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ORIGINAL ARTICLE
Year : 2014  |  Volume : 27  |  Issue : 1  |  Page : 50-54

Study of DNA damage in asphyxiated newborns


Department of Pediatrics, Faculty of Medicine, Menofiya University, Egypt

Date of Submission04-May-2013
Date of Acceptance28-Jul-2013
Date of Web Publication20-May-2014

Correspondence Address:
Hany M. El-Boshy
Msc, Samanoud, 36793, Gharbia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.132739

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  Abstract 

Objective
The present study was carried out to assess the presence of DNA damage in asphyxiated newborns.
Background
Perinatal asphyxia is one of the major causes of neonatal mortality. It results from lack of oxygen before, during, or after birth.
Patients and methods
Thirty asphyxiated and 20 nonasphyxiated neonates were included in this case-control study. Blood samples were collected from a peripheral vein into heparinized tubes and stored at 10°C away from light to prevent DNA damage and were processed within 24 h to avoid further DNA damage. Estimation of DNA damage in peripheral leukocytes by DNA fragmentation assay was carried out by DNA extraction and then gel electrophoresis.
Results
Significant DNA damage was found (56.7%) in asphyxiated newborns and positively correlated with severity and clinical complications of hypoxic-ischemic encephalopathy.
Conclusion
The study indicates the presence of DNA damage in asphyxiated newborns.

Keywords: DNA fragmentation, neonatal mortality, neonatal hypoxia


How to cite this article:
Tawfik MA, AboElella SS, El-Boshy HM. Study of DNA damage in asphyxiated newborns. Menoufia Med J 2014;27:50-4

How to cite this URL:
Tawfik MA, AboElella SS, El-Boshy HM. Study of DNA damage in asphyxiated newborns. Menoufia Med J [serial online] 2014 [cited 2020 Sep 20];27:50-4. Available from: http://www.mmj.eg.net/text.asp?2014/27/1/50/132739


  Introduction Top


Perinatal asphyxia is one of the major causes of neonatal mortality in developing countries. Perinatal asphyxia is characterized by impaired gas exchange, which leads to hypoxemia, hypercarbia, and metabolic acidosis.

Impaired gas exchange and insufficient perfusion to vital organs leads to severe neurological insult, resulting in death and long-term disability [1]. Neonatal encephalopathy because of perinatal hypoxic ischemia occurs in one to three per 1000 births at term [2]. The molecular mechanism behind the neurological deficit has not been fully evaluated. Two mechanisms of DNA damage have been suggested. It is envisaged that H2O2, which crosses the biological membrane, can easily penetrate the nucleus and react with ions of iron or copper to form a hydroxyl radical (OH) [3]. Another explanation is the ability of oxidative stress to cause DNA damage by triggering a series of metabolic events within the cell that lead to an elevation in nuclease enzymes that cleave the DNA backbone. The intracellular free Ca + interacts with Ca + -dependent endonuclease, leading to fragmentation of DNA, resembling the mechanism of apoptosis (programmed cell death) [4]. Hypothermia is the only effective neuroprotective therapy currently available for the treatment of neonatal encephalopathy, and is safe and easy to administer [5].


  Patients and methods Top


This study was carried out on 50 full-term newborns of at least 38 weeks old. They were divided into two groups. The patient group included 30 full-term newborns with perinatal asphyxia and the control group included 20 nonasphyxiated babies matched for gestational age and weight. The diagnosis of hypoxic-ischemic encephalopathy (HIE) was made on the basis of the following criteria [6]:

  1. At least one of the following:

    1. Apgar scores less than 5 at 10 min,
    2. metabolic acidosis (base deficit ≥16 mEq/l in the cord blood or in the first hour in arterial blood gases),
    3. assisted ventilation for at least 5 min, or
    4. cesarean section because of fetal distress.
    5. Need for assisted ventilation at birth;
    6. Lethargy/stupor, hypotonia, weak/absent sucking reflex, and abnormal reflexes;
    7. At least one more organ involvement in addition to encephalopathy.


Congenital malformation syndromes, septicemia, and jaundice are excluded.

All the neonates in this study were subjected to detailed assessment of history and a complete physical examination of all systems with a special focus on neurological examination. Full blood count and assessment of blood urea, and creatinine and serum Ca were performed. Blood samples were collected from a peripheral vein into heparinized tubes and stored at 10°C away from light and were processed within 24 h to avoid further DNA damage. Estimation of DNA damage in peripheral leukocytes by a DNA fragmentation assay was carried out by DNA extraction and then gel electrophoresis.


