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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 45  |  Issue : 2  |  Page : 77-80

Lymphocyte DNA damage in children with iron-deficiency anemia: a case–control study


Department of Pediatrics, K S Hedge Medical Academy, Derlakatte, Mangalore, Karnataka, India

Date of Submission23-Aug-2019
Date of Acceptance04-Dec-2019
Date of Web Publication29-Dec-2020

Correspondence Address:
Smriti Sinha
Department of Pediatrics, K S Hegde Medical Academy, Derlakatte, Mangalore 575018
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_32_19

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  Abstract 


Context Iron-deficiency anemia (IDA) is a widespread yet one of the most neglected micronutrient deficiency disorder worldwide.
Aims The objective was to evaluate lymphocyte DNA damage in children with anemia.
Materials and methods This was a prospective case–control study. A total of 80 infants and children aged six months to twelve years were included. Fifty had IDA, whereas 30 were controls. DNA damage scoring using alkaline comet assay was done in all children. Percentage and mean and/or SEM were calculated. Comparison between cases and controls was done using Student’s t test. Analysis of variance test was used to compare the groups within the cases and controls. Pearson correlation coefficient was performed to relate the hemoglobin levels with the DNA damage. Statistical software was used for analysis.
Results DNA damage scoring was significant in all the parameters in the iron-deficiency group with respect to the tail length and percentages of DNA in tail and olive moment, with a P value of 0.006, 0.002, and 0.038, respectively. A statistically significant negative correlation was found between hemoglobin levels and percentage of DNA in tail (r=−0.280; P=0.012) as well as olive moment (r=−0.240; P=0.032)
Conclusion IDA was associated significantly with lymphocyte DNA damage. A significant negative correlation between hemoglobin levels and percentage of DNA and olive moment was also elucidated. The authors conclude that early intervention is needed in even mild cases of IDA.

Keywords: alkaline comet assay, DNA breaks, microcytic hypochromic anemia, olive tail moment, oxidative stress


How to cite this article:
Varghese AP, Sinha S, Sindgikar SP, Shenoy RD, Shenoy V. Lymphocyte DNA damage in children with iron-deficiency anemia: a case–control study. Egypt J Haematol 2020;45:77-80

How to cite this URL:
Varghese AP, Sinha S, Sindgikar SP, Shenoy RD, Shenoy V. Lymphocyte DNA damage in children with iron-deficiency anemia: a case–control study. Egypt J Haematol [serial online] 2020 [cited 2021 Jul 31];45:77-80. Available from: http://www.ehj.eg.net/text.asp?2020/45/2/77/305399




  Introduction Top


Iron deficiency and the resulting anemia affects more than 3.5 billion people in the developing countries. Anemia accounted for 68.4 million years of life and disability in 2010, despite the decrease in anemia prevalence from 40.2% in 1990 to 32.9% in 2010 [1]. Of the global anemia burden, 37.5% is present in South Asia, including India [2]. According to NFHS IV, the under-five anemia prevalence rate varies from 45–70% in the state of Karnataka in India, having an average prevalence rate of 60% [3].

Iron, having multiple roles in human body, including electron transport and cellular respiration, is by far the most abundant transition metal in human body. The transition metal ions, being able to undergo facile l-electron oxidation/reduction, are an important chemical partner for producing biological-free radicals [4].

Iron in particular can undergo Fenton reaction with less reactive agents to produce hydroxyl radicals[5]:



These radicals once formed may cause oxidative damage to the cells, greater susceptibility to hemolysis due to cell membrane damage, increased membrane rigidity, and less deformability and impairment of nucleic acid synthesis in bone marrow. Human body counteracts this free iron in body through various physiological mechanisms [6].

Metal ion imbalance in various micronutrient deficiencies leads to DNA damage in cells when the metal is in transition state, by increasing the oxidative stress. Guanine particularly is very susceptible to reactive oxygen species-induced DNA damage. Some of this damage is repaired via respective enzymes, but some escape repair, leading to permanent damage. Measuring the DNA damage and quantifying it is an important parameter to find whether patients with iron-deficiency anemia (IDA) are prone to oxidative stress [7]. The single cell electrophoresis, or ‘comet’, assay for DNA damage is widely used for this purpose, and was judged to be one of the few adequate methods for assessing the oxidative stress and DNA effect. This assay uses the amount of DNA breaks to quantify the DNA damage [8]. It is visualized using fluorescence microscopy, and damage is scored using either visual or computerized image analysis. This simple and rapid technique requires a small number of any eukaryotic cells per sample and is sensitive even to low amounts of DNA damage.

There are scarce data relating the hemoglobin levels and peripheral DNA damage (as per comet assay) in pediatric population. Thus, the present study was designed to study the relation between IDA and DNA damage in children and to correlate it with hemoglobin levels.


