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Chocolate Vaping

Cara Sherwood and Scott Boitano of the University of Arizona published this study a few years before vaping related deaths starting making the news.  These authors noted that although some of the flavorings in electronic cigarettes are approved for human ingestion, they may not be safely inhaled.   Diacetyl, an artificial butter flavoring, may be toxic when inhaled but not when ingested.

Sherwood and Boitano used the following small molecule flavorants

  • 2,5-dimethylpyarzine (chocolate, nutty flavor)
  • damascenone (apple, citrus, wine-like)
  • linalool (floral, spice);
  • α-ionone (fruity, raspberry)
  • ethyl maltol (caramel)
  • furaneol (strawberry, sweet)
  • and vanillin (vanilla)

16HBE14o- cells, a SV40 transformed human bronchial epithelial cell line.  These cells have CFTR chloride channels that release chloride into the lumen of the airway when they are opened by phosophorylation by protein kinase A  (PKA).

CFTR_vaping
Sherwood and Boitano (2016) studied the affects of flavored vaping compounds on a bronchiolar cell line. By use of the adenylate cyclase inhibitor forskolin, chocolate vaping compound 2,5-dimethylpyrazine affect was  attributed to a protein kinase A-dependent (PKA) activation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel.

In this model, Na+ secretion (not shown) follows Cl- secretion.  Water then follows NaCl secretion.  This overall mechanism serves to clear the airways of noxious substances.  Chloride secretion in response to Ecigarette flavoring compounds may signal a subtoxic response.

These authors used a xCELLigence (ACEA Biosciences, San Diego, CA) real time cell analyzer to monitor mesure electcal impendence of  16HBE14o- cells  plated in 96 well E-plates.  Relative impedance is expressed as a Cell Index where:

Cell Index = (Zi-Z0)/15Ω;

  • Zi is impedance at a given time point during the experiment (i.e., post ATP addition)
  • Z0 is impedance before the addition of agonist.

Impedence_vaping

A dramatic decrease in impedance (complex resistence) can be indicative of cell death.  When the cells die, they are no longer there to resist electrical current.

Activation of  G-protein coupled receptor Gq, such as occurs following ATP activation of purinergic receptors, results in an increase in Cell Index.

This image was adapted from Lazarowski and Boucher 2009.   The Gq subunit of heterotrimeric  G protein was added based on previous work of Sherwood (2009).  Heterotrimeric G proteins are composed of Gα and Gβγ subunits.  Gαq activates phospholipase C that ulitmately results in an increase in intracellular calicum.  Protein kinase C (PKC) is activated by calcium.  Adenylate cyclase is positively and negatively  modulated by Gαs and Gαi, respectively.

vaping_purinergic
Binding of agonists to A2b and P2Y2 receptors causes dissociation of heterotrimeric G protein proteins that go on to modulate other cellular functions

How ATP activation of  P2Y2 by ATP increases impedance was not fully discussed.  Did the increase in calcium result in cell spreading?

Cell Index responses to e-liquid flavorings were recorded every 15 min for 24 h. Physiological responses to ATP and forskolin were recorded every 30 s for 4 h following a 24 h exposure to select e-liquid flavorings.

 

vaping_realtimecytotox
A highly stylized adaptation of real time response of cultured bronchiolar cells to two vaping flavors from Sherwood and Boitano (2016), Figure 1. Vanillin was considered non toxic.

This highly stylized exert from Sherwood and Boitano (2016) was increased 2x in Paint and subjected to a Gaussian blue in the image manipulation program Gimp.   Axes of the graphs were reentered for clarity.  The objective was to highlight the ability of this high throughput assay to detect compounds that interact with airway epithelials.  Other toxicology assays are will be necessary.  Sherwood and Boitano (2016) also used pharmacological approaches to deduce that 2,5-dimethylpryazine (chocolate) acted on adylate cyclase.

Perhaps, this clever, high throughput study of Sherwood and Boitano could be repeated with more complex formulations.  Lechasseur and others (2019) looked at particle size and where they might deposit within the airways.

 

Wölkart and coworkers tested the hypothesis that very similar e-cigarette flavors would be cytotoxic to endothelial cells of blood vessels.  The following flavors were not toxic in amounts found in e-cigarettes.

 

  • 2,5-dimethylpyarzine (chocolate, nutty flavor)
  • Acetylpyridine  (popcorn, etc)
  • Isoamylacetate (banana)
  • Cinnamaldehyde (cinnamon)
  • Eugenol  (cloves)
  • Diacetyl  butter)
  • and vanillin (vanilla)

The hypothesis that these compounds could cause vasoconstriction by scavenging nitric oxide (NO) from blood  vessel endothelial cells was also tested.  If anything, these compounds cause endothelium independent vasorelaxation of blood vessel smooth muscle?  What does this mean for brochiole smooth muscle?

vaping_smoothmuscle
The diameters of blood vessels (left) and bronchioles (right) are controlled by smooth muscle contraction and relaxation. Endothelial cells may release nitric oxide (NO) that causes blood vessels to relax.

If relaxation was endothelial cell independent, does this mean that flavor compound(S) were causing relaxation in their own right?  With an EC50 of about 0.5 mM, eugenol was the most potent vasorelaxant among the tested compounds, 2,5-dimethylpyarzine had a higher  EC50 .

  1. If compounds like 2,5-dimethylpyarzine cause epithelial cells to peel off the fibroblast surface as per the cell culture system of Sherwood and Boitano, does more 2,5-dimethylpyarzine have access to  airway smooth muscle once it works its way past the fibroblasts?
  2. If airway smooth muscle relaxes, will more vaping aerosols be able to make their way deeper into the lungs of e-cigarette smokers?

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References

Lechasseur A, Altmejd S, Turgeon N, Buonanno G, Morawska L, Brunet D, Duchaine C, Morissette MC. (2019) Variations in coil temperature/power and e-liquid constituents change size and lung deposition of particles emitted by an electronic cigarette. Physiol Rep. 2019 May;7(10):e14093  Free Paper

 

Sherwood CL, Boitano S. (2016) Airway epithelial cell exposure to distinct e-cigarette liquid flavorings reveals toxicity thresholds and activation of CFTR by the chocolate flavoring 2,5-dimethypyrazine. Respir Res. 2016 May 17;17(1):57. doi: 10.1186/s12931-016-0369-9.  Free Paper

Wölkart G, Kollau A, Stessel H, Russwurm M, Koesling D, Schrammel A, Schmidt K, Mayer B. (2019) Effects of flavoring compounds used in electronic cigarette refill liquids on endothelial and vascular function. PLoS One. 2019 Sep 9;14(9):e0222152. Free Paper 

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