February 24, 2019

Stanton A. Glantz, PhD

Evidence on how e-cigs cause lung and heart disease and, now, cancer, presented at SRNT meeting

There were several interesting scientific presentations at the Society for Research on Nicotine and Tobacco meeting held from February 20-23, 2019 in San Francisco dealing with the biology of how e-cigarettes cause disease.  It is becoming clearer and clearer that e-cigarettes are not just cigarettes that deliver less of some bad things, which is how everyone, including me, used to think about them.  The reality is that e-cigarettes are different from cigarettes.  While they damage the body through some mechanisms in common with cigarettes, it is now clear that e-cigarettes also have adverse effects through channels not affected by cigarettes (and vice versa).  The effects even transcend generations; exposure while pregnant compromises blood function in adult offspring.

These effects occur at the levels of exposure that real-world e-cigarettes generate and can be substantial.  Evidence is also appearing demonstrating that e-cigarettes are implicated in cancer, something most people, including me, previously did not think was a substantial risk.

The available of these cellular and molecular screens is allowing us to understand not just that e-cigarettes cause lung and cardiovascular disease and, likely cancer, but what the precise biological mechanisms are.

Here are my summaries of the presentations I saw, followed by the abstracts. 

Lung disease

Since lungs are in contact to air, which has many bacteria and viruses floating around, an important function of the lungs, in addition to gas exchange, is an immune function to destroy these potentially dangerous insults.  One way that the lungs do this is with enzymes called proteases which break up the proteins in these invaders.  At the same time, it is important to keep these proteases under control so they do not attack the proteins in the lungs themselves. So, your lungs also include protease inhibitors to keep things under control.  One of the ways that smoking damages lungs is by disrupting this balance.  Ghosh et al (abstract PA4-1) took samples from smokers, vapers (e-cigs only) and non-users and showed that e-cigarettes do the same thing that cigarettes do.  Nicotine was specifically implicated.

It is becoming clearer and clearer that the flavors in e-cigarettes are toxic to lungs.  Jabba et al (abstract PA4-2) showed that flavors degrade while being stored to produce chemicals called acetals, which are very stable. These acetals irritate lungs and kill human lung cells. 

Cardiovascular disease

Smoking and e-cigarette use interfere with arteries’ ability to dilate (enlarge) so they can accommodate increase blood flow when the body needs it.  A key step in this process is stimulation production of nitric oxide (NO) by the lining of the arteries (the endothelium).  Mohammadi et al (abstract PA15-2) took blood samples from non-users, smokers, and (exclusive) e-cigarette users and exposed isolated endothelial cells to the blood.  They found that the blood from e-cigarette users depressed production of NO even more than in smokers.

Lee et al (abstract PA15-4) examined the effects of e-cigarette liquids with differing nicotine levels and flavors on isolated endothelial cells.  They identified specific cellular changes, including cell death, in the cells that tended to increase with increasing nicotine concentration, with differences for different flavors (with cinnamon being the most potent.)

Another way that smoking, passive smoking, and e-cigarettes increase the risk of cardiovascular disease and trigger heart attacks is by activating platelets.  (Activate platelets form clots when you cut yourself, but when they are inappropriately activated they can tear up the liming of arteries and block arteries in the heart or brain to cause heart attacks or strokes.)  This effect is due to the immediate exposure of people or animals to the agents in the smoke or aerosol, effects which go away after the exposure ends.  Now Karim et al (abstract PA15-1) have shown that there can be lifelong effects in children of mice who are exposed to e-cigarette aerosol while the moms are pregnant.  They exposed pregnant mice to e-cigarette aerosol, then ended the exposure when they gave birth.  They then measured platelet function in the adult offspring and found hyperactive platelets.  What this means is that, in addition to any immediate effects on platelet activation, e-cigarette aerosol is doing genetic damage to the offspring.  To my knowledge, this is the first evidence that e-cigarettes have epi-genetic effects, i.e., do damage that crosses generations. 

Cancer

It is well-established that continuing to smoking reduces the efficacy of cancer therapy.  Manyanga et al (abstract PA4-5) assessed the response of human throat cancer cells to cisplatin, a chemotherapy that works by killing the cancer cells.  They found that exposing the cancer cells to e-cigarette aerosol made them resistant to cisplatin, i.e., fewer cells died.

