In Vitro Systems Toxicology Assessment of a Candidate ...
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Supporting Information
For
In Vitro Systems Toxicology Assessment of a Candidate Modified Risk Tobacco Product Shows
Reduced Toxicity Compared to a Reduced Cigarette
by
Ignacio Gonzalez-Suarez*, Florian Martin, Diego Marescotti, Emmanuel Guedj, Stefano Acali, Stephanie Johne, Remi Dulize, Karine Baumer, Dariusz Peric, Didier Goedertier, Stefan Frentzel,
Nikolai V. Ivanov, Carole Mathis, Julia Hoeng, Manuel C. Peitsch
Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000 Neuchâtel, Switzerland* Corresponding author
Table S1. List of biological networks and sub-networks.
Table S2. Selected AE, TPM and GVP concentrations for 3R4F and THS 2.2 used for the exposure
of NHBE cells to generate gene expression data.
Figure S1. Carbonyl levels in AE and GVP from THS 2.2 aerosol and 3R4F smoke.
Figure S2. Cell viability across fractions.
Figure S3. HCS results in NHBE cells exposed for 4h or 24h to AE from 3R4F smoke and THS 2.2
aerosol.
Figure S4. HCS results in NHBE cells exposed for 4h or 24h to TPM from 3R4F smoke and THS
2.2 aerosol.
Figure S5. HCS results in NHBE cells exposed for 4h or 24h to GVP from 3R4F smoke and THS
2.2 aerosol.
Figure S6. Cell cycle analysis results in NHBE cells exposed for 24h to AE, TPM and GVP from
3R4F smoke and THS 2.2 aerosol.
Figure S7. Biological Impact Factor (BIF).
Figure S8. NPA heatmaps for NHBE cells exposed for 4 h to one common dose of 3R4F smoke and
THS 2.2 aerosol fractions.
Networks SubnetworksCalcium
Cell cycle
Cell interaction
Clock
Epigenetics
Growth factor
Hedgehog
Jak Stat
Mapk
Notch
Nuclear receptors
PGE2
Wnt
Drug metabolism response
Endoplasmic reticulum stress
Hypoxic stress
NFE2L2 signaling
Osmotic stress
Oxidative stress
Caspase cascade
ER stress-induced apoptosis
MAPK signaling
NFkB signaling
PKC signaling
Prosurvival mitochondrial signaling
TNFR1F signaling
ATG induction of autophagy
mTOR signaling
Nutrient transporter signature
Components afecting TP53 activity
Components affecting TP63 activity
Components affecting TP73 activity
DNA damage to G1-S checkpoint
DNA damage to G2-M checkpoint
Double-strand break response
Inhibition of DNA repair
TP53 transcriptional signature
Fas activation
Gene signature
Oncogene-induced senescence
Regulation by tumor suppressors
Regulation of p16INK expression
Replicative senescence
Stress-induced premature senescence
Transcriptional regulation of the SASP
Epithelial barrier defense
Epithelial proinflammatory signaling
Mucus hypersecretion
Tissue damage
Senescence
Inflammation
Cell
Proliferacion
Cell stress
Apoptosis
Autophagy
DNA damage
Necroptosis
Table S1
Table S1. List of biological networks and sub-networks
3R4F 100* 25 13
THS2.2 350 100 25
3R4F 100* 25 13
THS2.2 350 100 25 13
3R4F 32* 25 13
THS2.2 38 32 25
CONCENTRATION (puff / l)
AE
GVP
TPM
Table S2
Table S2. Selected AE, TPM and GVP concentrations for 3R4F and THS 2.2 used for
the exposure of NHBE cells to generate gene expression data
Figure S1
0
10
20
30
40
50
sbPBS GVP
µg
/ st
ick
FORMALDEHYDE
3R4F P1
0250500750
100012501500
sbPBS GVP
µg
/ st
ick
ACETALDEHYDE
3R4F P1
0
250
500
750
sbPBS GVP
µg
/ st
ick
ACETONE
3R4F P1
0
50
100
150
sbPBS GVP
µg
/ st
ick
ACROLEIN
3R4F P1
0
10
20
30
sbPBS GVP
µg
/ st
ick
BUTYRALDEHYDE
3R4F P1
0
50
100
150
sbPBS GVP
µg
/ st
ick
MEK
3R4F P1
0
25
50
75
sbPBS GVP
µg
/ st
ick
CROTONALDEHYDE
3R4F P1
0
25
50
75
100
sbPBS GVP
µg
/ st
ick
PROPIONALDEHYDE
3R4F P1
A B
C D
E F
G H
Figure S1. Carbonyl levels in AE and GVP from THS 2.2 aerosol and 3R4F smoke: A,
Acetaldehyde; B, Acetone; C, Acrolein; D, Propionaldehyde; E, MEK; F, Crotonaldehyde; G,
Formaldehyde; H, Butyraldehyde. Values are expressed as µg of carbonyl per one 3R4F
cigarette or THS 2.2 stick and represent average and SEM from eight determinations performed on
eight different smoke/aerosol generations on eight different days. * indicates p <0.0001 vs. 3R4F
(reference cigarette) by the paired t-test.
