Nucleus https://doi.org/10.1007/s13237-018-0250-y ORIGINAL ARTICLE DNA damage in the peripheral blood lymphocytes of asthmatic patients in relation to disease progression Manisha Saini1 · Abhay S. Yadav1 Received: 24 March 2018 / Accepted: 9 October 2018 © Archana Sharma Foundation of Calcutta 2018 Abstract With increased incidences in recent times especially in the urban population, asthma represents a major global concern. The inflammatory reaction of the airways in asthmatic patients leads to generation of reactive oxygen intermediates which inter- fere with the basic structure of DNA causing genomic instability. The present study was aimed to assess the DNA damage in the peripheral blood lymphocytes in asthmatic subjects. Blood samples from 95 asthmatic subjects and 57 controls were analysed for DNA damage using Comet assay. Statistical analysis was done using student’s t test and ANOVA. p < 0.05 was considered significant. Significant elevation was observed in % tail DNA (p < 0.05) in asthmatic subjects as compared to their non-asthmatic counterparts. Tail length, tail and olive moment were also markedly higher in asthmatics subjects (p < 0.05). The DNA damage was evidently higher in patients with severe asthma as compared to those with mild and moderate asthma (p < 0.05). The genomic instability was positively correlated with the age of subjects and severity of asthma which implied association of DNA damage with asthma and its progression with duration and severity. Keywords Asthma · Comet assay · Oxidative stress · DNA damage Introduction inflammation of the airways leads to increased oxidative stress. There are several evidences that the reactive oxy- Asthma, a chronic inflammatory disorder of the airways, gen species (ROS) that are produced during inflammatory along with COPD is the third leading cause of worldwide reactions in asthma further interfere with the structural mortality [19]. In 2012, asthma along with other respira- molecules in the body like proteins, lipids and DNA and tory diseases was reported to cause 4 million deaths glob- cause their dysfunction. Further the physiological antioxi- ally [7]. It is estimated that there may be 100 million more dant system that is equipped to provide the defence against asthmatics in addition by 2025 [3]. There are more than 300 the increased oxidative forces is also impaired in asthma million individuals affected with asthma worldwide and the due to inflammation, poor diet or lifestyle that also leads to prevalence has increased considerably in the recent years, increased oxidative stress and exacerbates the disease [14]. especially in children [16, 24]. In India around 4% popula- The reactive oxygen species generate a large number of oxi- tion is affected with asthma [15]. The chronic inflammation dative modifications in DNA, including strand breaks and in asthma is associated with airways hyper-responsiveness base oxidations, leading to DNA damage [2, 5, 12]. that leads to recurrent episodes of wheezing, breathlessness, The Single cell gel electrophoresis (SCGE)/comet assay chest tightness, coughing particularly at night or in the early is widely used to detect single- and double-strand breaks, morning [15, 16]. protection and repair of DNA at the level of individual cells It is now well accepted that oxidative stress is an impor- [8–10, 20, 25]. The damaged or broken fragments of DNA tant component as well as consequence of asthma pathogen- move under the influence of electrophoretic field and form a esis that leads to DNA damage [4, 6, 22] as the increased comet like structure. The increase or decrease in DNA dam- age eventually leads to increase or decrease in head DNA * Manisha Saini (%), tail DNA (%), tail length, tail moment, olive moment manishasaini683@gmail.com and tail area, so all these parameters can be used to detect the level of DNA damage [23]. 1 Department of Zoology, Kurukshetra University, Kurukshetra, India 13 Vol.:(0123456789)  Nucleus The clinical, immunological and biochemical aspects of Statistical analysis asthma and the association of asthma with oxidative stress are now well understood but the association of the genomic Unpaired student’s t test and ANOVA modified for Dun- damage and asthma has not been studied profoundly so far. can’s multiple range post hoc tests were used for analyzing So the present study was undertaken to assess DNA dam- data. Correlation between various parameters was studied age in the peripheral blood lymphocytes of the asthmatic using Pearson correlation analysis with the help of SPSS subjects and the influence of progression of disease on the v16. p < 0.05 was considered significant. DNA damage using the comet assay. Results Materials and methods The subjects were categorized according to their age, gen- der, smoking, drinking habits, dietary habits and exposure to Subjects and sample collection biomass fuel smoke. A significant difference was observed in the Spirometric measurements (FVC, FEV1, FEV1/ The present investigation included 152 subjects of which FVC, PEF and FEF25-75% predicted) of the asthmatic and 95 were asthmatics and 57 healthy controls. All the patients control subjects (Table 1). Figure 1a–g displays different were diagnosed on the basis of spirometry tests and clinical types of comets observed in the lymphocytes