The development of cancer requires genetic changes in cells to turn them from normal to cancer cells.  Besaratinia et al (abstract PA15-3) examined cells from inside the cheeks of exclusive smokers and vapers to look for genetic damage.  These cells are a sensitive measure because they turn over about every 2 weeks, so they provide a biological measure of recent exposure.  (In particular, the effects of being a former cigarette smoker disappear quickly.)  Even though e-cigarettes expose users to much lower levels of most carcinogens than cigarettes, they found that e-cigarettes also affected a large number of genes related to cancer (by either turning them on or turning them off inappropriately).  While smoking affected more genes, e-cigarette use affected some genes not affected by smoking.  A USC press release summarizing the study says “Both smokers and vapers showed abnormal expression, or deregulation, in a large number of genes linked to cancer development. Twenty-six percent of the deregulated genes in e-cig users were identical to those found in smokers. Some deregulated genes found in e-cig users, but not in smokers, are nevertheless implicated in lung cancer, esophageal cancer, bladder cancer, ovarian cancer and leukemia.”

HERE ARE THE ABSTRACTS (all are available here):

Lung Disease

PA4-1

E-CIGARETTE USE CAUSES ENHANCED LUNG PROTEASE LEVELS

Arunava Ghosh, Ph.D1, Raymond C. Coakley, MD1, Eric S. Davis1, Andrew Ghio, MD2, Marianne S. Muhlebach, MD1, Charles R. Jr. Esther, MD, Ph.D1, Neil E. Alexis, Ph.D1, Robert Tarran, Ph.D1. 1University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 2Environmental Protection Agency (EPA), Chapel Hill, NC, USA.

 

Introduction: Proteolysis is a key aspect of the lung’s innate immune system. However, excessive proteolysis is causative for bronchiectasis, emphysema and several types of cancer and as such, protease activity is normally tightly regulated by a series of protease inhibitors. Chronic combustible tobacco use increases protease levels in the lung lumen, which contributes to the development of chronic lung disease. However, knowledge of the impact of e-cigarette exposure (vaping) on proteolysis is still limited. We analyzed the effects of vaping on pulmonary protease-antiprotease balance to assess the impact of e-cigarette use on lung.

Methods: We analyzed bronchoalveolar lavage fluid (BALF) from healthy non-smokers, cigarette smokers (smoker) and e-cigarette users (vaper) for pro-tease activity to identify cysteine, serine and matrix metalloproteinases (MMPs). We also performed western blotting and zymography of concentrated BALF to evaluate anti-protease levels along with validation of the protease assays. We further used in vitro exposure of e-liquid component to blood neutrophils and bronchoalveolar lavage (BAL) macrophages to validate in vivo observations.

Results: We found that elastase and MMP-2/9 activity were significantly enhanced in both smokers and vapers. Western blotting and zymography data confirmed these findings. No change was observed in the level of antiproteases tested. Nicotine increased intracellular calcium levels in both blood neutrophils and BAL macrophages in a dose-dependent fashion. Elastase release from blood-derived neutrophils was nicotine dependent. Additionally, nicotine caused MMP-2/9 release from BAL macrophages with an EC50 of 37.4 nM. Our observations suggest that vaping potentiates [increases] protease release from inflammatory cells and disrupts protease-antiprotease balance.

Conclusion: Chronic vaping may contribute towards enhanced protease levels in lung and may act as an etiological factor for pulmonary diseases. We propose further investigation on the effects of long-term use of these products.

 

PA4-2

FLAVOR SOLVENT ADDUCTS IN ELECTRONIC CIGARETTE LIQUIDS ARE MODULATORS OF RESPIRATORY IRRITANT RECEPTORS AND CYTOTOXIC TO HUMAN LUNG EPITHELIAL CELLS

Sairam V. Jabba, DVM, PhD1, Ana I. Caceres, PhD1, Hanno C. Erythropel, PhD2, Julie B. Zimmerman, PhD2, Sven-Eric Jordt, PhD1. 1Duke University School of Medicine, Durham, NC, USA, 2Yale University, New Haven, CT, USA.

