Flavor Classification/Categorization and Differential Toxicity of Oral Nicotine Pouches (ONPs) in Oral Gingival Epithelial Cells and Bronchial Epithelial Cells

Abstract

Oral nicotine pouches (ONPs) are a modern form of smokeless tobacco products sold by several brands in the U.S., which comprise a significant portion of non-combustible nicotine-containing product (NCNP) sales to date. ONPs are available in various flavors and may contain either tobacco-derived nicotine (TDN) or tobacco-free nicotine (TFN). The growth in popularity of these products has raised concerns that flavored ONPs may cause adverse oral health effects and promote systemic toxic effects due to nicotine and other ONP by-products being absorbed into the circulatory system through oral mucosa. We hypothesized that flavored ONPs are unsafe and likely to cause oral and pulmonary inflammation in oral and respiratory epithelial cells. Before analyzing the effects of ONPs, we first classified ONPs sold in the U.S. based on their flavor and the flavor category to which they belonged using a wheel diagram. Human gingival epithelial cells (HGEP) were treated with flavored ONP extracts of tobacco (original, smooth), menthol (wintergreen and cool cider), and fruit flavor (americana and citrus), each from the TDN and TFN groups. The levels of ONP-induced inflammatory cytokine release (TNF-α, IL-6, and IL-8) by ELISA, cellular reactive oxygen species (ROS) production by CellRox Green, and cytotoxicity by lactate dehydrogenase (LDH) release assay in HGEP cells were assessed. Flavored ONP extracts elicited differential toxicities in a dose- and extract-dependent manner in HGEP cells 24 h post-treatment. Both fruit TDN and TFN extracts resulted in the greatest cytotoxicity. Tobacco- and fruit-flavored, but not menthol-flavored, ONPs resulted in increased ROS production 4 h post-treatment. Flavored ONPs led to differential cytokine release (TNF-α, IL-6, and IL-8) which varied by flavor (menthol, tobacco, or fruit) and nicotine (TDN vs. TFN) 24 h post-treatment. Menthol-flavored ONPs led to the most significant TNF-α release; fruit TFN resulted in the most significant IL-6 release; and fruit TDN and tobacco TFN led to the highest release of IL-8. Subsequently, human bronchial epithelial cells (16-HBE and BEAS-2B) were also treated with flavored ONP extracts, and similar assays were evaluated. Here, the lowest concentration treatments displayed increased cytotoxicity. The most striking response was observed among cells treated with spearmint and tobacco flavored ONPs. Our data suggest that flavored ONPs are unsafe and likely to cause systemic and local toxicological responses during chronic usage.

Keywords: cytotoxicity; inflammation; oral nicotine pouches; periodontal problems; pulmonary health.

Figure 1

Wheel-based Classification/Categorization of Natural/Synthetic Oral Nicotine Pouches (ONPs)/products commonly sold in the U.S. (A) Flavor wheel for Natural/Snus Oral Nicotine Pouch (B) Flavor wheel for Synthetic oral Nicotine Pouch. The nicotine concentration of all smoke-free nicotine-based pouches ranges from 3 mg to 8 mg per pouch; mint/menthol and fruit are two of the most widely sold flavors in the U.S. The flavors of each pouch product in the diagram are color-coded by flavor category, Bright green Color represents ONPs of mint/menthol flavors, Light blue for fruit flavors, Red color mixed flavors, Pink color ONPs available in drink flavors, Yellow color for Tobacco flavors ONPs, parrot green color ONPs available in dessert flavors, Sky blue ONPs in aroma flavors and Orange represents ONPs available in flavor of spices. The inner wheel represents the most common flavors, and the outer wheel represents specific flavors.

Figure 1

Wheel-based Classification/Categorization of Natural/Synthetic Oral Nicotine Pouches (ONPs)/products commonly sold in the U.S. (A) Flavor wheel for Natural/Snus Oral Nicotine Pouch (B) Flavor wheel for Synthetic oral Nicotine Pouch. The nicotine concentration of all smoke-free nicotine-based pouches ranges from 3 mg to 8 mg per pouch; mint/menthol and fruit are two of the most widely sold flavors in the U.S. The flavors of each pouch product in the diagram are color-coded by flavor category, Bright green Color represents ONPs of mint/menthol flavors, Light blue for fruit flavors, Red color mixed flavors, Pink color ONPs available in drink flavors, Yellow color for Tobacco flavors ONPs, parrot green color ONPs available in dessert flavors, Sky blue ONPs in aroma flavors and Orange represents ONPs available in flavor of spices. The inner wheel represents the most common flavors, and the outer wheel represents specific flavors.

