IN VITRO PRO-OXIDANT AND CYTOTOXIC EFFECTS OF AEROSOLS FROM A THIRD-GENERATION ELECTRONIC CIGARETTE IN HUMAN ORAL CELL LINES
Open Access
- Author:
- Urena, Jose
- Graduate Program:
- Food Science
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- June 13, 2019
- Committee Members:
- Joshua D. Lambert, Thesis Advisor/Co-Advisor
Alexey Silakov, Committee Member
Ryan J. Elias, Committee Member - Keywords:
- electronic cigarette
carbonyls
free radicals
human oral cells
cytotoxicity
oxidative stress
in vitro
air-liquid interface
atomizer - Abstract:
- Electronic cigarettes (ECs) were created as an alternative to conventional cigarettes and have become popular among both smokers and former smokers who seek effective smoking cessation tools. These devices are also appealing to adolescents and adults who have never smoked cigarettes due to factors such as flavors, curiosity, and social influences. ECs vary widely in design and are generally categorized into four generations based on design, price, battery output voltage, aerosol production and customizability. The different EC generations provide users with diverse experiences. As EC users become more comfortable and knowledgeable with first-generation EC devices, they tend to transition to second- and third-generation devices that allow for greater customizability and aerosol production. Former smokers prefer third-generation ECs, because these devices relieve nicotine cravings better than older generations. ECs operate by vaporizing a fluid (EC liquid) consisting of propylene glycol, glycerol, flavorings, and nicotine by resistive heating to deliver aerosol to the user. The heating element, or atomizer, is typically a replaceable, metal object that contains a cotton or silica wicking material that absorbs and delivers EC liquid to a resistive heating wire, or coil, during the heating process. This process differs from conventional cigarette smoking which burns tobacco to deliver nicotine in the smoke. The popularity, rapid introduction, and unknown health effects of ECs have prompted toxicological studies to determine the health impacts of these devices marketed as “safer alternatives” to conventional cigarettes. In spite of this, there is a growing body of evidence that suggests EC aerosol inhalation may cause adverse biological effects. Chemical analysis has demonstrated that EC aerosol contains heavy metals from the heating element, flavor chemicals from the EC liquid, and thermal degradation products such as reactive carbonyls (formaldehyde, acetaldehyde, and acrolein) and free radicals formed from the degradation of EC liquid solvents. ECs that generate larger volumes of aerosol and operate at higher temperatures produce increased levels of thermal degradation products. In vitro studies indicate that EC liquid, EC aerosol condensate, and EC aerosol cause reductions in cell viability and increases in oxidative stress, DNA damage, and inflammation in dose- and flavor-dependent manners. In addition, studies investigating that toxicity of EC aerosol demonstrate that aerosols generated at higher power settings are more toxic to cells. Current in vitro research has provided valuable toxicological data on the potential adverse biological effects of EC aerosol. However, the EC market is constantly evolving and more research is needed to properly assess the potential harm of ECs. The role of nicotine, reactive carbonyls, and free radicals in the toxicity of EC aerosols in vitro is unknown. Cell culture studies have used the air-liquid interface (ALI) to expose respiratory cells to EC aerosol in a physiologically-relevant approach, but the application of this model to cell types found in the oral cavity, a site exposed to high levels of EC aerosol, is lacking. The emissions and in vitro toxicity of newer, third-generation ECs have not been adequately studied. Therefore, we proposed to investigate the cytotoxicity and oxidative stress in human oral cells exposed to EC aerosol generated by a third-generation EC device at the air-liquid interface. We tested the hypothesis that EC aerosols generated by a third-generation EC device using a panel of commercial EC liquids can reduce cell viability and increase oxidative stress in human oral cells treated at the ALI and that toxicity is associated with carbonyl production, radical generation, and atomizer degradation. In our first objective, we quantified formaldehyde, acetaldehyde, acrolein, and free radicals in EC aerosol generated by a third-generation EC device and panel of commercial EC liquids. We also characterized the flavoring compounds in the commercial EC liquids and investigated the effect of nicotine concentration on carbonyl and radical production. We found that all EC aerosols generated from new atomizers contained each carbonyl of interest. Formaldehyde was the predominant carbonyl in EC aerosol (15.10-53.45 