Microsoft PowerPoint - RASS Webinar_051320 Part
1_LuMeasurement of exogenous and endogenous DNA adducts
via mass spectrometry and stable isotope labeled
chemicals for exposure
Gillings School of Global Public Health
Director, UNC Biomarker Mass Spectrometry Facility
University of North Carolina at Chapel Hill
May 13, 2020
[email protected]
DNA Adducts: Biomarkers of Exposure for Risk Assessment
• 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,
detoxification and DNA repair.
•
Frequently used as biomarkers in risk
assessment of chemical exposure.
• DNA adducts are expected to be
linear at low doses. An exception is
when identical adducts are formed
endogenously.
Toxicol Sci. 2011, 120, S130–S145
How to distinguish endogenous and exogenous adducts?
•
We developed sensitive mass spec methods, coupled with the use of
stable isotope labeled chemicals for exposure, to differentiate DNA
adducts originating from both endogenous and exogenous sources.
Animal Exposure with
Stable isotope
labeled Chemicals (13C, 15N etc)
Exposure
MS/MS platforms
UPLCMS/MS
Adapted for IARC monograph 88
glutathione
DNADNA CLs
Leng, J.P., Liu, C.W., Hartwell, H.J., Lu, K. Archieves in
Toxicology, 2019, 93(3):763-773 Liu CW, Tian X, Hartwell HJ, Leng
J1, Chi L, Lu. K. Chem. Res. Toxicol., 2018,31(5):350-357 Lu, K.,
Collins, L.B, Ru, H.Y., Bermudez,E., Swenberg, J.A. Toxicological
Sciences, 2010, 116,441 Lu, K., Moeller,B., Doyle-Eisele.M,
McDonald J., Swenberg, J.A. Chem. Res. Toxicol., 2011, 24,159 Lu,
K., Boysen, G., Gao, L., Collins, L., and Swenberg, J.A. Chem. Res.
Toxicol., 2008, 21,1586 Lu, K., Craft S., Nakamura J., Moeller BC,
Swenberg, J.A. Chem. Res. Toxicol., 2012, 25(3): 664–675. Lu, K.,
Ye, W.J., Gold, A., Ball, L.M. and Swenberg, J.A. J. Am. Chem.
Soc., 2009,131, 3414 Lu, K., Ye, W.J., Collins B., Gold, A., Ball,
L.M., Swenberg, J.A. J. Am. Chem. Soc., 2010, 132, 3388
Rats: 15ppm formaldehyde induced 50% incidence of nasal carcinomas
after 2 yearexposure (10ppm formaldehyde caused 22%
incidence).
Endogenous formaldehyde pathways
Develop formaldehydespecific DNA adduct biomarkers
Inhalation only exposure
mass=3 Da
RT: 7.60
Time (min)
RT: 7.58
(fmol) Detected
10 10.8±0.7 108 6 10 10.2±0.5 102 5
20 22.5±0.9 112 4 20 21.6±1.2 108 5
40 42.3±0.4 105 0.1 40 40.9±1.4 102 3
Accuracy and precision of the LC/MS/MS-SRM analysis of monoadducts
of formaldehyde*
*Rat hepatic DNA samples were spiked in triplicate with the
indicated amounts of N2-CH3-dG or N6-CH3-dA + Mean±SD
50200 µg isolated DNA
Enzyme digestion
Centrifugation with Millipore filters
Fraction collection by HPLC
LCESIMS/MSSRM
N2CH3dG: m/z 282.2→m/z 166.1
N213CD2HdG: m/z 285.2→m/z 169.1
[13C1015N5]N2CH3dG: m/z 297.2→m/z 176.1
Incubated with 50 mM NaCNBH3 for 6 h at 37°C. Add 80 fmol of
[13C10
15N5]-N2-CH3-dG internal standard.
Digested by DNase I in Tris buffer for 10min at 37°C, followed by
alkaline phosphatase and phosphodiesterases for additional
1h.
