Influenza infection induces host DNA damage and dynamic DNA damage
responses during tissue regeneration
Na Li1,3, Marcus Parrish2, Tze Khee Chan1,4, Lu Yin1, Prashant Rai1,3, Yamada
Yoshiyuki1, Nona Abolhassani2, Kong Bing Tan5, Orsolya Kiraly1, Vincent TK
Chow3, Bevin P. Engelward2#
1 Singapore-MIT Alliance for Research and Technology
2 Department of Bioengineering, Massachusetts Institute of Technology
3 Department of Microbiology, National University of Singapore
4 Department of Pharmacology, National University of Singapore
5 Department of Pathology, National University of Singapore
# Corresponding author. Address: 16-743, Massachusetts Institute of Technology,
77 Massachusetts Ave., Cambridge, MA 02139. Phone: (617) 258-0260. Fax: (617)
258-0499. E-mail: bevin@mit.edu.
Supplementary Methods and Materials:
Alkaline comet assay
The alkaline comet assay was performed as previously described [1]. Briefly, gels were
lysed overnight at 4°C (10 mM Tris–HCl, 100 mM Na2EDTA, 2.5 M NaCl, pH 10 with 1%
Triton X-100 added 20 min before use). After lysis, gels were transferred to an
electrophoresis chamber filled with alkaline unwinding buffer (0.3 M NaOH and 1 mM
Na2EDTA) for 40 min at 4°C. Electrophoresis was conducted with the same buffer at
4°C for 30 min, 1 V/cm and 300 mA.
Percent tail DNA was measured by custom
software written in MATLAB (The Mathworks) [23].
Neutral comet assay
The neutral comet assay was performed as previously described [2]. Briefly, gels were
lysed overnight at 43°C (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1% NLauroylsarcosine, pH 9.5 with 0.5% Triton X-100 and 10% DMSO added 20 min before
use). After lysis, gels were washed twice for 60 min at 4°C with the neutral
electrophoresis buffer (90 mM Tris, 90 mM Boric Acid, 2 mM Na 2EDTA, pH 8.5). The
gels were transferred to an electrophoresis chamber filled with the neutral
electrophoresis buffer for 60 min at 4°C. Electrophoresis was conducted at 4°C for 60
min at 0.6 V/cm and 6 mA. Tail length (µm) was quantified by custom software written in
MATLAB (The Mathworks) [23].
Virus titration with plaque assay
MDCK cells were seeded at a density of 2 x 105 cells per well in a 24 well plate
overnight in MEM containing 10% FBS. Next day, cells were washed twice with PBS. 10
folds serially diluted lung homogenate were then inoculated onto MDCK cells which are
approximately 80% confluent in 24 well plates [3]. Infected MDCK cells were then
cultured for 48 - 72 hours in serum free MEM (Gibco) with 2mg/ml TPCK trypsin and 0.2%
BSA. Cells were then fixed with formalin and stained with 1% crystal violet. Plaques
forming units enumerated were normalized with protein concentration of lung
homogenate estimated with Bradford assay.
ELISA quantification of nuclear 8-OH-dG
Animals used for ELISA quantification were handled based on methods described in the
main paper. Nucleotides from nuclear DNA were extracted from frozen lungs (less than
50 mg) from uninfected (mock infected with PBS) and infected mice based on
modification of previously published protocol [4]. First, nuclear DNA was first isolated
from tissues using DNeasy Blood & Tissue kit (Qiagen), in the presence of deaminase
inhibitors coformycin (5 ng/ml) and tetrahydrouridine (THU, 50 µg/ml), and antioxidants
butylated hydroxytoluene (BHT, 100 µM) and desferroxamine (DFO, 100 µM) in every
step to prevent adventitious DNA damage during sample processing. Briefly, tissues
were lyzed using 360 µl of modified Buffer ATL (containing coformycin, THU, BHT, and
DFO) with Qiagen Tissuelyser and stainless steel beads at maximum oscillation speed
for 2 min at 4⁰C in a 2 ml microcentrifuge tube. Tubes were spun down at full speed for
5 min to settle foam, and the process of lysis was repeated once. Then, 40 µl of
proteinase K was added based on manufacturer’s protocol and samples were incubated
at 56⁰C for 1 h in a water-bath.
Samples were added with 400 µl of Buffer AL
containing the appropriate inhibitors and antioxidants, mixed and added to another 400
µl of pure ethanol, and vortexed. Samples were placed in DNeasy mini spin column and
washed with washing buffers based on manufacturer’s protocol. Washing buffers were
also modified by adding the appropriate concentrations of deaminase inhibitors and
antioxidants to control for oxidation and alkylation artifacts. Finally, isolated DNA was
eluted with nuclease-free water.
