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

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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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TNF,

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Eq-nic units

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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