  Materials and methods Top


Study population

This study included 50 children between the age group of 6 months to 12 years with anemia, and 30 age-matched and sex-matched controls without anemia. This study was conducted over a period of 2 years at a tertiary care teaching hospital in coastal India. After the approval of the study by the Institution’s Ethics Committee, an informed written consent was taken from all the concerned participants.

Anemia in children was defined as per WHO: hemoglobin less than 11 g/dl for children aged 6 months to 6 years and less than 12 g/dl for children between 6 and 12 years. Mild anemia, moderate anemia, and severe anemia were defined as less than 7, 7–10, and greater than 10 g/dl, respectively. IDA was diagnosed when the children with anemia had microcytic hypochromic anemia in peripheral smear with mean corpuscular volume less than 80 fl and/or mean corpuscular hemoglobin concentration less than 32 g/dl with an increased red cell distribution width (RDW) greater than 17% and serum ferritin less than or equal to 12 g/dl.

Children having non-nutritional anemia, chronic systemic illnesses, or on oral iron supplements, chemotherapy, or radiotherapy were excluded.

Data collection and sampling

A detailed history was noted for symptoms of IDA and its co-morbidities. A complete physical examination was done to look for features of anemia, malnutrition, and co-existent micronutrients deficiencies. Two blood samples were taken from each participant. Overall, 2 ml of EDTA sample was sent for hematological investigations like hemoglobin, peripheral smear, red cell indices, and RDW. This was done with an automated cell counter (Coulter SKTS; Beckman Coulter, Brea, California, USA). The second plain blood sample was collected for comet assay and analyzed immediately.

Within an hour of collection, the sample was mixed with histopaque and centrifuged. The reagent and low-melting-point agarose gel were prepared followed by lymphocyte isolation from the buffy coat. After staining with ethidium bromide, the migro gel slides were subjected to electrophoresis at pH greater than 13. Visualization of DNA damage was done using a fluorescence microscope, under a green filter [9]. Images were taken by Q capture pro and analyzed using comet assay software to quantitate the length of DNA migration and percentage of migrated DNA. Scoring was done according to length of the tail and percentage DNA in tail and olive tail moment. Tail length denotes the total length of damaged DNA migration from the body of the nuclear core. Head percentage DNA is the percentage of the ratio of head optical intensity to the total optical intensity (head+tail). Tail percentage DNA is the total DNA minus the head percentage DNA. Olive tail moment is calculated by multiplying tail length and total percentage of DNA in the tail. Tail moment incorporates the minutest of DNA damage (tail length) along with the percentage DNA in the tail (intensity of the tail).

Statistical analysis

Software package SPSS version 16.0 (IBM Corporation, Armonk, New York, USA) was employed for the statistical analysis. Percentage and mean and/or SEM were calculated. Comparison between cases and controls was done using Student’s t test. Values were expressed as 95% confidence interval derived from SEM. Analysis of variance test was used to compare the groups within the cases and controls. Pearson’s correlation coefficient was performed to relate the hemoglobin levels with the DNA damage. Significance was attributed only if P values were less than or equal to 0.05.


  Results Top


Demographic and laboratory data of the participants are shown in [Table 1]. The median age among cases and controls was 45 and 50 months, respectively. Cases were more symptomatic with respect to comorbid systemic illnesses, physical symptoms of anemia, and presence of micronutrient deficiencies (P=0.416). Overall, 48% of cases (n=24) also had disturbances of growth like malnutrition as compared with controls (P=0.005). Mean hemoglobin among anemic children was 8.78±1.22 g/dl. Moreover, 10% of children (n=5) had severe anemia (<7 g/dl), moderate anemia was found in 74% (n=37), and 16% (n=8) had mild anemia. The mean RDW for the cases and controls were 17.31±2.36 and 13.65±1.76, respectively. Most cases had microcytic hypochromic peripheral blood picture, whereas the controls all were normocytic normochromic.
Table 1 Demographic and laboratory data of participants

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[Table 2] shows the three parameters in alkaline comet assay among anemic children and controls. Children with IDA had a significantly higher lymphocyte DNA damage compared with the healthy controls. The mean tail length for cases was as high as 18.50 compared with controls who had a tail length of 6.38. The percentages of DNA in tail for cases and controls were 12.72 and 1.19, respectively. The mean olive moment for cases was 3.37, whereas for the controls was 1.41. All three aforementioned parameters of comet assay between cases and controls were statistically significant, with a P value of 0.006, 0.002, and 0.038, respectively.
Table 2 The parameters in alkaline comet assay among patients with iron-deficiency anemia and controls

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The olive moment and percentage of DNA had significant negative correlation with the hemoglobin levels (r=−0.240, P=0.032, and r=−0.280, P=0.012, respectively). However, the hemoglobin level did not correlate with the tail length (r=−0.193 and P=0.087) ([Figure 1]).
Figure 1 The correlation of lymphocyte DNA damage and hemoglobin levels in anemic children. The hemoglobin level was negatively correlated with lymphocyte DNA damage with respect to percentage of DNA and olive moment and was found to be statistically significant.