AE AE
AE AE
AE AE
AE AE
Figure S2
Figure S2. Cell viability across fractions. NHBE cells were exposed for 24h to AE, TPM or GVP
fractions from (A) 3R4F smoke or (B) THS 2.2 aerosol and cell viability was measured using a
real-time cellular analysis system. Values represent average ± SEM of at least three independent
experiments. Values are normalized to vehicle control. R.U. relative units.
P1 (GVP)
10 100 10000.0
0.5
1.0
1.5
Concentration (puff/l)
Imp
ed
an
ce (
R.U
.)SbPBS (3R4F)
0.001 0.01 0.1 10.0
0.5
1.0
1.5
Concentration (puff/l)
Imp
ed
an
ce (
R.U
.)
3R4F
1 10 100 1000
Concentration (puff / l)
1.5
1.0
0.5
0.5
Cell
via
bilit
y (
R.U
.)
THS2.2
10 100 1000
Concentration (puff / l)
1.5
1.0
0.5
0.5
Cell
via
bilit
y (
R.U
.)
GVP (3R4F)
0.001 0.01 0.1 10.0
0.5
1.0
1.5
Concentration
Cell v
iab
ilit
y (
R.U
.)
3R4F TPM
1 10 100 10000.0
0.5
1.0
1.5
Puff/L
TPM
GVP
AE
P1 TPM
10 100 10000.0
0.5
1.0
1.5
Puff/L
P1 sbPBS
10 100 10000.0
0.5
1.0
1.5
puff/l
TPM
GVP
AE
A
B
A B
D
C
E
F G H
I J
Figure S3. HCS results in NHBE cells exposed for 4h or 24h to AE from 3R4F smoke and
THS 2.2 aerosol. Values were normalized to the vehicle control and represent average ± SEM of
at least three independent experiments (n ⩾ 3) with independent smoke/aerosol generations on
different days. The error bars represent SEM. ∗ Significant different from the vehicle control.
Figure S3
Figure S4. HCS results in NHBE cells exposed for 4h or 24h to TPM from 3R4F smoke and
THS 2.2 aerosol. Values were normalized to the vehicle control and represent average ± SEM of
at least three independent experiments (n ⩾ 3) with independent smoke/aerosol generations on
different days. The error bars represent SEM. ∗ Significant different from the vehicle control.
A B
D
C
E
F G H
I J
Figure S4
Figure S5. HCS results in NHBE cells exposed for 4h or 24h to GVP from 3R4F smoke and
THS 2.2 aerosol. Values were normalized to the vehicle control and represent average ± SEM of
at least three independent experiments (n ⩾ 3) with independent smoke/aerosol generations on
different days. The error bars represent the SEM. ∗ Significant different from the vehicle control.