Figure 2

Protocol for preparation of TDN (natural) and TFN (synthetic) nicotine pouches extract for cell exposures. (A) Extracts of oral nicotine pouches were prepared by incubating pouches in PBS (1:10 w/v) for 1 h on a shaker (500 rpm) at 37 °C. Extracts were centrifuged and then filtered through 0.45 micron sterile filters and denoted 100% for treatments. (B) The appearance of freshly extracted TDN (natural) and TFN (synthetic) extracts are shown.

Figure 3

Differential cytotoxicity among oral epithelial cells upon treatment with different flavored oral smokeless nicotine products: (A) Diagrammatical representation of the experimental workflow. (B–D) Cytotoxicity Assay on oral epithelial cells: human gingival epithelium progenitors cells (HGEPp; gingival epithelial cells) were treated with different doses (0.25%, 0.5%, 1%, 3%, and 10%) of oral nicotine extracts categorizing natural/synthetic tobacco, menthol, and fruit flavors from different brands (Snus, Zyn, Grizzly, Lucy and ON). Followed by 24 h exposure, conditioned media were used for the lactate dehydrogenase (LDH) assay. Data represented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control. n = 3.

Figure 4

Differential cytotoxicity due to exposure of flavored oral smokeless nicotine products in bronchial epithelial cells. (A) BEAS-2B cells were seeded and treated with spearmint-flavored oral product extracts from two different brands. Following 24 h exposure, conditioned media was used for the lactate dehydrogenase (LDH) measurements. (B) 16HBE cells were seeded and treated with ON! Original, Rogue mango, or Velo black cherry-flavored nicotine pouch extracts. Following 24 h exposure, conditioned media was used LDH measurements. Data represented as means *** p < 0.001 compared to control. (Mean ± SEM, n = 3.)

Figure 5

ROS production in Human gingival epithelium progenitors (HGEPp). HGEPp cells were treated at 3% dosage with extracts of oral pouches categorized with natural/synthetic tobacco, menthol, and fruit flavors) belonging to different brands (Snus, Zyn, Grizzly, Lucy, and ON) and incubated for 4 h. Followed by 4 h exposure, cells were exposed to a 5 µM CellRox Green Reagent. Then, were fixed with PFA and nuclei were counterstained with Hoechst 33,342 stain. (A) Fluorescence images display the release of ROS and nuclei (blue) in HGEPp. (B) Quantitative analysis of fluorescence. Data are represented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. Images were captured by Cytation 5 imaging reader (BioTek) reader and CellROX fluorescent signals were quantified using Image J software (n = 3). Scale bar = 200 μm.

Figure 6

Production of ROS was measured in 16 HBE cells after treatment with different flavors of nicotine pouches. 16HBE cells were serum-deprived and were treated with ON! Original, Rogue mango, or Velo black cherry flavored nicotine pouch extracts and incubated for 4 h. Following the incubation oxidative stress measurements were carried out using CellROX Green Reagent. (A) Fluorescence images displaying the production of ROS and nuclear morphology (blue) in 16HBE cells treated with different flavors of nicotine pouch extracts at doses of 0.25% and 1%. (B) Quantitative analysis of fluorescence in 16 HBE cells. Data are represented as mean ± SEM. *** p < 0.001. Images were captured by Cytation 5 imaging reader (BioTek) reader and CellROX fluorescent signals were quantified using Image J software (n = 3). Scale bar = 200 μm.

Figure 7

Inflammatory mediator response by oral nicotine products in oral epithelial cells: HGEPp cells were treated at oral nicotine pouches extracts categorized with natural/synthetic tobacco, menthol, and fruit flavors) of different brands (Snus, Zyn, Grizzly, Lucy, and ON). Following 24 h exposure, conditioned media was used to perform ELISAs. (A) Protein levels of TNF-α determined by ELISA. (B) Protein levels of IL-6 determined by ELISA. (C) Protein levels of IL-8 determined by ELISA. Data represented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to negative control (untreated) n = 3.

Figure 8

Inflammatory mediator response due to flavoring nicotine oral products in bronchial epithelial cells. (A) BEAS-2B cells were treated with spearmint-flavored nicotine pouches from Zyn and Skoal brands. Following 24 h exposure, conditioned media was used for IL-8 measurements. (B) 16HBE cells were treated with different oral nicotine pouch flavors ON! original, Rogue mango, or Velo black cherry and incubated for 24 h. Following 24 h exposure, conditioned media was used for IL-6 measurement. Results are represented graphically.

Figure 9

Schematic demonstration of the effect of Oral Nicotine Pouch on Oral/lung Epithelial Cells. Chewing of Oral Nicotine Pouch could lead to oral epithelial injury by releasing ROS via activation of inflammatory cytokines such as TNF-α, IL-6, and IL-8 which could further lead to oral and lung-related problems.