Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Perspective
Systems toxicology assessment of the biological impact of a candidate Modified Risk Tobacco Product on human organotypic oral epithelial cultures Filippo Zanetti, Alain Sewer, Carole Mathis, Anita R. Iskandar, Radina Kostadinova, Walter K. Schlage, Patrice Leroy, Shoaib Majeed, Emmanuel Guedj, Keyur Trivedi, Florian Martin, Ashraf Elamin, Céline Merg, Nikolai V. Ivanov, Stefan Frentzel, Manuel C Peitsch, and Julia Hoeng Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00174 • Publication Date (Web): 12 Jul 2016 Downloaded from http://pubs.acs.org on July 27, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Chemical Research in Toxicology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 52
Chemical Research in Toxicology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Chemical Research in Toxicology
In vivo buccal epithelium
Buccal mucosa
In vitro buccal cultures
Page 2 of 52
Stratified squamous epitelial layer
Basement membrane
Gene expression and miRNA profiling
Histopathological analysis
Lamina propria
Cytotoxicity (AK-assay) CYP1A1/1B1 activity
3R4F
Sys-Tox Assessment
3R4F cigarettes
Profiling of secreted inflammatory markers
em
t s y s n o i t u n / distrib
Dilutio
Smoking Machines
37
THS2.2 Electrically heated tobacco product / tobacco stick
Aerosol
trumpet
n o i t a v i t l u C le u d o m e bas
(=aerosol inlet)
holder
Climatic Chamber
tissue insert
ACS Paragon Plus Environment
48-well plate
Exposure Module
Page 3 of 52 aerosol inlet
Figure 1
Chemical Research in Toxicology
1 tissue culture 2 well 3 4 5 culture 6 insert 7 8 organotypic 9 tissue 10 11 membrane 12 13 14 15 medium 16 17 18 19 EXPOSURE (28min) 20 21 ACS Paragon Plus Environment 22 4h 24h 48h 23Post-Exposure Time Points 24
Endpoints Histology AK cytotoxicity -
CYP1A1/1B1 activity Cytokines Secretion mRNA and miRNA
72h
Figure 2
mean ± SEM
Cytotoxicity (%)
8 mean ± SEM
Cytotoxicity (%)
6 4 2 0
8 6 4 2
(Air) (0.32)(0.51) (Air) (0.31)(0.46)(1.09)
3R4F
(Air) (0.32)(0.51) (Air) (0.31)(0.46)(1.09)
THS2.2
3R4F
48 h Post-Exposure
THS2.2
72 h Post-Exposure
8 6
mean ± SEM
mean ± SEM
Cytotoxicity (%)
24 h Post-Exposure
0
Cytotoxicity (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
4 h Post-Exposure
Chemical Research in Toxicology
4 2 0
8 6 4 2 0
(Air) (0.32)(0.51) (Air) (0.31)(0.46)(1.09)
3R4F
THS2.2
(Air) (0.32)(0.51) (Air) (0.31)(0.46)(1.09)
ACS Paragon Plus Environment
3R4F
THS2.2
Page 4 of 52
A
Page 5 of 52
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Chemical Research in Toxicology
Figure 3
48 h Post-Exposure
3R4F (Air)
THS2.2 (Air) 200 µm
3R4F (0.32)
THS2.2 (0.31)
3R4F (0.51)
THS2.2 (0.46)
Apical side Basal side Membrane
B
THS2.2 (1.09)
72 h Post-Exposure
3R4F (Air)
THS2.2 (Air) 200 µm
3R4F (0.32)
THS2.2 (0.31)
3R4F (0.51)
THS2.2 (0.46)
ACS Paragon Plus Environment
THS2.2 (1.09)
A
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 4