A B
D
C
E
F G H
I J
Figure S5
Vehicle 6 13 25 50 100 200
Polyploid 0.10 ± 0.03 0.08 ± 0.02 0.10 ± 0.01 0.10 ± 0.02 0.07 ± 0.01 0.06 ± 0.01 0.01 ± 0.00
Apoptotic 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.01 0.01 ± 0.00 0.02 ± 0.00 0.05 ± 0.08 0.70 ± 0.06
S-phase 0.08 ± 0.01 0.09 ± 0.03 0.06 ± 0.03 0.02 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
G2-M 0.28 ± 0.02 0.27 ± 0.02 0.29 ± 0.02 0.39 ± 0.04 0.38 ± 0.04 0.33 ± 0.05 0.09 ± 0.03
G0-G1 0.54 ± 0.04 0.55 ± 0.04 0.54 ± 0.02 0.48 ± 0.04 0.53 ± 0.04 0.56 ± 0.05 0.20 ± 0.03
Vehicle 35 70 140 200 300 350
Polyploid 0.12 ± 0.00 0.09 ± 0.05 0.09 ± 0.06 0.12 ± 0.08 0.12 ± 0.07 0.13 ± 0.07 0.10 ± 0.05
Apoptotic 0.01 ±0.00 0.02 ±0.02 0.02 ±0.02 0.02 ±0.02 0.05 ±0.06 0.01 ±0.00 0.01 ±0.00
S-phase 0.08 ±0.03 0.11 ±0.07 0.10 ±0.07 0.07 ±0.06 0.04 ±0.04 0.01 ±0.01 0.00 ±0.00
G2-M 0.29 ±0.01 0.28 ±0.04 0.26 ±0.04 0.29 ±0.03 0.34 ±0.01 0.40 ±0.01 0.41 ±0.02
G0-G1 0.50 ±0.02 0.50 ±0.01 0.53 ±0.01 0.51 ±0.03 0.45 ±0.07 0.45 ±0.06 0.47 ±0.04
Vehicle 6 13 25 50 100 200
Polyploid 0.05 ± 0.03 0.07 ± 0.03 0.08 ± 0.03 0.07 ± 0.04 0.05 ± 0.02 0.03 ± 0.03 0.01 ± 0.00
Apoptotic 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.08 ± 0.10 0.32 ± 0.49 0.82 ± 0.03
S-phase 0.10 ± 0.02 0.07 ± 0.03 0.03 ± 0.03 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
G2-M 0.26 ± 0.03 0.26 ± 0.02 0.32 ± 0.08 0.37 ± 0.01 0.32 ± 0.02 0.22 ± 0.17 0.05 ± 0.01
G0-G1 0.58 ± 0.05 0.59 ± 0.04 0.56 ± 0.02 0.54 ± 0.03 0.55 ± 0.07 0.43 ± 0.29 0.12 ± 0.02
Vehicle 35 70 140 200 300 350
Polyploid 0.05 ± 0.02 0.07 ± 0.03 0.08 ± 0.04 0.09 ± 0.04 0.09 ± 0.04 0.08 ± 0.05 0.06 ± 0.03
Apoptotic 0.02 ±0.03 0.02 ±0.02 0.02 ±0.02 0.02 ±0.02 0.02 ±0.02 0.03 ±0.02 0.03 ±0.01
S-phase 0.12 ±0.05 0.10 ±0.05 0.09 ±0.05 0.05 ±0.04 0.02 ±0.02 0.00 ±0.00 0.00 ±0.00
G2-M 0.27 ±0.05 0.27 ±0.03 0.27 ±0.02 0.35 ±0.06 0.39 ±0.02 0.37 ±0.03 0.35 ±0.04
G0-G1 0.53 ±0.02 0.55 ±0.03 0.54 ±0.03 0.49 ±0.01 0.48 ±0.03 0.52 ±0.06 0.56 ±0.06
3R4F (TPM) THS 2.2 (TPM)
Vehicle 8 16 32 42 50 64
Polyploid 0.03 ± 0.01 0.04 ± 0.02 0.05 ± 0.02 0.07 ± 0.03 0.03 ± 0.01 0.01 ± 0.01 0.01 ± 0.00
Apoptotic 0.01 ± 0.00 0.00 ± 0.00 0.01 ± 0.00 0.01 ± 0.01 0.34 ± 0.19 0.72 ± 0.05 0.75 ± 0.00
S-phase 0.04 ± 0.02 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
G2-M 0.24 ± 0.02 0.32 ± 0.06 0.37 ± 0.02 0.36 ± 0.02 0.23 ± 0.06 0.11 ± 0.03 0.09 ± 0.00
