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G. L. Prasad 1
Wolfgang Zacharias 2
Subhashini Arimilli 3 1
DIFFERENTIATING THE EFFECTS
OF EXPOSURE TO COMBUSTIBLE AND
NON-COMBUSTIBLE TOBACCO PRODUCT
PREPARATIONS USING IN VITRO AND EX VIVO
MODELS 1 Research and Development, R.J. Reynolds Tobacco Company,
Winston-Salem, NC USA 2 Department of Medicine and Department of Pharmacology and Toxicology, University of Louisville
School of Medicine, Louisville, KY 3 Department of Microbiology and Immunology, Wake Forest University Baptist Health, Winston-
Salem, NC
CORESTA Congress 12-16 October 2014 Québec City, Canada ST 17
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Cellular Effects of Cigarette Smoke (CS)
• Physiologically, exposure to CS elicits direct effects at
local areas of exposure and indirect systemic effects.
• Many of those effects have been fairly well understood in
many cellular models.
• Exposure to CS or its constituent phases results in a
range of adverse responses including: – Cytotoxicity
– Genotoxicity
– Oxidative stress
– Inflammatory responses
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• ST constitutes a large diverse group of oral tobacco
products consumed globally.
– Moist Snuff is the predominant form of ST in the USA.
• US epidemiological data indicate that the risk of lung
cancer, COPD, CVD and oral cancer are significantly
reduced in ST users relative to smokers.
• The cellular effects of exposure to smokeless tobacco (ST)
are less clear and appear to vary.
– Hence, we initiated a series of studies to comparatively
evaluate the effects of CS and ST in cell culture.
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Smokeless Tobacco (ST)
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Objective of This Presentation
To present an integrated summary of those studies on the
effects of combustible and non-combustible tobacco.
Reference Tobacco Product Preparations (TPPs)
3R4F reference cigarettes
2S3 Moist Snuff
In vitro and ex vivo studies
Oral cavity cells
Hematopoietic cells
Biological responses
Cytotoxicity
Inflammation
DNA damage
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Approach
• Combustible TPPs from 3R4F reference cigarettes – Total Particulate Matter (TPM) – Whole Smoke Conditioned Medium (WS-CM)
• Non-combustible TPPs – Extracts of 2S3 Moist snuff in Complete Artificial Saliva (ST/CAS)
• Equi-nicotine paradigm: Nicotine content of the TPPs used to report exposure/dosing
• Cell types – Human oral cavity (HGEC, HGF, 101A) cells to assess local effects – Human hematopoietic cells (examples: HL60 and PBMCs) to assess
systemic effects
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Results of these studies have been published
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Chemical Analyses of Tobacco Preparations
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Representative analyses of TPPs
BDL; Below Detection Level
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Cytotoxicity of TPPs in Hematopoietic Cells
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EC50 values, given in µg/mL of equi-nicotine units at 24h, as measured by positive
staining with 7-amino-actinomycin-D (7-AAD)
Cytotoxic potential in hematopoietic cells: WS-CM > TPM > ST/CAS > nicotine.
PBMC HL60 THP1
TPM 2.67±0.15 1.55 ±0.31 1.48 ±0.07
WS-CM 1.55 ±0.07 1.62 ±0.07 2.5 ±0.5
ST/CAS >700 >700 >700
Nicotine 1650 ±70
(10mM)
1420 ±52
(8mM)
1800 ±52
(11mM)
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Cytotoxic Effects of TPPs in Oral Cavity Cells
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TPP /
Culture (h)
101A cells HGECs
(24h) (48h) (24h) (48h)
TPM 14 10 >24 4
WS-CM 5.1 5.5 >21.2 >21.2
ST/CAS >474 >474 >474 >474
EC50 values, given in µg/mL of equi-nicotine units at 24h and 48h, as measured
by sulforhodamine B assay
Cell type specific effects: Human gingival epithelial cells (HGECs) are relatively
more resistant than 101A cells.
ST/CAS is the least cytotoxic TPP, based on 101 cell data.
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TPP-induced DNA Damage in THP-1 Cells
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DNA damage was quantified by flow cytometric detection of phosphorylated histone
H2AX protein in TPP-treated THP-1 macrophage cells.
Combustible TPPs caused significantly more DNA damage than ST/CAS and nicotine,
based on equi-nicotine dosing.
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ST/CAS
Nicotine
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TPP-induced DNA Damage in Oral Cavity Cells
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Positive staining for –H2AX is
indicative of DNA damage
• TPM treatment resulted in DNA damage.
