analysis of cell exposure to arsenic and smokeless tobacco 1 Epigenomics and Mechanisms Branch International Agency for Research on Cancer WHO Lyon France 2 Faculty of Science Charles University Prague Czech Republic ID: 930368
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Slide1
Integrative
toxicogenomics analysis of cell exposure to arsenic and smokeless tobacco
1 Epigenomics and Mechanisms Branch, International Agency for Research on Cancer WHO, Lyon, France 2 Faculty of Science, Charles University, Prague, Czech Republic3 Early Detection, Prevention, and Infections Branch, International Agency for Research on Cancer WHO, Lyon, France4 Centre of Excellence in Mycotoxicology and Public Health, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium
Das S1, Thakur S1,2, Cros MP1, Cuenin C1,Venuti A3, Sirand C3, Claeys L1,4, Renard C1, Cahais V1, Keita S, Herceg Z1, Korenjak M1, Zavadil J1
HYPOTHESISCo-exposure of arsenic and SLT leads to the increased genotoxic effects which can contribute to higher oral cancer incidence.OBJECTIVEIdentify in vitro the combinatorial effects of co-exposure of arsenic and smokeless tobacco on DNA integrity, cellular proliferation, epigenetic and gene expression changes using human oral cells and primary mouse cells.
Slide2clonal expansion
dilution cloning/cell sorting
crisis stage
primary cells
SA
SLT
SA+SLT
barrier bypass
clonal expansion
senescence
Hupki
MEF (mouse)
NOK (Human)
0 months
3-7 months
Cell exposure /clonal expansion - experimental design
Exposures:
2-8 weeks of repeated exposure
Single cell subcloning (NOKS)/Immortalization (
Hupki
MEF)
Total Timeline for generation of clones for Whole exome sequencing
APPROACH
Evaluate the effects of
sodium
arsenite
(NaAsO
2
, SA)
and
sadagura
(SLT)
on cellular metabolic and phenotypic states: MTT/MTS assays, cell cycle and apoptosis assays
Genomic DNA:
γ
H2AX, Comet Assay, Whole-
Exome
Sequencing for mutational signature analysis
Transcriptome:
mRNA expression (RNA-seq)
Epigenome:
Illumina 850K Array DNA
methylome
profiling
Slide3Each experimental condition: N=3
SASLT24h48hNOKS
Hupki MEFNOKSHupki MEF
Cell Viability
SA+SLTNOKSHupki MEF
Exposure to SA and SLT, singly or combined, resulted in decreased cell metabolic activity
Test compound concentrations
Cytotoxic responses to SA and/or SLT exposure
Slide4NOKs
Hupki MEF
SASLTSA+SLTIncreased
genotoxicity by γH2AXSASLTSA+SLT
24h48hCell cycle analysis in NOKsSA
SLT
SA+SLT
24h
48h
SA, SLT and SA+SLT adversely affect cell metabolic activity / viability
SA, SLT and SA+SLT induce DNA damage in NOKs and
Hupki
MEF
Cell DNA content flow
cytometry
suggests possible cell death following SA treatment
ONGOING
Transcriptome
and
epigenome
remodeling analyses will unravel early and late-stage effects of the SA and SLT exposure
in vitro
CONCLUSIONS
Genotoxic
response and increase in sub-G0 population
Increased genotoxicity by Comet
assay in NOKs
*
*
*
Untreated control
Untreated control
Unt
Pos
ctrl (AA)
SA
SLT
SA+SLT
Tail DNA percentage
24h
48h
Unt
Pos
ctrl (AA)
SA
SLT
SA+SLT
Slide5Acknowledgements
Epigenomics & Mechanisms Branch
IARC WHO, Lyon, FranceJiri ZAVADIL (Postdoctoral Supervisor)Michael KORENJAKMarie-Pierre CROSVincent CAHAISClaire RENARDZdenko HERCEGCyrille CUENINStephane KEITAIARC & Charles University, Prague, Czech RepublicShefali THAKURGhent University, Ghent, BelgiumLiesel CLAEYS
Funding: IARC Postdoctoral Fellowship 2019-2021