RASS Webinar 051320 Part 1 Lu - toxicology.org
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Measurement of exogenous and endogenous DNA adducts via mass spectrometry and stable isotope labeled chemicals for exposure Kun Lu, Associate Professor Department of Environmental Sciences and Engineering Gillings School of Global Public Health Director, UNC Biomarker Mass Spectrometry Facility University of North Carolina at Chapel Hill May 13, 2020 [email protected]
Transcript of RASS Webinar 051320 Part 1 Lu - toxicology.org
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
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