 

Significance: The major ingredients of the liquids used in E-cigarettes are nicotine, the solvents propylene glycol (PG) and vegetable glycerin (VG), and flavorants. Some of the popular flavors in E-liquids are cherry, cinnamon and vanilla flavors which are reactive aldehydes that may undergo chemical reactions with other E-liquid constituents under storage conditions. Using Gas Chromatography (GC) to chemically analyze E-liquids and their vapors, we recently discovered the presence of novel flavor-solvent (PG) adducts called aldehyde-PG acetals. These aldehyde-PG acetals were formed in the E-liquids after mixing of the parent E-liquid constituents and at normal storage conditions. The toxicological properties of these PG-acetals have not been studied systematically. Flavor aldehydes cause respiratory irritation and pain through activation of irritant receptors expressed in sensory neurons innervating the airways called the Transient Receptor Potential (TRP) ion-channels (TRPA1 and TRPV1). It is unknown whether the detected aldehyde-PG acetals also activate these irritant pathways.

Methods and Results: Using calcium microfluorimetry in cultured HEK 293t cells transfected with cloned human TRPA1 or TRPV1, we observed that the many aldehyde-PG acetals triggered robust activation of these TRP channels, some more efficaciously than their parent flavor aldehyde. While some PG acetals are categorized as GRAS (Generally Recognized As Safe) for scent (perfume) and food applications, their cellular and systemic toxicities, especially in the respiratory system, remain to be examined. Using BEAS-2B cells, a normal human bronchial epithelial cell line, as a cellular model, we conducted a range of cellular toxicity assays and transcriptional approaches to examine inflammatory transcriptional responses. In live/dead and LDH assay, some aldehyde-PG acetals demonstrated more cytotoxicity than parent aldehydes at several concentrations tested. Gene expression analysis demonstrate that PG acetals induce a transcriptional program leading to expression of several pro-inflammatory genes.

Conclusions: Our data demonstrates that chemical reaction products formed at normal storage conditions in E-liquids like aldehyde-PG acetals have potent pharmacological and toxicological effects that are different from parent constituents.

Cardiovascular disease

PA15-2

RELATIVE ENDOTHELIAL TOXICITY OF TOBACCO SMOKE AND E-CIGARETTE AEROSOL: A FUNCTIONAL AND MECHANISTICAL ASSESSMENT

Leila Mohammadi, MD, PhD, Ronak Derakhshandeh, Daniel Han, Abel Huang, Adam Whitlatch, Suzaynn Schick, PhD, Matthew Springer, PhD. UCSF, San Francisco, CA, USA.

Background: Smoking cigarettes decreases expression of eNOS in the endothelium, resulting in lower nitric oxide (NO) secretion and decreased flow-mediated dilation in the conducting arteries. In contrast, the effects of e-cigarettes (e-cigs) on endothelial function are just beginning to be studied and the mechanisms of action are unclear.

Aim: To test the hypothesis that circulating factors from e-cigarette users decrease endothelial eNOS protein levels and NO secretion in primary endothelial cell cultures, relative to nonsmokers and cigarette smokers.

Methods: 36 healthy individuals were recruited and grouped as nonsmokers (n=12), cigarette smokers (n=7), and e-cig users (n=17). Human umbilical vein endothelial cells (HUVECs) were cultured at 20,000 cells per well in 24-well culture plate. Serum samples from individual subjects and endothelial growth media were added at a 1:1 ratio to confluent cells and incubated for 12 hours (basal condition), followed by fresh medium containing VEGF at 50 ng/ml for 30 min (stimulated condition). The amount of NO liberated from cells was measured in culture supernatants by the chemiluminescence method using a NO analyzer. Endothelial NO synthase (eNOS) protein levels in HUVECs was measured in cell lysates by ELISA. NO and eNOS results were normalized to cell number.

Results: The cells treated with serum from e-cig users vs. nonsmokers produced less NO upon stimulation (P<.03) and contained less eNOS protein (P<.03). eNOS protein level was lower in the e-cig user serum group than in the cigarette smoker serum group (P<.05), although stimulated NO production was decreased less by e-cig user serum than by smoker serum. Lower eNOS levels in the cigarette group vs. non-smoker group was not significant, but stimulated NO was significantly lower in the cigarette group vs. nonsmoker (P<.006). Conclusion: Exposure of cultured endothelial cells to circulating factors from e-cig users, relative to non-users, leads to lower eNOS protein levels and decreased NO production.

