Evaluating electronic cigarette cytotoxicity and inflammatory responses in vitro

INTRODUCTION Cigarette smoking poses many health risks and can cause chronic obstructive pulmonary disease (COPD), cardiovascular disease, cancer of the lung and other organs. Smokers can substantially reduce their risks of these diseases by quitting, but nicotine addiction makes this difficult. Alternatives, such as electronic cigarettes (e-cigarettes), may provide a similar dose of nicotine, but expose users to fewer toxic chemicals than traditional cigarettes and may still be harmful especially for dual users, therefore, we sought to develop bioassays that can assess the potential toxicity and inflammatory response induced by e-cigarette liquids (e-liquids) with and without flavors. METHODS E-liquids with varying nicotine content and flavors were aerosolized through growth media and exposed to human bronchial epithelial cell line (BEAS-2B) and human monocyte-macrophage cell line (THP-1) in vitro. Cytotoxicity in response to e-cigarette aerosols was measured by MTT assay in BEAS-2B cells and inflammatory response was measured by TNF-α, IL-6, IL-8, and MCP-1 released from THP-1 cells. In addition, the oxidative stress marker, REDD1, and impact on phagocytosis, was assessed following exposure of BEAS-2B and THP-1 derived macrophages, respectively. Cigarette smoke extract was used as a positive control with known cytotoxicity and impairment of inflammatory response. RESULTS E-cigarette aerosols induced moderate cellular toxicity in bronchial epithelial cells. Our data also show that low nicotine levels are less damaging to the bronchial epithelial cells, and flavors in e-liquids influence the combined inflammatory response markers, phagocytosis, and REDD1 when examined in vitro. CONCLUSIONS Our in vitro bioassays can be utilized to effectively measure flavor and nicotine-induced effects of e-cigarettes on combined inflammatory response and cytotoxicity in human macrophages and human bronchial epithelial cells, respectively.

What did this study look at?
• Smoking can increase the risk of developing smoking-related conditions including: -Asthma, a lung condition caused by swelling of the breathing tubes; -Chronic obstructive pulmonary disease, a group of lung conditions that cause breathing difficulties; -Cardiovascular diseases, a group of conditions that affect the heart and blood vessels; and -Diabetes, a condition that causes the level of sugar in the blood to become too high.
• Helping smokers to stop smoking is important. It lowers the risk of death and disability caused by smoking-related conditions.
• When people smoke, they become addicted to a chemical called nicotine.
• Varenicline, bupropion, and nicotine replacement therapies are medicines that help people to stop smoking.
-People take varenicline and bupropion as tablets. The medicines act on different types of receptors in the brain that play a part in addiction to nicotine.
-They work in a similar way to nicotine and help reduce the cravings and withdrawal symptoms that people experience when they stop smoking.
While taking varenicline, smoking cigarettes can be less enjoyable. -Nicotine patches are a type of nicotine replacement therapy that people apply to the skin. They allow a small amount of nicotine to enter the body without the added chemicals found in cigarettes.
• In the EAGLES study, researchers looked at how safe and effective varenicline, bupropion, and nicotine patches were in smokers with and without mental health problems.
-All 3 medicines helped people to stop smoking, but more people stopped smoking when they took varenicline.
• Few studies have looked at how well the medicines work for people with different smoking-related conditions.
• In this study, researchers looked at how effective varenicline, bupropion, and nicotine patches were in smokers with smoking-related conditions who took part in EAGLES. They looked at: -The number of people who stopped smoking while using the medicines -If people had anything about them (such as where they lived, or what medicine they took during the study) that could help to predict if they were likely to stop smoking -Any side effects that people experienced during the study.
A side effect is something (expected or unexpected) that a person feels was caused by a medicine or treatment they take.

Additional information
More information can be found in the scientific article of this study, which you can access here: For more information on this study, please visit: Where is this study in the drug development timeline?
• Varenicline, bupropion, and nicotine patches are approved treatments to help people to stop smoking.

