DNA damage in smokers with Chronic Obstructive Pulmonary Disease| Tabaccologia

Abstract

Introduction: Chronic Obstructive Pulmonary Disease (COPD) is a complex respiratory condition with significant health implications, often associated with cigarette smoking but also involving non-smoking factors. This study focused on the analysis of DNA damage as a potential mechanism of lung injury.

Materials and methods: In a study involving 80 elderly COPD patients, participants were categorized into three groups based on their smoking habits: current smokers, former smokers, and non-smokers. The comet assay was used to quantify DNA damage. Additionally, we investigated whether oxygen therapy could influence the level of DNA damage in the patients.

Results: Current smoking patients exhibited significantly higher levels of DNA damage compared to former smokers and non-smokers, confirming the role of cigarette smoking in such damage.Moreover, former smokers displayed persistent DNA damage despite having quit smoking. Surprisingly, oxygen therapy had no significant impact on DNA damage in COPD patients.

Conclusion: This study confirms the significant association between cigarette smoking and DNA damage in COPD patients. DNA damage appears to persist in former smokers, highlighting the need for a deeper understanding of the involved mechanisms. These findings underscore the importance of further research to address this issue and its implications for human health.

Introduction

Chronic Obstructive Pulmonary Disease (COPD) is a significant contributor to global mortality and disability. It is a complex condition characterized by persistent airway obstruction and respiratory symptoms. The primary causes of COPD involve exposure to inhaled particulate matter, including cigarette smoke and air pollutants, along with genetic, developmental, and social factors [1]. While the condition is often associated with tobacco smoking, it is estimated that 25-45% of COPD patients have never smoked and can still develop COPD, possibly due to passive smoking, occupational exposure, air pollution, and a history of past lung infections, including tuberculosis [2]. Additionally, COPD is linked to increased oxidative stress in the lower airways, which can cause DNA damage and lung carcinogenesis [3]. A recent review [4] summarizing the extent of DNA damage in prevalent diseases reveals the following: The overall findings of these studies indicate a higher frequency of DNA strand breaks in COPD patients compared to control groups. A meta-analytic analysis reported significantly elevated levels of DNA strand breaks in the group of COPD patients (SMD = 1.29, 95% CI: 0.69, 1.90). After accounting for current smoking and other potential confounding factors, the odds ratio for high levels of DNA strand breaks in relation to increasing COPD severity was reduced. However, the effect in the adjusted analyses was relatively modest (e.g., the odds ratio was 1.008 for the increase in % DNA in the tail per increment in COPD severity group). DNA damage triggers various cellular responses, allowing cells to repair or adapt to the damage or activate programmed cell death, ultimately eliminating cells with potentially harmful mutations [5]. While tobacco smoking is a well-documented source of potentially mutagenic and carcinogenic compounds, and smokers are considered a relevant study group with significant mutagenic exposure, results regarding DNA damage assessed by the comet assay in smokers present conflicting outcomes [6-8]. The situation may be different when evaluating DNA damage in patients with COPD, divided into smokers and non-smokers. Numerous studies have demonstrated an increase in oxidative stress in COPD patients, including both current and former smokers, compared to healthy controls [9]. Therefore, we evaluated DNA damage using the comet assay in various COPD patients, categorized as current smokers, former smokers, and non-smokers.

Materials and methods

An observational cohort study was carried out in 87 patients aged 70 years or older suffering from severe COPD and admitted to the Pulmonary Rehabilitation (PR) Unit of the IRCCS San Raffaele Roma between January 2013 and December 2015 for a comprehensive 3-weeks PR program. Peripheral blood samples were collected and stored at −80 °C at admission and after 3 weeks of PR. Additional detail of the study population can be found in Russo et al. [10]. The study was approved by the ethics committee of the IRCCS San Raffaele Roma (Prot. 15/2013), and all participants signed the consent to participate in the study at admission. All patients were stratified into three groups according to their smoking history, i.e., according to National center for health statistics (NHIS - Adult Tobacco Use - Glossary, 2019), current smoker: an adult who has smoked 100 cigarettes in his or her lifetime and who currently smokes cigarettes; former smoker: an adult who has smoked at least 100 cigarettes in his or her lifetime but who had quit smoking at the time of interview; and never-smoker: an adult who has never smoked, or who has smoked less than 100 cigarettes in his or her lifetime. These categories were confirmed after measuring the presence of cotinine in the urine samples. Consequently, we assigned subjects based on the positivity of the test rather than solely relying on their self-declarations. Urine specimens were taken at the admission into plastic containers that were subsequently frozen at −20 °C. In the urine samples, levels of cotinine were analyzed using Instant Cotinine Testing Kit by Sure Screen (Eazzi, Edinburgh, UK), certified to ISO 9001 and ISO 13485, and this drug test cassette was CE Marked in accordance with 98/79/EC. The complete detailed procedure for the assay can be found in our previous work [10]. In summary, following new guidelines for minimal required detailed information on reporting comet assay procedures and new technical information [11], damage of DNA from lymphocytes was evaluated after lymphocytes lysation, DNA denaturation, electrophoresis on agarose gel, and staining, using the Comet assay IV software version 4 (Instem, London, UK). Tail intensity values (TI, % DNA in comet tail) were calculated from 100 comets counted for each individual.