Chemical Research in Toxicology
Page 6 of 52
48 h Post-Exposure
3R4F (Air)
THS2.2 (Air) 200µm
3R4F (0.32)
THS2.2 (0.31)
3R4F (0.51)
THS2.2 (0.46)
Apical side Basal side Membrane
B
THS2.2 (1.09)
72 h Post-Exposure THS2.2 (Air)
3R4F (Air) 200µm
3R4F (0.32)
THS2.2 (0.31)
3R4F (0.51)
THS2.2 (0.46)
THS2.2 (1.09) ACS Paragon Plus Environment
Normalized CYP1A1/1B1 activity (%) mean ± SEM
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
24 h Post-Exposure
Chemical Research in Toxicology
#
20 15
#
10
#
5 0
(Air) (0.32)(0.51) (Air) (0.31)(0.46)(1.09)
3R4F
Normalized CYP1A1/1B1 activity (%) mean ± SEM
Figure 5
Page 7 of 52
48 h Post-Exposure #
20 15
#
10 5 0
(Air) (0.32)(0.51) (Air) (0.31)(0.46)(1.09)
THS2.2ACS Paragon Plus Environment
3R4F
THS2.2
Figure 6
24 h Post-Exposure
1 1.8* 1.1 2 1.6* 3 1.6* 2.1* 1.0 4 2.2* 3.0* 1.2* 5 0.6* 1.0 6 0.6* 7 0.9 0.9 1.0 8 0.7 0.4* 1.0 9 7.1* 14.6* 1.0 10 1.2 0.9 11 1.2 12 0.7 0.6* 1.0 13 1.3 0.9 1.1 14 15(0.32) (0.51) (0.31) 16 17 3R4F 18 19 20 21 22* p-value < 0.05 of the 23 24 25
1.4* 1.4* 1.6* 1.0 1.2 1.1 1.6 1.3 1.3 1.5
1.4* 1.3 1.7* 0.8* 1.0 1.0 3.0* 1.1 1.0 1.7
(0.46) (1.09)
THS2.2
Chemical Research in Toxicology
48 h Post-Exposure 1.4* 1.6 2.3* 0.5* 0.8 0.3* 7.2* 0.9 0.8 1.0
1.4* 2.2* 2.5* 0.4* 0.8 0.2* 11.6* 1.1 0.7* 0.9
1.0 1.3* 1.3* 0.7 1.2 1.1 1.9* 1.3 1.2 1.9*
0.9 1.3 1.3 0.6* 1.3 1.0 1.5 1.3 1.2 2.2*
1.2 1.6 1.7* 0.5* 1.2 1.0 3.1 1.3 1.1 2.2
(0.32) (0.51) (0.31) (0.46) (1.09)
3R4F
THS2.2
72 h Post-Exposure 1.1* 2.7* 3.4* 0.4* 0.8 0.5* 5.3* 0.9 0.9 1.7
1.1 3.9* 3.0* 0.4* 0.7* 0.2* 8.9* 0.8 0.6* 1.0
1.0 1.0 1.2 0.8 1.0 0.9 1.1 1.0 1.2 1.2
0.9 0.8 1.3 0.5 1.1 0.9 1.0 1.1 1.2 1.3
VEGFA TGFA MMP-1 CXCL10 CXCL8 IL6 IL1B IL1A CSF2 CSF3
1.1 1.2 1.8* 0.5* 1.1 0.6 2.0 1.1 1.1 1.7
(0.32) (0.51) (0.31) (0.46) (1.09)
THS2.2
3R4F
Fold- Changes
fold change difference relative to their respective air controls ACS Paragon Plus Environment
Page 8 of 52
0.6
1.0
17.0
Figure 7
Page 9 of 52
Chemical Research in Toxicology Gene expression summary Biological Impact Factor (all 28 networks)
BIF NPA GSA DEG DE
Cell Fate (CFA − 5 networks) 1 Cell Proliferation (CPR − 15 networks) 2 Cellular Stress (CST − 6 networks) 3 Inflammation (IPN − 2 networks) 4 Apoptosis (CFA−1) Autophagy (CFA−2) 5 Necroptosis (CFA−3) 6 Response To DNA Damage (CFA−4) 7 Senescence (CFA−5) Calcium (CPR−1) 8 Cell Cycle (CPR−2) 9 Cell Interaction (CPR−3) 10 Clock (CPR−4) Epigenetics (CPR−5) 11 Growth Factor (CPR−6) 12 Hedgehog (CPR−7) 13 Hox (CPR−8) Jak Stat (CPR−9) 14 Mapk (CPR−10) 15 MTor (CPR−11) 16 Notch (CPR−12) Nuclear Receptors (CPR−13) 17 PGE2 (CPR−14) 18 Wnt (CPR−15) 19 Endoplasmic Reticulum Stress (CST−1) 20 Hypoxic Stress (CST−2) NFE2L2 Signaling (CST−3) 21 Osmotic Stress (CST−4) 22 Oxidative Stress (CST−5) 23 Xenobiotic Metabolism Response (CST−6) Epithelial Innate Immune Activation (IPN−1) 24 Tissue Damage (IPN−2) 25 KEGG collection − Q1,FDR
