Journal: Toxicology Reports

Article Title: Comparative toxicity of menthol- and tobacco-flavored electronic cigarette constituents inducing inflammation, epithelial barrier dysfunction, and nicotinic acetylcholine receptor modulation in the absence of nicotine

doi: 10.1016/j.toxrep.2026.102224

Figure Lengend Snippet: Menthol and tobacco flavoring chemicals caused BEAS-2B epithelial cell barrier dysfunction. BEAS-2B cells were grown in transwell inserts in complete medium. Once reached a monolayer and 80–85 % confluency, cells were serum deprived overnight. Around 90–95 % confluency, cells were treated with 100 μM (A) 98 % Menthone. (B) L -Menthone, (C). Carvone (D) WS-23 (E) Acetoin, (F) Vanillin, (G) PG/VG, and (H) Benzoic Acid. Transepithelial electrical resistance (TEER) and voltage (mV) data were collected pretreatment (0 hr), 6, 8, 20, and 24 hrs. following the treatments and the correlation of TEER and mV vs. time ± SEM are represented. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. untreated control., two-way ANOVA. N = 3 wells per chemical treatment.

Article Snippet: Human bronchial epithelial cells (BEAS-2B) (ATCC) were seeded on the apical side of 12 mm diameter polyester membrane transwell inserts with 0.4 μM pore size and 1.12 cm 2 surface area (Corning #3460) in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 1:1 (Gibco, Cat# 11320033) supplemented with 5 % fetal bovine serum (FBS), 15 mM HEPES, 1 % L -glutamine, and 1 % antibiotic-antimycotic.

Techniques: Control

Journal: Toxicology Reports

Article Title: Comparative toxicity of menthol- and tobacco-flavored electronic cigarette constituents inducing inflammation, epithelial barrier dysfunction, and nicotinic acetylcholine receptor modulation in the absence of nicotine

doi: 10.1016/j.toxrep.2026.102224

Figure Lengend Snippet: Menthol and tobacco flavoring constituents elicited an interleukin 6 cytokine response in lung epithelial cells. BEAS-2B cells cultured in transwells in complete media, 80–85 % confluency, and serum deprived overnight. Around 90–95 % confluency, cells were treated with 100µM L -Menthone, 98 % Menthone, Carvone, WS-23, Vanillin, Acetoin, Benzoic Acid, and PG/VG. Apical conditioned media was collected after the 24-h time point and IL-6 was quantified. (A) control, PG/VG, and Benzoic Acid-induced response, and (B) L -Menthone, 98 % Menthone, Carvone, WS-23, Vanillin, and Acetoin response compared to untreated control. IL-6 concentration in pg/mL ± SEM is represented, *p < 0.05. vs. control, one-way ANOVA. N = 3 wells per treatment.

Article Snippet: Human bronchial epithelial cells (BEAS-2B) (ATCC) were seeded on the apical side of 12 mm diameter polyester membrane transwell inserts with 0.4 μM pore size and 1.12 cm 2 surface area (Corning #3460) in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 1:1 (Gibco, Cat# 11320033) supplemented with 5 % fetal bovine serum (FBS), 15 mM HEPES, 1 % L -glutamine, and 1 % antibiotic-antimycotic.

Techniques: Cell Culture, Control, Concentration Assay

Journal: Toxicology Reports

Article Title: Comparative toxicity of menthol- and tobacco-flavored electronic cigarette constituents inducing inflammation, epithelial barrier dysfunction, and nicotinic acetylcholine receptor modulation in the absence of nicotine

doi: 10.1016/j.toxrep.2026.102224

Figure Lengend Snippet: Menthol and tobacco flavoring constituents elicited an interleukin-8 cytokine response in lung epithelial cells. BEAS-2B cells cultured in transwells in complete media, 80–85 % confluency, and serum deprived overnight. Around 90–95 % confluency, cells were treated with 100 μM l-menthone, 98 % menthone, carvone, WS-23, vanillin, acetoin, benzoic acid, and PG/VG. Apical conditioned media was collected after the 24-h time point and IL6 was quantified. (A) control, PG/VG, and Benzoic Acid-induced response, and (B) l-menthone, 98 % menthone, carvone, WS-23, vanillin, and acetoin response compared to untreated control. IL-8 concentration in pg/mL ± SEM is represented, *p < 0.05, and **p < 0.01 vs. untreated control. one-way ANOVA. N = 3 wells per treatment.