of the asth- symptoms like wheezing, recurrent episodes and measuring matic subjects during the present study. The % tail DNA the Forced Expiratory Volume in 1 second (FEV1) and other (37.15 ± 1.12), tail length (20.93 ± 0.91 µm), tail moment spirometric parameters like Forced Vital Capacity (FVC), (9.93 ± 0.55) and olive moment (6.73 ± 0.33) were all sig- Peak Expiratory Flow rate (PEF) etc. by the registered medi- nificantly elevated among the asthmatics in comparison to cal practitioner and the severity of disease was determined controls (p < 0.05) suggesting the increased genetic damage as per the global initiative for asthma guidelines [17]. For among the asthmatic subjects (Fig. 2). control, those individuals were selected who had no respira- tory symptoms or any other ailment. The study was con- ducted keeping in view the ethical guidelines of declaration of Helsinki for experiments involving human subjects [26]. Table 1  General, demographic and clinical variables of asthmatic and An appropriate written informed consent was taken from control subjects each subject prior taking samples and ethical clearance was Characteristics Asthmatics Controls obtained from Institutional Ethics Committee of Kuruk- N 95 57 shetra University, Kurukshetra. Age# 43.62 ± 1.66 39.16 ± 1.70 Questionnaire was obtained from each subject asking FVC (% predicted)* 71.24 ± 1.94 97.67 ± 1.51 their age, gender, dietary habits, smoking and drinking hab- FEV1 (% predicted)* 67.60 ± 2.15 96.56 ± 1.30 its and other parameters regarding their lifestyle. Smokers FEV1/FVC (% predicted)* 96.78 ± 1.47 104.49 ± 1.02 and alcoholic subjects were excluded from the study to avoid PEF (% predicted)* 53.52 ± 2.18 83.56 ± 2.56 conflicting results. Each patient’s clinical profile, Spiromet- FEF 25–75 (% predicted)* 46.75 ± 2.09 77.97 ± 2.87 ric measurements, history of allergy and treatment details Males/Females 22/73 24/33 was also obtained. Vegetarians/non-vegetarians 72/23 42/15 Education Nil/below secondary/sec- 30/20/33/12 20/17/12/8 Single cell gel electrophoresis (SCGE)/comet assay ondary/graduate Occupation Venous blood samples were collected from the subjects by Farmers/housewives/ 42/21/12/08/07/05 22/15/07/04/06/03 venipuncture by a registered medical practitioner and col- laborer/service/business/ lected in K ­ 2EDTA coated vials (Becton–Dickinson). The student samples were taken to the laboratory and processed follow- FVC Forced Vital Capacity, FEV1 Forced Expiratory Volume in ing the protocol of Dhawan et al. [11]. The comets were 1 Second, PEF Peak Expiratory Flow Rate, FEF 25–75% Forced visualized with Nikon Eclipse 90i Trinocular Research Expiratory Flow at 25–75% microscope at 100× magnification and scored with LUCIA The values of age, FVC, FEV1, FEV1/FVC, PEF and FEF 25–75 are Comet Software (version 6.22). 100 cells were scored per expressed as Mean ± SE of mean individual and % tail DNA, tail length, tail and olive moment # Non-significant (p > 0.05), Unpaired student’s T test were measured for evaluation of DNA damage. *Significant (p < 0.05), Unpaired student’s T test 13 Nucleus Fig. 1  Lymphocytes of the asth- matic subjects showing different types of comets: a Type 0, b Type 1, c Type 2, d Type 3, e, f Type 4 comet and g different types of comets The % tail DNA, tail length, tail and olive moment tail length (p < 0.01), tail and olive moment (p < 0.05) with were increased markedly among the patients with moder- the age of the patients (Table 4). ate and severe asthma than those with intermittent and mild asthma (Table 2). The comet parameters were also analyzed in respect to duration of disease and % tail DNA Discussion and tail length were evidently increased with increase in duration of asthma and maximum damage was observed Despite of abundant information available on asthma among the patients with ≥ 10 years of duration (Table 3). pathophysiology and treatment facilities, the incidences of A significant negative correlation of the comet param- asthma morbidity and mortality are continuously increas- eters with the measured FVC and FEV1 depicted an ing among children and adults which represent a major increase in the genomic instability with increased sever- global concern. It is a well established fact that asthma ity of asthma. Pearson correlation coefficient of various is a disorder of chronic inflammation of the airways and comet parameters with the duration of asthma was found associated with systemic oxidative stress but, very slight to be non-significant while it was significantly positive for has been known about the genetic damage associated with 13  Nucleus Peripheral blood lymphocytes were chosen to study DNA damage in the present investigation as blood is an easily available source and plays the central role in the oxidant- antioxidant balance in the body. Earlier studies have reported the association of asthma with DNA damage. Fortoul et al. [13] performed Single-cell gel electrophoresis assay of nasal epithelium and leukocytes from asthmatic and non-asthmatic subjects in Mexico City. The asthmatic subjects had more DNA breaks in their nasal epithelial cells than the non-asth- matics. Similarly increased oxidative DNA damage and its relation to oxidative stress in the asthmatics was reported by Al-Afaleg et al. [1] in the Saudi asthmatic