PA15-4

ASSESSING CARDIOVASCULAR RISKS ASSOCIATED WITH E-CIGARETTES WITH HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED ENDOTHELIAL CELLS

Won Hee Lee, PhD1, Yang Zhou, MS2, Sang-Ging Ong, PhD3, Lei Tian, PhD2, Natalie Baker2, Hye Ryeong Bae, BS2, Leila Mohammadi, PhD4, Matthew L. Springer, PhD4, Suzaynn F. Schick, PhD4, Aruni Bhatnagar, PhD5, Joseph C. Wu, MD, PhD2. 1Universi-ty of Arizona, Phoenix, AZ, USA, 2Stanford University, Stanford, CA, USA, 3University of Illinois College of Medicine, Chicago, IL, USA, 4University of California, San Francisco, CA, USA, 5University of Louisville, Louisville, KY, USA.

Significance: Marketed since 2007, electronic cigarettes (e-cigarettes) have experienced a tremendous increase in use despite the scarcity of scientific evidence pertaining to their safety. Unlike conventional cigarette smoking, the effects of e-cigarette products and their constituents on mediating vascular health remain poorly understood. Given the increasing popularity of flavored e-cigarettes, it is imperative to carefully study the ingredients, especially nicotine and different flavorings, so that potential health risks of e-cigarettes may be evaluated. The aim of the present study was to investigate the biological effects of e-cigarette flavored liquids on endothelial health. The inclusion of serum from smokers of e-cigarettes serves to further corroborate the in vitro results.

Methods: We used human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) and a high-throughput screening approach to assess endothelial integrity following exposure to 6 different e-liquids with varying nicotine concentrations and to serum collected from e-cigarette users.

Results: The cytotoxicity varied greatly among the e-liquids, with the cinnamon-flavored product being most potent that led to significantly decreased cell viability, increased reactive oxygen species (ROS) and caspase 3/7 activity, activation of oxidative stress-related pathway, and impaired tube formation, confirming endothelial dysfunction. Interestingly, we observed an increasing trend of endothelial toxicity associated with higher nicotine concentration, though the overall differences were not significant. In addition, after exposure of iPSC-ECs to serum of e-cigarette users, we observed increased ROS linked to endothelial dysfunction as indicated by interrupted angiogenesis in iPSC-ECs. We also noted a decrease in the number of platelets and an increase in inflammatory cytokine expression in serum of e-cigarette users.

Conclusions: Taken together, these results indicate that acute exposure to flavored e-liquids or e-cigarette use exacerbates endothelial dysfunction, which may alter the progression of cardiovascular diseases.

PA15-1

IN UTERO EXPOSURE TO E-CIGARETTES MODULATES PLATELET FUNCTION AND INCREASES THE RISK OF THROMBOGENESIS, IN MICE

Zubair A. Karim, PhD, Keziah Hernandez, MS, José O. Rivera, PharmD, Fadi T. Kha-sawneh, B.Pharm, PhD, Fatima Z. Alshbool, PharmD, PhD. The University of Texas at El Paso, El Paso, TX, USA.

Significance- Cardiovascular disease is the main cause of death in the United States with smoking being the primary preventable cause of premature death, and thrombosis being the main mechanism of cardiovascular mortality in smokers. We have previously shown that short term exposure, of adult mice, to electronic/e-cigarettes increases the risk of thrombosis, in part, by enhancing platelet function. However, whether in utero/prenatal exposure exerts similar detrimental health effects is yet to be investigated.

Methods- We employed a passive e-Vape TM vapor inhalation system, and performed whole-body e-cigarette exposures on pregnant mice (in utero) under exposure conditions that mimic real-life human exposure scenarios/conditions- and investigated the effects of e-cigarettes and clean air on platelet function and thrombogenesis.

Results- Our results show that platelets from in utero e-cigarette exposed mice are hyperactive with enhanced: aggregation, dense and alpha granule secretion, activation of the αIIbβ3 integrin, and phosphatidylserine expression, when compared to clean air exposed platelets. Furthermore, the in utero e-cigarette exposed mice exhibited a shortened thrombosis occlusion and bleeding times, in comparison to the clean air control mice.

Conclusion- Taken together, our data demonstrate for the first time that in utero exposure to e-cigarettes alters physiological hemostasis, and increases the risk of thrombogenic events. This is due, at least in part, to the hyperactive state of platelets. Thus, the negative health consequences of e-cigarette exposure should not be underestimated, and warrant further investigation.                                          

Cancer

PA4-5

EXPOSURE TO ELECTRONIC CIGARETTE AEROSOL INCREASES CISPLATIN RESISTANCE IN PHARYNGEAL CANCER CELLS

Jimmy Manyanga, Célia Bouharati, Balaji Sadhasivam, Vengatesh Ganapathy, Lurdes Queimado. The University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.