• Treatment with ST/CAS (at high equi-nicotine units)
or nicotine alone (565µg/mL) did not result in
detectable DNA damage by this method.
• Similar data were obtained with HGF and 101A
cells (not shown here).
Detection of –H2AX in
TPM-treated HGEC cells
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TPM-induced DNA Damage In Oral Cavity Cells -
Comet Assays
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HG
EC
1
01
A
HG
F
• TPM treatment caused marked DNA
damage in all three cell types. • Normal HGEC and HGF cells appear to be
more resistant than the malignant 101A
cells.
• ST/CAS induced DNA damage only at 565
µg/mL of equi-nicotine units.
• DNA damage in nicotine-treated cells (565
µg/mL) was not detected.
TPM-treated cells were subjected
to alkaline comet assays.
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Cigarette Smoking and Inflammation
• Smoking causes chronic inflammation, yet suppresses immune responses.
• Smokers are at increased risk for microbial infections and malignancies such as lung cancer. Smoking-induced immune suppression may be a contributing factor.
• Treatment of PBMCs with “smoke extracts” inhibits secretion of proinflammatory cytokines, in response to stimulation with Toll-Like Receptor (TLR) ligand, and cytolytic functions. – Useful ex vivo model.
• LPS used as a ligand for TLR-4
• Poly I:C used as a ligand for TLR-2
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WS-CM Suppresses Immune Responses in
PBMCs
• Treat PBMCs with WSCM or nicotine
• Stimulate with poly: IC or LPS
• Measure secreted cytokines
• WS-CM treatment suppresses the secretion
of TNF by the stimulated PBMCs.
• Nicotine treatment (below 500 µg/mL) did not
inhibit the cytokine secretion by cells.
• Similar results were obtained for several
other inflammatory cytokines.
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WS-CM Suppresses Immune Responses in
PBMCs
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• Treat PBMCs with WSCM
• Co-culture K562 cells (target cells)
• Measure cytolysis
• WS-CM treatment suppresses the killing of
target K562 cells by PBMCs.
• TPM also suppresses target cell killing.
• Nicotine treatment (below 500 µg/mL) did not
inhibit the target cell killing.
Collectively, these results show that WS-CM, not nicotine, suppresses proinflammatory
and immune surveillance functions of PBMCs.
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Summary
Collectively, these findings:
• Support the epidemiological evidence that
combustible tobacco products are more harmful
compared to the non-combustible products.
• Support evidence of a risk continuum across
tobacco product categories.
• Suggest that in vitro and ex vivo models may be
useful in evaluating combustible and non-
combustible tobacco products and nicotine.
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References
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• Fowler K, Hill JA, Bombick B, Prasad GL. Standardization of the preparation of smokeless tobacco extracts for assessment of
biological effects. CORESTA Congress (2010) Poster.
• Arimilli S, Damratoski BE, Bombick B, Borgerding MF, Prasad GL. Evaluation of cytotoxicity of different tobacco product
preparations. Regulatory Toxicology and Pharmacology (2012) 64: 350-360.
http://www.sciencedirect.com/science/article/pii/S0273230012001833
• Arimilli S, Damratoski BE, Prasad GL. Combustible and non-combustible tobacco product preparations differentially regulate
peripheral blood mononuclear cell functions. Toxicology in Vitro (2013) 27: 1992-2004.
http://www.ncbi.nlm.nih.gov/pubmed/23851003
• Gao H, Prasad GL, Zacharias W. Differential cell-specific cytotoxic responses of oral cavity cells to tobacco preparations.
Toxicology in Vitro (2013) 27: 282-291. http://www.ncbi.nlm.nih.gov/pubmed/22960471
• Gao H, Prasad GL, Zacharias W. Combusted but not smokeless tobacco products cause DNA damage in oral cavity cells.
Environmental Toxicology and Pharmacology (2014) 37: 1079-1089. http://www.ncbi.nlm.nih.gov/pubmed/24780532
• Arimilli S, Damratoski BE, Prasad GL. Methods to evaluate cytotoxicity and immunosuppression of combustible tobacco product
preparations. Journal of Visualized Experiments (2014) In press.
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http://www.sciencedirect.com/science/article/pii/S0273230012001833http://www.ncbi.nlm.nih.gov/pubmed/23851003http://www.ncbi.nlm.nih.gov/pubmed/22960471http://www.ncbi.nlm.nih.gov/pubmed/24780532