To hydrolyze DNA to nucleotides, 30 µg of nuclear DNA was taken from each sample
and added to DNA hydrolysis cocktail based on previous publication with slight
modifications [4]. The resulting mixture contains 9 mM sodium acetate, 1 mM ZnCl2, 8
units nuclease P1, 8000 units Dnase I, THU (20 µg/mL), DFO (1.5 mM), BHT (500 μM),
and coformycin (10 ng/mL). DNA samples were hydrolyzed overnight at 37⁰C and then
added
to
DNA
dephosphorylation
cocktail
containing
alkaline
phosphatase,
phosphodiesterase I and coformycin based on previous publication [4]. Resulting
deoxynucleosides were quantified with DNA / RNA Oxidative Damage EIA Kit (Caymen)
based on manufacturer’s protocol, and normalized by the concentration of nuclear DNA
originally present in each sample mixture.
Mass spectrometry measurement of modified bases
Animals used for mass spectrometry experiments were handled in accordance to
protocol approved by the Institutional Animal Care and Use Committee (MIT CAC).
Influenza A PR8 virus was obtained from ATCC. Six- to 8 week- old female C57/Bl6
(Jackson Labs) mice were instilled intra-tracheally with PBS or 20 PFU of PR8 influenza
virus in PBS. The mice were euthanized at 0, 3, 5, 7, 9, 11, 13, 15, or 17 days following
treatment. The lungs were harvested and flash frozen in liquid nitrogen. The tissues
were transferred to a 13 ml round tube, mechanically homogenized in lysis buffer and
the DNA extracted through centrifugation for genomic DNA isolation (Roche).
Antioxidants and deaminase inhibitors were added to the lysis buffer, protein
precipitation solution, and water: THU (50 µg/mL), DFO (0.1 mM), BHT (100 μM), and
pentostatin (0.2 µg/mL). DNA etheno (ε) adducts 1, N6-Etheno-2’-deoxyadenosine (εdA)
and 1, N2-Etheno-2’-deoxyguanosine (εdG), 8-OH-dG, and hypoxanthine were
quantified as described previously [5]. Measurements were on DNA prepared from 3
animals per group, with the following exceptions: 3 dpi was from two samples for εdA, 8OH-dG, and εdG, 13 dpi was from four samples for εdA, and 0, 5 and 9 dpi for
hypoxanthine were from 2 samples.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and
quantification
Paraffin sections were dewaxed and treated with Proteinase K (20 ug/ml in TE Buffer,
pH 8.0) for 30 minutes at 37⁰C, washed and then stained with In Situ Cell Death
Fluorescein Detection Kit (Roche). Briefly, tissue sections were washed two times in
PBS and incubated with TUNEL labeling mixture for 1 hour at 37⁰C. Lastly sections
were washed, stained with DAPI and mounted with ProLong gold antifade (Life
Technologies).
TUNEL-stained sections were scanned with Mirax Midi slide scanner (Carl Zeiss). 10
randomly chosen 40x digital images were cropped from scanned lung sections and
saved as TIF files. Selection was made nonbiased by switching off the TUNEL (FITC)
channel on Pannoramic viewer (3DHISTECH) used to access Mirax midi files. The
number of TUNEL positive nuclei on each image were then quantified automatically
using Imaris by segmenting DAPI channel to enumerate the number of nuclei. This is
followed with quantification of nuclei overlapped with FITC channel (TUNEL-positive
cells).
References:
1. Wood DK, Weingeist DM, Bhatia SN, Engelward BP (2010) Single cell trapping and DNA damage
analysis using microwell arrays. Proceedings of the National Academy of Sciences of the United States of
America 107 (22):10008-10013. doi:10.1073/pnas.1004056107
2. Weingeist DM, Ge J, Wood DK, Mutamba JT, Huang Q, Rowland EA, Yaffe MB, Floyd S, Engelward BP
(2013) Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA
repair inhibitors. Cell cycle 12 (6):907-915. doi:10.4161/cc.23880
3. Li N, Yin L, Thevenin D, Yamada Y, Limmon G, Chen J, Chow VT, Engelman DM, Engelward BP (2013)
Peptide targeting and imaging of damaged lung tissue in influenza-infected mice. Future microbiology 8
(2):257-269. doi:10.2217/fmb.12.134
4. Taghizadeh K, McFaline JL, Pang B, Sullivan M, Dong M, Plummer E, Dedon PC (2008) Quantification of
DNA damage products resulting from deamination, oxidation and reaction with products of lipid
peroxidation by liquid chromatography isotope dilution tandem mass spectrometry. Nature protocols 3
(8):1287-1298. doi:10.1038/nprot.2008.119
5. Pang B, Zhou X, Yu H, Dong M, Taghizadeh K, Wishnok JS, Tannenbaum SR, Dedon PC (2007) Lipid
peroxidation dominates the chemistry of DNA adduct formation in a mouse model of inflammation.
Carcinogenesis 28 (8):1807-1813. doi:10.1093/carcin/bgm037