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  Discussion Top


Various studies have tried to correlate oxidative stress and lymphocyte DNA damage with iron deficiency, but the studies in pediatric population have been precious few. Fewer still have been studies correlating DNA damage severity with the hemoglobin levels.

In the present study, we found that there is a significant association between the presence of anemia and the lymphocyte DNA damage on all the parameters. When correlating the DNA damage with the hemoglobin levels, the olive moment and percentage DNA in tail had a significant negative correlation suggesting lower the hemoglobin falls, more is the DNA damage and hence the long-term sequelae.

Coghetto Baccin et al. [10] in their study on older patients correlated IDA with higher catalase and superoxide dismutase levels compared with age-matched controls. They concluded that the patients with IDA have to deal with higher chronic oxidative stress even in a higher age group.

Few studies have also tried to correlate the effect of iron therapy on the reversal of DNA damage or oxidative stress, but the results have not been consistent. In their study, Aksu et al. [7] compared the oxidative stress and DNA breaks before and after 12 weeks of iron therapy in 27 children with IDA. They found a significant increase in both the number of DNA strands break and Fpg-sensitive sites after treatment with iron for twelve weeks compared with the baseline values.On the contrary, recent studies done by Yoo et al. [6] and Akça et al. [11] stated that the children with IDA have a higher total oxidative stress compared with healthy children and that their total oxidative stress levels significantly decreased after treatment for IDA.

In a previous study, Aslan et al. [12] concluded that the patients with IDA have both increased oxidative stress and DNA damage. Like the present study, they also found that hemoglobin levels have a negative correlation with DNA damage and positive correlation with antioxidant capacity, implying some role of oxidative stress in IDA pathogenesis.

DNA breaks can occur owing to multiple causes other than IDA. The main limitation of the present study is the other confounding factors influencing these end points, like passive smoke exposure, environmental pollutants, and background radiation exposure. These were not matched or excluded. Rigorous sampling methods were not employed. Further scope of this study is to apply this to a larger sample size of children with systematic sampling and randomization. The reversal of DNA damage after treatment can also be studied. The action of adding antioxidants like vitamin C along with oral iron therapy can also be studied.

In our present study, we found a significant association between this DNA damage in lymphocytes and presence of IDA. Furthermore, we also found a negative correlation between the amount of DNA damage and the hemoglobin levels. This fact may have an important role in pathogenesis and should also highlight the necessity of early diagnosis and treatment of IDA.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Kotecha PV. Nutritional anemia in young children with focus on Asia and India. Indian J Community Med 2011; 36:8.  Back to cited text no. 1
    
2.
Kassebaum NJ, Jasrasaria R, Naghavi M, Wulf SK, Johns N, Lozano R et al. A systematic analysis of global anemia burden from1990 to 2010. Blood 2014; 123:615–624.  Back to cited text no. 2
    
3.
International Institute of Population Sciences (IIPS) and MoHFW. National Family Health Survey (NFHS-4), 2015-2016. Mumbai, India.  Back to cited text no. 3
    
4.
McCord JM. Iron, free radicals, and oxidative injury. Semin Hematol 1998; 35:5–12.  Back to cited text no. 4
    
5.
Jabs T. Reactive oxygen intermediates as mediators of programmed cell death in plants and animal. Biochem Pharmacol 1999; 57:231–245.  Back to cited text no. 5
    
6.
Yoo JH, Maeng HY, Sun YK, Kim YA, Park DW, Park TS et al. Oxidative status in iron‐deficiency anemia. J Clin Lab Anal 2009; 23:319–323.  Back to cited text no. 6
    
7.
Aksu BY, Hasbal C, Himmetoglu S, Dincer Y, Koc EE, Hatipoglu S et al. Leukocyte DNA damage in children with iron deficiency anemia: effect of iron supplementation. Eur J Pediatr 2010; 169:951–956.  Back to cited text no. 7
    
8.
Collins AR. The comet assay for DNA damage and repair. Mol Biotechnol 2004; 26:249–261.  Back to cited text no. 8
    
9.
Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988; 175:184–191.  Back to cited text no. 9
    
10.
Coghetto Baccin A, Lauerman Lazzaretti L, Duarte Martins Brandao V, Manfredini V, Peralba MC, Silviera Benfato M. Oxidative stress in older patients with iron deficiency anemia. J Nutr Health Ageing 2009; 13:666–670.  Back to cited text no. 10
    
11.
Akça H, Polat A, Koca C. Determination of total oxidative stress and total antioxidant capacity before and after the treatment of iron-deficiency anemia. J Clin Lab Anal 2013; 27:227–230.  Back to cited text no. 11
    
12.
Aslan M, Horoz M, Kocyigit A, Ozgonul S, Celik M, Celik H et al. Lymphocyte DNA damage and oxidative stress in patients with iron deficiency anemia. Mutat Res 2006; 601:144–149.  Back to cited text no. 12
    


    Figures

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    Tables

  [Table 1], [Table 2]



 

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