15
20
Formaldehyde Exposure Dose(ppm)
N2-hydroxymethyl-dG Adduct Half-life Study
t1/2 = 63 hours
7
ln (Exogenous Adducts/10 dG)
1
0
-1
-2
Y= -0.011x – 0.46
R2 = 0.771
0
20
40
Hours
Days
60
80
n=5 per time point
Mean ± SD
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
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
Primate Femoral Bone Marrow
Endogenous and Exogenous Adducts
RT: 10.52
312 µg
DNA
10000000
5000000
4000000
178 µg
DNA
3000000
2000000
1000000
0
0
40000
Exogenous
285.2 → 169.1 m/z
35000
4E4
40000
Intensity
25000
20000
15000
6E4
Exogenous
285.2 → 169.1
m/z
50000
30000
Intensity
7E6
Endogenous
282.2 → 166.1
m/z
5000000
15000000
Intensity
6000000
Intensity
20000000
RT: 10.62
2E7
Endogenous
282.2 → 166.1 m/z
30000
20000
10000
10000
5000
0
3000000
0
RT: 10.52
Internal Standard
297.2 → 176.1 m/z
2500000
3E6
Internal
Standard
297.2 → 176.1
m/z
1600000
1400000
2000000
1200000
Intensity
Intensity
RT: 10.62
1800000
1500000
2E6
1000000
800000
600000
1000000
400000
500000
200000
0
0
8
9
10
Time (min)
11
12
1.9 ppm 13CD2O
8
9
No Exogenous
Adducts
Detected with
5-10 fold >DNA
10
Time (min)
11
12
6.1 ppm 13CD2O
Note: We
used ~2030 ug for
nasal
tissue
Adduct Numbers in Primate Bone
Marrow
Exposure
concentrati
on
Exogenous
adducts/107
dG
Endogenous
adducts/107
dG
1.9 ppm
nd
17.48 ± 2.61
6.1 ppm
nd
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
Survival (% control)
100
10
1
0
0
20
40
60
80
Formaldehyde (µ
µ M)
28
Ridpath, JR et al (2007) Cancer Res
DNA adduct analysis
Endogenous
296.1 → 180.1 m/z
s
ou
n
e
og
d
en
ex
og
en
ou
s
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
Nucleosides
DAD1 B, Sig=254,4 Ref=360,100 (BEN\10192011_AA_CALCURVE 2011-10-20 11-10-08\101911_AA_002.D)
1.
1.
Isolate DNA using Nucleobond anion
exchange columns
Reduce DNA with NaCNBH3 at 37°C, 6 hours
with phosphate buffer, pH 7.2
mAU
3.685
2000
13.146
1500
14.769
1000
2.
16.598
Digest DNA
N2-ethyl-dG
500
1.
2.
3.
3.
4.
Enyzmes: AP, PDE, DNAse
Tris/MgCl2, pH 7.2, IS - 5 fmol
60 min with 10 min pre-incubation w/
DNAse at 37°C
0
5
10
15
20
25
30
35
40
Fraction Collection Chromatogram
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
5.
6.
Dry in speedvac
Re-dissolve in 10 µL water, inject 5 µL
Waters nanoUPLC and Thermo Quantum Ultra
45
min
UPLC-MS/MS Chromatograms
Endogenous
296.1 → 180.1 m/z
60
40
20
0
40
20
0
RT: 16.58
AA: 36539
RT: 15.70
AA: 2064135
60
40
20
IS
301.1 → 185.1 m/z
1.9E5
100
60
40
20
0
2.9E5
RT: 15.69
AA: 2462242
80
60
40
20
0
14
15
16
Time (min)
17
18
RT: 15.68
AA: 471225815
3.4E7
RT: 15.67
AA: 3544716
2.9E5
40
20
80
60
40
20
0
16.66
5.5E5
60
100
80
RT: 15.67
AA: 7391301
80
0
1.0E5
0
16.55
RT: 15.75
AA: 1707053
Relative Abundance
Relative Abundance
80
40
100
60
100
60
20
100
Relative Abundance
80
Exogenous
298.1 → 182.1 m/z
8.1E5
80
0
6.0E3
Relative Abundance
Relative Abundance
100
RT: 15.69
AA: 10457675
100
2.0 mM [13C2]Acetaldehyde
100
Relative Abundance
80
4.7E5
Relative Abundance
Relative Abundance
100
RT: 15.76
AA: 5557894
0.01 mM [13C2]Acetaldehyde
Relative Abundance
Control TK6 Cells
80
60
40
20
0
13
14
15
16
Time (min)
17
18
13
14
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
Sum Adducts/107 dG
500
100
50
10
Endogenous Mean
5
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
% MN
5
35
2
Vehicle Control
1
0.5
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
Number per Cell
Abasic sites
50,000
OHEtG
3,000
7-(2-Oxoethyl)guanine
3,000
8-oxodG
2,400
Formaldehyde
1,000-4,000
Acetaldehyde
1,000
7-Methylguanine
1,200
AcrdG
120
M1dG
60
N2,3-Ethenoguanine
36
1N2-Etheno dG
30
1N6-Etheno dA
12
Total
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
-6
Mutant Fraction (x10 )
5
q1
4
3
min
median
tk locus
n=87
max
q3
hprt locus
n=34
2
1
0
TK6
AHH-1
Cell Line
95th
Penman and Crespi, Environ Mol Mut 10:35-60, 1987
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•
•
•
•
•
•
•
•
•
•
•
•
•
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Patricia Upton
Betsy Bermudez
Ed Bermudez
Jacob McDonald
Melanie Doyle-Eisele
Andrew Gigliotti
Julia Rager
Rebecca Fry
•
•
•
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Formaldehyde Council
FormaCare-CEFIC
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Hamner Institutes for Health
Sciences
• Lovelace Respiratory Research
Institute
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Program (P42-ES 5948)
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Health and Susceptibility (P30
ES 10126)