Article Snippet: Human bronchial epithelial cells (BEAS-2B) (ATCC) were seeded on the apical side of 12 mm diameter polyester membrane transwell inserts with 0.4 μM pore size and 1.12 cm 2 surface area (Corning #3460) in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 1:1 (Gibco, Cat# 11320033) supplemented with 5 % fetal bovine serum (FBS), 15 mM HEPES, 1 % L -glutamine, and 1 % antibiotic-antimycotic.

Techniques: Cell Culture, Control, Concentration Assay

Journal: Toxicology Reports

Article Title: Comparative toxicity of menthol- and tobacco-flavored electronic cigarette constituents inducing inflammation, epithelial barrier dysfunction, and nicotinic acetylcholine receptor modulation in the absence of nicotine

doi: 10.1016/j.toxrep.2026.102224

Figure Lengend Snippet: Menthol and tobacco flavoring constituents caused minimum cytotoxicity in BEAS-2B cells. BEAS-2B cells cultured in transwells in complete media, at 80–85 % confluency, and serum deprived overnight. Around 90–95 % confluency, Cells were treated with 100 μM l-menthone, 98 % menthone, carvone, WS-23, vanillin, acetoin, benzoic acid, and PG/VG. At the 24-h time point, cells were collected and stained with acridine orange and propidium iodide and the live, cell, and total cells were counted using CellDrop automatic cell counter. Cytotoxicity ± SEM is represented. *p < 0.05 vs. control, one-way ANOVA, N = 3 wells per treatment.

Article Snippet: Human bronchial epithelial cells (BEAS-2B) (ATCC) were seeded on the apical side of 12 mm diameter polyester membrane transwell inserts with 0.4 μM pore size and 1.12 cm 2 surface area (Corning #3460) in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 1:1 (Gibco, Cat# 11320033) supplemented with 5 % fetal bovine serum (FBS), 15 mM HEPES, 1 % L -glutamine, and 1 % antibiotic-antimycotic.

Techniques: Cell Culture, Staining, Control

Journal: Toxicology Reports

Article Title: Comparative toxicity of menthol- and tobacco-flavored electronic cigarette constituents inducing inflammation, epithelial barrier dysfunction, and nicotinic acetylcholine receptor modulation in the absence of nicotine

doi: 10.1016/j.toxrep.2026.102224

Figure Lengend Snippet: Menthol and tobacco flavoring constituents caused nicotinic acetylcholine receptor (nAchR) modulation in BEAS-2B lung epithelial cells. BEAS-2B cells cultured in transwells in complete media, 80–85 % confluency, and serum deprived overnight. Around 90–95 % confluency, cells were treated with 100 μM l-menthone, 98 % menthone, carvone, WS-23, vanillin, acetoin, benzoic acid, and PG/VG. At the 24-h time point, cells were collected, lysed, and after BCA protein estimation, 5 μg of protein were loaded to 10-well gel for SDS-gel electrophoresis. After cellulose membrane transfer and blocking, the membranes were probed with primary antibodies for nAchR1,4,5, and 7, with ß-actin loading control for normalization. The same membrane was sometimes re-probed up to 3 times with a different CHRNA. The blots with (A) Nicotinic Acetylcholine Receptors α1 expression with acetoin and PG/VG. (B) Nicotinic Acetylcholine Receptors α4 expression with carvone and WS-23. (C) Nicotinic Acetylcholine Receptors α5 expression with acetoin and PG/VG. (D) Nicotinic Acetylcholine Receptors α5 expression with l-menthone and 98 % menthone. (E) Nicotinic Acetylcholine Receptors α7 expression with carvone and WS-23. All respective CHRNA bands ß-actin are shown with their densitometry fold-change ± SEM. *p < 0.05 and ****p < 0.0001 vs. control, one-way ANOVA. N = 3 wells per chemical. Full blots are shown in the .

Article Snippet: Human bronchial epithelial cells (BEAS-2B) (ATCC) were seeded on the apical side of 12 mm diameter polyester membrane transwell inserts with 0.4 μM pore size and 1.12 cm 2 surface area (Corning #3460) in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 1:1 (Gibco, Cat# 11320033) supplemented with 5 % fetal bovine serum (FBS), 15 mM HEPES, 1 % L -glutamine, and 1 % antibiotic-antimycotic.

Techniques: Cell Culture, SDS-Gel, Electrophoresis, Membrane, Blocking Assay, Control, Expressing