This step removed enzymes and undigested DNA.
The fractions were dried by a speed vacuum.
13 Exogenous
Formaldehyde Exposure
Nuclear Pellets DNAzol Lysis and Proteinase K
digestion
Homogenization in sucrose buffer
b. DPC isolation
NucleosidePeptide Crosslinks
DNAProtein Crosslinks Dnase I Alkaline Phosphatase
Phosphodiesterase I Pronase
DNA Digestion
Peptide Digestion
DPC Isolation
Ethanol precipitation
Homogenized Sample
Stop reaction by adding acetic acid Add Internal Standard
NucleosideAmino Acid Crosslinks Quantification of dG by
HPLC-UV
d. DPC analysis
Offline HPLC purification
Nano LC-ESI-MS/MS Analysis
Experimental Workflow for DPC Measurement
An improved method for DPC detection using high resolution orbitrap
mass spec
Current method Previous method
The initial study: Rats exposed to 10 ppm [13CD2]
Formaldehyde
13CD2FA
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
A. nasal epithelium of a 1 day-exposed rat
B. nasal epithelium of a 5 day-exposed rat
C. bone marrow of a 5 day-exposed rat
D. spleen of a 5 day-exposed rat
LC-ESI-MS/MS SRM chromatograms of N2-Me-dG in typical tissues
Toxicological Sciences, 2010, 116,441
exogenous endogenous exogenous endogenous
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
Blood n.d. 1.28±0.38 n.d. 3.80±0.29
5 day
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
Blood n.d. 1.10±0.28 n.d. 3.66±0.78
Formaldehyde-induced monoadducts in tissues of rats exposed to 10
ppm [13CD2]-formaldehyde for 1 day or 5 days
1 Day 5 Days 0
0.3
0.6
0.9
1.2
1.5
1.8
8 10 12 14 Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
ln (E
xo ge
no us
A dd
uc ts
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0h 6h 12h 24h 48h 72h
Collect tissues at 6 time points postexposure
Nasal epithelium Nasal epithelium
N um
Steady State
0 20 40 60 80
1000 20 40 60 80
100 0
20 40 60 80
100 0
A rat in FEMA trailers for 6 h
Mean [Formaldehyde]= 77 ppb
R at
io o
0.7 ppm
2 ppm
5.8 ppm
9.1 ppm
15.2 ppm
Exposed to FA for
6h/day for 28 days
Air
Positive Control (10 ppm)
n Endogenous Exogenous Endogenous Exogenous Endogenous Exogenous
Endogenous Exogenous
Nasal Mucosa 3.23±0.85 ndb 3.59±0.90 nd 3.27±0.76 nd 3.48±0.83 nd
8
Bone Marrow 4.83±1.54 nd 4.32±1.21 nd 5.03±1.71 nd 4.42±0.69 nd
8
PBMC 2.64±1.03 nd 2.72±0.73 nd 2.80±1.11 nd 2.94±1.15 nd 8
Trachea 3.14±0.61 nd 3.23±1.02 nd 3.34±0.75 nd 3.23±0.47 nd 6
Liver 2.48±0.21 nd 2.57±0.31 nd 2.44±0.34 nd 2.60±0.76 nd 6
Hippo campus 2.35±0.56 nd 2.62±0.74 nd 2.52±0.82 nd 2.86±0.76 nd
5
Olfactory Bulbs 2.51±0.62 nd 2.74±1.05 nd 2.84±0.45 nd 2.59±0.38 nd
5
Cerebellum 2.45±0.76 nd 2.62±0.67 nd 2.46±0.43 nd 2.35±0.57 nd