µg/g liquid), followed by acetaldehyde (2.13-14.61 µg/g) and acrolein (1.94-7.06 µg/g), respectively. These carbonyl levels were similar to those found naturally in foods and lower than both conventional cigarette and Occupational Safety and Health Administration exposure limits. Radicals were present in EC aerosol generated from aged atomizers, but not in EC aerosol generated with new atomizers. EC aerosols generated from aged atomizers contained radical concentrations ranging from 3.24 to 8.04 pmol/g liquid. No significant relationship was observed between nicotine concentration and acetaldehyde, acrolein, and radical production. EC liquid with 12 mg/mL nicotine generated significantly lower levels of formaldehyde (18.19 µg/g liquid) compared to EC liquid with 6 mg/mL nicotine (44.75 µg/g). Carbonyl and radical levels were modulated by EC liquid flavor. Twelve unique flavoring chemicals were identified and semi-quantified in 8 EC liquids. Each flavor had a unique combination of flavor chemicals. In our second objective, we investigated the cytotoxicity and intracellular oxidative stress induced by EC aerosols generated by a third-generation EC and panel of commercial EC liquids in human oral cells treated at the ALI. We also studied the effect of nicotine concentration on cytotoxicity of EC aerosols and explored the role of oxidative stress on cytotoxicity using an antioxidant treatment. SCC-25 human oral squamous cell carcinoma cells and HGF-1 human gingival fibroblast cells were exposed to a physiologically-relevant EC aerosol exposure at the ALI using a custom-made smoking machine. In general, SCC-25 and HGF-1 cells responded similarly to EC aerosol. EC aerosol generated from 7 of the 8 EC liquids using a new atomizer had no significant cytotoxic effects in oral cells. Lava Flow EC aerosol without nicotine caused significant reductions (28%) in the viability of both cancerous and non-cancerous cells compared to air-treated controls, while Lava Flow EC aerosol with nicotine was not cytotoxic. Treatment of oral cells with a cytotoxic EC aerosol (Lava Flow) caused a significant increase (1.7- to 1.9-fold) in oxidative stress in both cell lines tested, regardless of nicotine content. No significant increase was observed for cells exposed to aerosols generated from non-cytotoxic liquid, Hawaiian Pog. Inclusion of an antioxidant, N-acetyl-L-cysteine (NAC), reduced EC aerosol-induced cytotoxicity in SCC-25 cells by 36%, indicating that cytotoxicity of EC aerosol was partially mediated by oxidative stress. EC aerosols generated from nicotine-free Lava Flow liquid and Lava Flow liquid containing 12 mg/mL nicotine reduced cell viability by 31% and 33% respectively. By contrast, EC aerosol from Lava Flow containing 6 mg/mL nicotine had no significant cytotoxic effects. In our third objective, we examined the impact of atomizer age on the levels of formaldehyde, acetaldehyde, acrolein, butyraldehyde, and free radicals in EC aerosol. In addition, we investigated the effect of atomizer age on the potential cytotoxicity of EC aerosol and explored the role of oxidative stress on cytotoxicity using an antioxidant treatment. An atomizer was used over a series of consecutive experiments to simulate continuous use by an EC user. Atomizer degradation was monitored by a decrease in EC liquid consumption during EC aerosol generation and charring on the cotton wick and coil. Carbonyl and radical production increased, while cell viability decreased as function of atomizer age. Levels of formaldehyde, acetaldehyde, and acrolein in EC aerosol generated by an aged atomizer were 237%, 211%, 2822% higher than those produced by a new atomizer. Butyraldehyde and radical concentrations increased from no detectable levels to 246 µg/g liquid and 6.76 pmol/g liquid, respectively. Inclusion of an antioxidant, NAC, did not reduce EC aerosol-induced cytotoxicity when EC aerosol was generated by an aged atomizer, suggesting the pro-oxidant capacity of the aerosol was too high to be overcome by the antioxidant. Overall, the present work demonstrates that third-generation ECs are not risk-free but may be potential harm reduction alternatives to conventional smoking with proper use and maintenance. EC aerosol produced from a third-generation EC device contained reactive carbonyls and radical species. Our results are consistent with previous studies exploring the cytotoxic and oxidative potential of EC aerosol, generated by first- and second-generation devices, on respiratory tract cell lines. Failure to replace atomizers frequently causes substantial cellular damage and exposes users to high levels of harmful carbonyls and radicals. Our toxicological data provide necessary information on the potential health impacts of ECs and EC aerosol to support future in vivo studies and aid in development of science-based regulations for the EC industry.