Little Cigars are More Toxic than Cigarettes and Uniquely Change the Airway Gene and Protein Expression

Abstract

Little cigars (LCs) are regulated differently than cigarettes, allowing them to be potentially targeted at youth/young adults. We exposed human bronchial epithelial cultures (HBECs) to air or whole tobacco smoke from cigarettes vs. LCs. Chronic smoke exposure increased the number of dead cells, lactate dehydrogenase release, and interleukin-8 (IL-8) secretion and decreased apical cilia, cystic fibrosis transmembrane conductance regulator (CFTR) protein levels, and transepithelial resistance. These adverse effects were significantly greater in LC-exposed HBECs than cigarette exposed cultures. LC-exposure also elicited unique gene expression changes and altered the proteomic profiles of airway apical secretions compared to cigarette-exposed HBECs. Gas chromatography-mass spectrometry (GC-MS) analysis indicated that LCs produced more chemicals than cigarettes, suggesting that the increased chemical load of LCs may be the cause of the greater toxicity. This is the first study of the biological effects of LCs on pulmonary epithelia and our observations strongly suggest that LCs pose a more severe danger to human health than cigarettes.

Figure 1. Chronic tobacco smoke causes enhanced cytotoxicity and inflammation.

(a) Left, light micrographs showing H&E stained human airway cultures and right, XZ confocal micrographs showing calcein-AM (green) and rhodamine dextran (red) stained live cells and airway surface liquid respectively. Bar graphs show mean changes as noted for (b) apical cilia abundance (n = 10), (c) transepithelial electrical resistance (n = 14) and (d) propidium iodide (PI) positive cells (n = 23), (e) percent cytotoxicity measured by percentage of LDH release (n = 20) and (f) IL-8 release (n = 20) into basolateral media following chronic smoke exposure (*p < 0.05, **p < 0.001, ***p < 0.0001).

Figure 2. Little cigar smoke exposure causes more cytotoxicity and inflammation than commercial cigarettes.

HBECs were chronically exposed to 10 × 35 ml puffs of smoke per day for five days from Kentucky, Marlboro, Camel cigarettes and Swisher Sweets little cigar. (a) LDH release as a marker of cytotoxicity and (b) and IL-8 release into the basal media. ^*Denotes p < 0.05 different to air. All data points represent n = 6.

Figure 3. Chronic LC exposure induces significantly greater airway surface liquid (ASL) dehydration than cigarette exposure.

(a) Representative XZ confocal micrographs of ASL (red) labeled with rhodamine-dextran. Scale bar is 10 μm. 0′ denotes ASL before fifth day exposure and 30′ indicates ASL after 30 minutes of fifth day exposure. (b) Mean ASL height measured by XZ confocal microscopy after chronic (5 day) exposure to air, Kentucky cigarettes or LCs (n = 10). (c) HBECs were exposed to smoke or air for 5 days, lysed and Western blots were then run and probed for CFTR, β-ENaC and actin as indicated. Bands corresponding to the relevant protein sizes (kD) are shown in the figure. (d) Mean integrated densitometry for CFTR and β-ENaC respectively after normalization to the actin loading control (n = 10 blots). (*p < 0.05, **p < 0.001, ***p < 0.0001).

Figure 4. Chronic LC exposure results in unique changes in gene expression.

HBECs derived from 3 individual donors were exposed to chronic smoke from Kentucky cigarettes vs. Swisher Sweets LCs for 5 days vs. air controls and probed with the Nanostring pan-cancer-immune gene panel. (a) Heat map showing genes altered across the groups (q-value <0.01, fold change >2). Color codes represent normalized expression levels of the genes after standardization across samples with mean = 0 and standard deviation = 1. (b) Venn diagram comparing the number of altered genes in the tobacco-exposed groups relative to air exposure (q-value <0.1, fold change > 2). (c) Pie chart representing altered genes representing biological processes following LC smoke compared to Kentucky cigarette exposure. (d) Pie chart representing protein classes corresponding to altered genes following LC smoke compared to Kentucky cigarette exposure.

Figure 5. LC smoke exposure causes greater changes to the ASL proteome than cigarette smoke exposure.

(a) Heat map of significantly changed proteins relative to air controls. Significance was set at p ≤ 0.001. (b) Venn diagram showing proteins that are upregulated in each group. (c) Pie chart representing the biological process classification for significantly changing proteins of all exposure groups. (d) Reactome map showing the functional enrichment (FE) of proteins with significant increases after Kentucky cigarette and LC exposure.

Figure 6. LC combustion yields different chemicals than cigarette combustion.

(a) Heat map showing presence (red) or absence (white) of chemical compounds from mass spectrometry of tar particles in different tobacco products. (b) Gas Chromatogram showing peaks representing different compounds in the collected tar phase from different tobacco products.

Figure 7. Little cigars have more chemical constituents in their tar phase than Kentucky research cigarettes.

Whole tobacco smoke tar particles were analyzed by GC-MS and 30 chemicals were found to be common to all the tobacco products. 49 unique chemical entities were identified in tar particles from all 3 little cigars. In contrast, 6 unique chemicals were identified in Kentucky cigarettes.