5
Lung 5.25±3.23 nd 3.72±2.20 nd 4.79±3.22 nd 5.06±2.51 nd 7
No exogenous formaldehyde DNA adducts (adducts/107 dG) in rat
tissues exposed to [13CD2]-formaldehyde (1, 30, 300 ppb) for 28
days
Tissues Air control 1 ppb 30 ppb 300 ppb
n Endogenous Exogenous Endogenous Exogenous Endogenous Exogenous
Endogenous Exogenous
Nasal Mucosa 2.66±0.54 ndb 2.77±0.61 nd 3.01±0.85 nd 2.85±0.74 nd
8
Bone Marrow 2.19±0.46 nd 2.28±0.55 nd 1.98±0.42 nd 2.45±0.48 nd
8
PBMC 1.96±0.66 nd 2.08±0.56 nd 1.88±0.64 nd 1.93±0.85 nd 8
Trachea 1.52±0.70 nd 2.30±1.03 nd 2.41±0.83 nd 1.99±0.57 nd 8
Liver 7.27±1.66 nd 8.03±1.46 nd 7.93±1.58 nd 7.13±1.58 nd 8
Hippo campus 1.81±0.46 nd 1.87±0.41 nd 1.63±0.51 nd 1.94±0.39 nd
5
Olfactory Bulbs 1.69±0.37 nd 2.55±0.40 nd 1.89±0.34 nd 2.04±0.42 nd
5
Cerebellum 2.71±0.87 nd 2.37±0.68 nd 2.39±1.60 nd 2.33±0.73 nd
5
Lung 4.07±1.11 nd 3.99±0.61 nd 3.34±0.67 nd 3.48±0.65 nd 8
No exogenous formaldehyde DNA-protein crosslinks (DPCs/108 dG) in
rat tissues exposed to [13CD2]-formaldehyde (1, 30, 300 ppb) for 28
days
Archives of Toxicology, 2019, 93(3):763773
Our methodology applicable to other chemicals that cause
identical DNA adducts as endogenous sources: vinyl acetate as
another example
Target signals
Mean endogenous level
13.0 13.5 14.0 14.5 15.0 15.5 16.0 Time (min)
0
50000
100000
150000
The rich dataset from key references
•
Leng, J.P., Liu, C.W., Hartwell, H.J., Lu, K. Archives of Toxicology, 2019, 93(3):763773
• Liu CW, Tian X, Hartwell HJ, Leng
J, Chi L, Lu. K. Chem. Res. Toxicol., 2018,31(5):350357
•
Lai Y, Yu R, Hartwell HJ, Moeller BC, Bodnar
WM, Swenberg
JA. Cancer Research, 2016, 76(9):265261
•
Yu R, Lai Y, Hartwell HJ, Moeller BC, DoyleEisele
M, Kracko D, Bodnar WM, Starr TB, Swenberg
JA, Toxicol Sci. 2015, 146(1):17082
• Swenberg JA, Moeller BC, Lu K, Rager
JE, Fry RC, Starr TB. Toxicol
Pathol. 2013, 41(2):1819
•
Lu, K., Craft S., Nakamura J., Moeller BC, Swenberg, J.A. Chem. Res. Toxicol., 2012, 25(3): 664–675.
• Moeller BC, Lu K, DoyleEisele
M, McDonald J, Gigliotti A, Swenberg
JA. Chem Res Toxicol. 2011, 24(2):1624.
• Swenberg
JA, Lu K, Moeller BC, Gao L, Upton PB, Nakamura J, Starr TB.
Toxicol Sci. 2011, 120, S13045.
•
Lu, K., Moeller,B., DoyleEisele.M, McDonald J., Swenberg, J.A. Chem. Res. Toxicol., 2011, 24,159
•
Lu, K., Collins, L.B, Ru, H.Y., Bermudez,E., Swenberg, J.A. Toxicological Sciences, 2010, 116,441
•
Lu, K., Ye, W.J., Collins B., Gold, A., Ball, L.M., Swenberg, J.A. J. Am. Chem. Soc., 2010, 132, 3388
•
Lu, K., Ye, W.J., Gold, A., Ball, L.M. and Swenberg, J.A. J. Am. Chem. Soc., 2009,131, 3414