Significance: Tobacco smoking is the main risk factor for lung and head and neck cancer. Moreover, continued smoking after cancer diagnosis increases drug resistance and reduces overall survival rate by approximately 50%. Aware of the challenges of smoking cessation and the negative consequences of continued smoking, cancer patients are questioning clinicians on whether they should switch to electronic cigarettes, which are being promoted as a safer alternative to tobacco smoking. To help answer that question, we examined the effects of electronic cigarette aerosol exposure on cisplatin resistance in head and neck cancer cells. Methods: Pharyngeal cancer epithelial cells were exposed for 48 hours to electronic cigarette aerosol extracts at doses yielding less than 40 ng/ml of nicotine. Mainstream (MS) smoke was used as a positive control. To assess cisplatin chemosensitivity, cancer cells were subsequently treated with cisplatin (0.1-100 μM) for 48 h in the presence of electronic cigarette aerosol extracts. Cell viability was assessed by the MTT assay. Data were analyzed by student’s t-tests and one-way analysis of variance (ANOVA). To determine the half-maximal inhibitory concentration (IC50) values, data were analyzed using GraphPad Prism software.

Results and Conclusions: Cancer cells exposed to electronic cigarette aerosol, at doses similar to those observed in experienced vapers, and treated with cisplatin showed a significant increase in cell viability compared to those not exposed to extracts. The concentrations of cisplatin needed to induce a 50% reduction in cell growth relative to no cisplatin control cells (IC50 values) were also significantly increased in the presence of electronic cigarette aerosol extracts. These observations suggest that exposure to electronic cigarette aerosol might increase chemotherapy resistance. Additional studies are needed to fully assess the impact of electronic cigarette use in cancer patients. Nonetheless, our data emphasize the urgent need to further evaluate electronic cigarette safety to ensure evidence-based public health policies and regulations.

Grant support: This work was supported by the Oklahoma Tobacco Research Center, NIH/NCI (R33), Presbyterian Health Foundation, and OCAST. Dr. Queimado holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology.

PA15-3

VAPING DEREGULATES CANCER-RELATED GENES AND ASSOCIATED MOLECULAR PATHWAYS IN THE ORAL EPITHELIUM

Ahmad Besaratinia, M.P.H., Ph.D., Stella Tommasi, Ph.D., Andrew W. Caliri, B.S., Amanda Caceres, B.S., Debra E. Moreno, B.S., Meng Li, Ph.D., Yibu Chen, Ph.D., Kimberly D. Siegmund, Ph.D.. University of Southern California - Keck School of Medicine, Los Angeles, CA,

USA.Electronic cigarettes (e-cigs) are promoted as safe alternatives to tobacco cigarettes. E-cigs are increasingly popular among adult smokers and adolescent never smokers. Despite the known presence of carcinogens in e-cig liquid and vapor, the cancer-causing potential of e-cig use (otherwise known as vaping) is not known. We have investigated the extent of gene deregulation and the affected pathways in oral cells of e-cig users and cigarette smokers as compared to nonsmokers. Interrogation of the oral transcriptome by RNA-seq analysis showed numerous aberrantly expressed transcripts in both e-cig users and smokers relative to nonsmokers; although smokers had ~50% more differentially expressed transcripts than vapers (1,726 vs. 1,152). Molecular pathway and functional network analyses revealed that cancer was the top disease associated with the deregulated genes in both e-cig users and smokers (~62 vs. 79%). Examination of the canonical pathways and networks that were modulated in either e-cig users or smokers identified the ‘Wnt/Ca+ pathway’ in vapers and the ‘integrin signaling pathway in smokers as the most affected pathways. Amongst the overlapping functional pathways that were impacted in both e-cig users and smokers, the ‘Rho family GTPases signaling pathway’ was the top disrupted pathway, although the number of affected targets was three times higher in smokers than vapers. We conclude that vaping, similarly to smoking, alters the expression of crucial cancer-related genes, though to a different extent and via both overlapping and unique pathways as compared to smoking. Our findings have significant implications for public health and tobacco regulatory science.

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