patients. Zeyrek et al. [28] reported DNA damage in children with asthma bronchiale and its association with oxidative and antioxida- tive measurements. Increased production of ROS leading to an imbalance between the oxidative forces and the anti- Fig. 2  Comparison of various comet parameters among asthmatic and control subjects. *Significant (p < 0.05), Unpaired student’s t test oxidant defense systems favoring an oxidative damage was found to play a role in the pathogenesis of asthma. The find- ings indicated that lymphocyte DNA damage level increases asthma. Several studies have been done in the past regard- in children with asthma [28]. ing the oxidant-antioxidant imbalance and oxidative stress DNA damage in children with asthma was studied by as a cause and consequence of asthma [4, 6, 21, 22, 27] Hasbal et al. [18]. Before and after 8 weeks of anti-asthmatic but there is a lack of research reports about the genomic therapy blood samples were taken, DNA strand breaks and damage caused due to asthma especially in north Indian Fpg-sensitive sites in peripheral leukocytes were determined population. by comet assay. DNA strand breaks and Fpg-sensitive sites Table 2  Comparison of comet Variables Level of asthma severity parameters among patients with increased severity of asthma Intermittent Mild Moderate Severe N 16 25 27 27 % Tail DNA* 28.37 ± 1.70C 31.68 ± 1.43C 38.68 ± 2.12B 45.88 ± 1.67A Tail length* 20.23 ± 2.78B 17.62 ± 1.11B 22.08 ± 1.89A 23.25 ± 1.59A Tail moment* 7.96 ± 1.35B 7.23 ± 0.65B 11.31 ± 0.93A 12.23 ± 1.17A Olive moment* 5.51 ± 0.75B 5.11 ± 0.38B 7.45 ± 0.56A 8.23 ± 0.71A Values are expressed as Mean ± SE of mean *Significant (p < 0.05), ANOVA The alphabets in superscript depict the significant difference. Values with different alphabets in superscript, along a row show significant difference (p < 0.05), ANOVA with Duncan’s post hoc test Table 3  Comparison of various Variables Duration of asthma (years) comet parameters among asthmatic patients in relation <1 1–5 5–10 ≥ 10 with duration of asthma N 22 27 21 25 % Tail DNA* 36.29 ± 2.78B 33.45 ± 1.40B 38.96 ± 1.80A 39.05 ± 2.25A Tail length* 18.18 ± 1.70B 20.93 ± 1.02AB 20.96 ± 1.74AB 23.75 ± 2.18A Tail moment 8.46 ± 1.12A 10.22 ± 0.90A 12.03 ± 1.85A 11.25 ± 0.95A Olive moment 6.19 ± 0.64A 6.84 ± 0.44A 7.62 ± 0.87A 7.59 ± 0.57A Values are expressed as Mean ± S.E. of mean *Significant (p < 0.05), ANOVA The alphabets in superscript depict the significant difference. Values with different alphabets in superscript, along a row show significant difference (p < 0.05), ANOVA with Duncan’s post hoc test 13 Nucleus Table 4  Correlation of comet Correlates Pearson’s coefficient of correlation (r) parameters with FVC, FEV1, age and duration of disease in FVC (% predicted) FEV1 (% predicted) Age Duration asthmatic subjects % Tail DNA − 0.473** − 0.360** 0.101 0.052 Tail length − 0.038 − 0.029 0.242** 0.014 Tail moment − 0.212* − 0.142 0.231* 0.036 Olive moment − 0.251** − 0.186* 0.188* 0.042 **Pearson’s correlation is significant at the 0.01 level (1-tailed) *Pearson’s correlation is significant at the 0.05 level (1-tailed) in the asthma group were found to be increased as com- From the present study, it can be summarized that asthma pared with control group. Evidences from the earlier studies is associated with DNA damage and the increased DNA suggest the association of asthma with genomic instability damage in asthma can be attributed to disease progression and the present findings correlate well with these studies. as a consequence of reduced antioxidant level and increased The increased DNA damage might be a component in the oxidative stress with duration and severity which further exacerbation of asthma and a consequence of the increased causes genomic instability and DNA damage. oxidant injury as well. The present findings are in accordance with the above Acknowledgements Financial assistance received from Haryana State Council for Science and Technology, Panchkula, in the form of JRF mentioned studies. Besides increased DNA damage to Manisha Saini is gratefully acknowledged. We are grateful to Dr. observed in asthmatic subjects in comparison to controls, Madan Khanna and Dr. Subhash Garg for providing blood samples, and the present study also reported increased DNA damage to the authorities of Kurukshetra University for providing Laboratory among the asthmatic patients with increased severity and and other necessary facilities. duration of asthma depicting a strong association of disease progression and DNA damage. The strong negative correla- Compliance with ethical standards tion of comet parameters with measured FVC and FEV1 of Conflict of interest Authors declare that they have no conflict of inter- asthma patients during the current investigation indicated est. that exacerbation of asthma elevated the level of genetic damage which was probably due to the increased oxidative Ethical approval All procedures performed in the present study were stress with increased exacerbation of asthma that has been in accordance with the ethical standards of the Institutional Ethics Committee, KUK and with the 1964 Helsinki declaration and its later reported in a previous study by Yadav and Saini [27]. Dur- amendments (2013 Helsinki declaration). 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