G0-G1 0.67 ± 0.03 0.64 ± 0.07 0.57 ± 0.04 0.55 ± 0.06 0.39 ± 0.13 0.16 ± 0.02 0.14 ± 0.00
Vehicle 75 150 225 300 375 450
Polyploid 0.04 ± 0.02 0.06 ± 0.04 0.08 ± 0.04 0.08 ± 0.04 0.07 ± 0.04 0.09 ± 0.05 0.07 ± 0.03
Apoptotic 0.01 ±0.01 0.00 ±0.00 0.00 ±0.00 0.01 ±0.01 0.01 ±0.01 0.01 ±0.01 0.17 ±0.27
S-phase 0.05 ±0.02 0.03 ±0.03 0.01 ±0.01 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00
G2-M 0.25 ±0.04 0.26 ±0.03 0.28 ±0.02 0.32 ±0.02 0.35 ±0.01 0.34 ±0.03 0.30 ±0.09
G0-G1 0.65 ±0.06 0.64 ±0.07 0.63 ±0.05 0.60 ±0.05 0.57 ±0.04 0.56 ±0.06 0.45 ±0.17
3R4F (GVP) THS 2.2 (GVP)
A3R4F (AE) THS 2.2 (AE)
B
C
Figure S6. Cell cycle analysis results in NHBE cells exposed for 24h to AE (A), TPM (B) and
GVP (C) from 3R4F smoke and THS 2.2 aerosol. Values are expressed as cell percentage and
represent the average of three independent experiments with independent smoke/aerosol
generations on different days. Specific values of percentage of cells in different phases (polyploid,
apoptotic, S-phase, G2-M, G0-G1) for each fraction concentration (indicated in bold in puffs/L),
are expressed as average ± standard deviation in the table below each graph. The percentage of
cells in S-phase is highlighted in orange.
Figure S6
GVP AE TPM GVP AE TPM
3R4F THS2.2
GVP AE TPM
THS2.2
100%
80%
60%
40%
20%
BIOLOGICAL IMPACT FACTOR (BIF)
Figure S7. Biological Impact Factor (BIF). Relative BIF values for 3R4F and THS2.2 fractions
at one common dose (25 puffs/L). The percentages give the relative biological impact that is
derived from the cumulated network perturbations caused by the treatment relative to the
reference (3R4F TPM), which is defined as the treatment comparison showing the highest level of
perturbation.
Figure S7
Oxidative Stress
Osmotic Stress
NFE2L2 Signaling
Xenobiotic Metabolism Response
Senescence
Response to DNA Damage
Tissue Damage
Necroptosis
Wnt
PGE2
Nuclear Receptors
Mapk
Jak Stat
Growth Factor
Autophagy
Cell Cycle
25 Puffs / L
Epithelial Immune Activation
Notch
mTOR
hOX
Hedgehog
Calcium
Cell interaction
Epigenetics
Clock
3R4F THS2.2
CEL
L FA
TEP
RO
LIFE
RA
TIO
NC
ELL
STR
ESS
INFL
AM
MA
TIO
N
3R4F THS 2.2
GV
P
AE
TPM
GV
P
AE
TPM
Endoplasmic reticulum stress
Hypoxic stress
Apoptosis
Figure S8. NPA heatmaps for NHBE cells exposed for 4h to one common dose of 3R4F smoke
and THS 2.2 aerosol fractions. Significantly perturbed networks are indicated as *. Darker colors
indicate higher NPA scores.
Figure S8