Clinical trial phases
Phase 3 A large group of patients with a speci c condition take a medicine to see how well it works and how safe it is

Approval
All of the results are reviewed by agencies that approve medicines and, if approved, the medicine is made available for doctors to prescribe to patients, or for patients to buy

Phase 1
Healthy volunteers take a medicine so researchers can look at how safe it is and how it works in the body

Phase 2
A small number of patients with a speci c condition take a medicine to see how well it works, what dose should be taken, and how safe it is EAGLES is a Phase 4 clinical trial

Phase 4
After a medicine is approved, researchers do studies in different groups of people not included in earlier trials, and over long periods of time, to monitor how effective and safe it is Who took part in this study?
• Adults aged 18-75 years old from 16 countries took part in EAGLES.
• They could take part if they smoked at least 10 cigarettes every day and they wanted to stop smoking. • On average, people with a smoking-related condition were 52 years of age.
• On average, people without a smoking-related condition were 45 years of age.
• People with smoking-related conditions had smoked for an average of 34 years.
• Otherwise healthy smokers (smokers without smoking-related conditions) had smoked for an average of 27 years. What were the results of the study?
• Compared with otherwise healthy smokers, people with smoking-related conditions: -Were older -Had smoked for longer -Had tried to stop smoking more often.
• They were also more likely to: -Have mental health problems -Live in the United States -Have used stop-smoking medicines before.
How many people stopped smoking while taking the medicines?
• Researchers recorded the percentage of people who stopped smoking completely for a certain period of time.

Researchers looked at:
People received medicines to help them stop smoking

Weeks 9-12
The percentage of people who stopped smoking for the last 4 weeks of treatment (weeks 9-12)

Weeks 9-24
The percentage of people who stopped smoking for the last 4 weeks of treatment plus 12 weeks after treatment stopped (weeks 9-24) weeks 4 weeks 16

Additional information
More information can be found in the scientific article of this study, which you can access here: For more information on this study, please visit: • The percentage of people who stopped smoking for weeks 9-12 and weeks 9-24 was lower for those with smoking-related conditions compared with otherwise healthy smokers.
-When researchers looked at each smoking-related condition separately, this difference didn't apply to: Smokers with diabetes at weeks 9-12 or weeks 9-24 Smokers with cardiovascular disease at weeks 9-12.
• More people stopped smoking if they were taking varenicline or bupropion or using nicotine patches than if they were taking placebo. This included people with and without a smoking-related condition.
• More people (with or without a smoking-related condition) stopped smoking if they were taking varenicline than if they were taking bupropion or using nicotine patches.
-Among smokers with each of the different smoking-related conditions, more people stopped smoking if they were taking varenicline than if they were taking bupropion or using nicotine patches.
The percentage of people who stopped smoking for weeks 9-24 was similar for smokers with asthma if they took varenicline or bupropion.
• People took their medicine for a similar length of time: -Whether or not they had a smoking-related condition; and -Whichever medicine they were taking.
What factors may help to predict whether people will be able to stop smoking?
• Researchers used mathematical models to see if people had anything about them at the start of the study that may help predict if they were more likely to stop smoking. These included: -If they had a smoking-related disease -If they had mental health problems -Which medicine they took in the study -Where they lived.
• Taking a medicine to help them stop smoking was the most useful factor for predicting which people would stop smoking. Researchers also looked at the percentage of people with each smoking-related condition who stopped smoking

Additional information
More information can be found in the scientific article of this study, which you can access here: For more information on this study, please visit: • People were more likely to stop smoking for weeks 9-12 and 9-24 if they lived outside the United States, and were of older age.
• Having tried to stop smoking before helped to predict that people would stop smoking for weeks 9-12, but not for longer periods (weeks 9-24).
• People with a smoking-related condition or with mental health problems were less likely to stop smoking.
What were the side effects of the treatments? The sponsors would like to thank all of the people who took part in this study. • In this study, 4 in every 100 people with and without a smoking-related condition reported a problem related to their mental health.
• Researchers weighed people at the start of the study, at week 12, and at week 24.
-At week 12 and week 24, the average amount of weight that people gained was less than 2 kg, whether or not they had a smoking-related condition and whichever medicine they took to help them stop smoking.

Additional information
More information can be found in the scientific article of this study, which you can access here: For more information on this study, please visit: What were the main conclusions reported by the researchers?
• People who had already developed certain smoking-related conditions were less likely to stop smoking than those without those conditions.
• Varenicline, bupropion, and nicotine patches were effective and safe in helping people with smoking-related conditions to stop smoking.
-More people stopped smoking when they took varenicline than those who took bupropion or used nicotine patches.
• Taking one of the medicines was the best predictor of whether people would stop smoking during weeks 9-12 and weeks 9-24.
• Side effects while taking medicines to help stop smoking were similar in people with and without smoking-related conditions.
• The results from this study show that people with smoking-related conditions may benefit from taking medicines to help them stop smoking. This may be helpful for smokers and their doctors when making treatment decisions.