Results

A total of 80 elderly patients were enrolled, with an average age of 71.6 ± 8.7 years (SD), including 45 females and 35 males, after providing informed consent. These patients presented with various stages of chronic obstructive pulmonary disease (COPD) at the time of hospitalization.

The patients were grouped according to their smoking habits into current smokers (15 patients), former smokers (55 patients), and non-smokers (10 patients). Notably, the former smokers had quit smoking at least one year prior, potentially retaining residual risks associated with their previous smoking habits.

DNA damage was quantified in terms of Tail Intensity (%). Table 1 shows that the tail intensity (19.92 ± 7.57) was increased in all COPD patients. As previously reported by Bonassi et al. in 2021, individuals without underlying medical conditions exhibited a tail intensity (%) of 7.4 ± 8.8. Interestingly, this value increased to 10.5 ± 11.2 for individuals over the age of 60 in a sample of 1,329 subjects. COPD is an inflammatory disease, which is why non-smokers show elevated levels of DNA damage (tail intensity 14.43 ± 4.11). Former smokers have slightly higher DNA damage (19.75 ± 7.76) compared to non-smokers. However, current smokers have significantly higher DNA damage (24.19 ± 6.22).

Additionally, since the patients were receiving oxygen therapy, we conducted an analysis to assess whether oxygen had an impact on DNA, and we found that there was no significant difference (Table 1). Therefore, the observed DNA damage is not associated with oxygen but rather with smoking.

Conclusions

Patients with COPD exhibit significantly higher tail intensity values compared to healthy subjects. As reported by Bonassi et al. in 2021 [11], the average tail intensity (%) in healthy subjects was 7.4 ± 8.8. Interestingly, this value increased to 10.5 ± 11.2 in individuals over the age of 60, based on a sample of 1,329 subjects. In patients with COPD, DNA damage may be a key factor in the complications associated with this respiratory disease, especially in smokers. We examined DNA damage levels in elderly patients with COPD, dividing them into groups of current smokers, former smokers, and non-smokers. Our results revealed a significant difference in DNA damage levels among these groups, confirming the relationship between cigarette smoking and DNA damage, as current smokers exhibited significantly higher DNA damage levels compared to former smokers and non-smokers. This suggests that exposure to cigarette smoke represents a significant risk factor for DNA damage in patients with COPD. Furthermore, we observed that even former smokers, who had quit smoking for at least a year, had higher levels of DNA damage compared to non-smokers. This suggests that DNA damage caused by cigarette smoking can persist over time even after quitting. We also examined the effect of oxygen therapy, considering that it might cause DNA damage, but our results indicated no significant difference in DNA damage between patients receiving oxygen therapy and those who were not. This suggests that the DNA damage we observed is not associated with oxygen. Ongoing research is crucial to gain a comprehensive understanding of the intricate biological effects of cigarette smoking and its potential involvement in DNA damage. Further investigations are needed to fully comprehend the mechanisms and consequences of smoking’s actions on human health. However, it is essential to emphasize that smoking habits are highly hazardous to individuals and public health. The detrimental effects of smoking are negative health outcomes associated with smoking, including lung diseases, heart diseases, cancer, and many other health conditions. These harmful effects pose a significant public health threat. The implementation and enforcement of effective public health policies are essential in addressing the harmful effects of smoking. This can include the adoption of laws or regulations, the creation of smoking prevention and cessation programs, or the implementation of awareness campaigns. The enforcement aspect involves the rigorous implementation of these policies and measures to ensure that smoking-related laws and rules are adhered to and that there are consequences for those who violate them. Furthermore, providing accessible support and resources for those who wish to quit smoking is fundamental to improving public health outcomes.

Figures and tables

References

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