  Results Top


The results of this study are shown in [Table 1],[Table 2],[Table 3],[Table 4],[Table 5] and [Table 6].
Table 1: Demographic data of all studied neonates and their mothers

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Table 2: Clinical examination of patients

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Table 3: Laboratory data of studied neonates

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Table 4: DNA damage in patients

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Table 5: Relation between DNA damage and type of delivery, muscle tone, and convulsions

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Table 6: Relation between DNA damage and other laboratory investigations

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In terms of the demographic data of the neonates studied, the gestational age of the cases was 38.7 ± 1.1 weeks, which was not significantly different (P > 0.05) from the controls (38.6 ± 1.2 weeks). The percentage of males was 56.7% in the patient group and 75% in the control group. There was a high incidence of delivery complications in patients compared with the controls; also, there were highly significant lower Apgar scores at 1 and 10 min in patients compared with controls. There was no difference between patients and controls in the mode of delivery [Table 1]. Clinical examination of patients' central nervous system showed a high incidence of seizures (46.7%), flaccidity and hypotonia (53.3%), and nonelicited reflexes (76.7%). Examination of the respiratory system of patients showed a significantly high incidence of respiratory distress to different degrees in patients (100%). Examination of the gastrointestinal system of our patients showed a high incidence of feeding intolerance (53.3%) [Table 2].

In this study, laboratory investigation showed that hemoglobin levels were significantly lower in patients than in the controls (P < 0.05) and leukocytic counts were significantly higher in patients than in the controls (P < 0.05). There was no statistically significant difference in the platelet count between patients and controls. Serum Ca levels were significantly low in patients when compared with the controls (P < 0.05). Urea and creatinine were significantly higher in patients than in the controls [Table 3].

In terms of DNA changes, our results showed that there were significant differences between the two groups of the study in gel results, with positive results in patients with perinatal asphyxia (56.7%) [Table 4].

There were positive correlations between DNA damage and muscle tone affection. DNA damage occurs more with hypotonic muscle tone (100%) and floppy muscle tone (75%). Also, there were positive correlations between DNA damage and convulsions (78.6%) [Table 5]. In this study, the serum calcium level was highly significantly decreased in patients with DNA damage compared with patients with no DNA damage (P < 0.05); also, serum urea and creatinine levels were significantly increased in patients with DNA damage compared with patients with no DNA damage (P < 0.05) [Table 6].


  Discussion Top


Perinatal hypoxia-ischemia is the most common cause of neurologic disease during the neonatal period. HIE is associated with high mortality and morbidity rates. In this study, we focused on studying DNA damage in patients exposed to hypoxia during the perinatal period. In our study, there was no difference between the patient and the control group in demographic data (gestational age, sex, mother age, parity, mode of delivery), but there was a significant incidence of delivery complications in patients (63%) compared with the controls (0%). In our study, a significantly lower Apgar score at 1 and 10 min was found in cases when compared with controls. The American Academy of Pediatrics (AAP) [7] declared that the Apgar score is a rapid means of evaluating an infant's cardiopulmonary and neurological functions at set intervals after birth (routinely at 1 and 5 min). Clinical examination of our patients' central nervous system showed a significantly high incidence of seizures (53.3%), flaccidity and hypotonia (53.3%), and nonelicited reflexes (76.7%). Zanelli et al. [8] found that infants with moderate HIE are lethargic, with significant hypotonia and reduced deep tendon reflexes. Moreover, they reported that generalized hypotonia and depressed deep tendon reflexes are common in severe HIE. Examination of the respiratory system of patients shows a significantly high incidence of respiratory distress to different degrees in patients (100%). Risso et al. [9] found that the incidence of respiratory distress syndrome, need for mechanical ventilation support, and inotrope therapy were significantly higher (P < 0.05, for all) in the perinatal asphyxia group. Examination of the gastrointestinal system of our patients showed a high incidence of feeding intolerance (53.3%). Zanelli et al. [8] found that in most cases of HIE (particularly in moderately severe and severe ones), the infant is restricted to nothing by mouth during the first 3 days of life or until the general level of alertness and consciousness improves. This is caused by transient disturbances in intestinal motor activity patterns, which may result from loss of neural regulation or inhibition of motor control [10]. In terms of DNA changes, our results showed that there were significant differences between the two groups of the study in gel results, with positive results in patients with perinatal asphyxia (56.7%), whereas in the control group, it was 0%. This could be attributed to the effects of hypoxia and ischemia, which are the primary physiological processes that trigger HIE [11]. Supporting our study, Manoj et al. [12] found severe DNA damage in peripheral leukocytes as well as increased serum lipid peroxidation among patients with perinatal asphyxia. We found that there were positive correlations between DNA damage and muscle tone affection. DNA damage occurs more with hypotonic muscle tone (100%) and floppy muscle tone (75%). According to the criteria described by Sarnat and Sarnat [13], muscle tone reflects the degree of severity of HIE: Hypotonic muscle tone in moderate HIE and flaccidity in severe HIE, which indicates a positive correlation between DNA damage with the severity of asphyxia. These results are in agreement with those of Manoj and colleagues, who found that there were associations of DNA damage and lipid peroxidation with the severity and clinical complications of asphyxia. Also, we found that DNA damage correlated positively with convulsions (78.6%). This is also in agreement with the study of Manoj and colleagues, who found that DNA damage was highly significantly associated with convulsions.

Our laboratory investigations of the groups studied included a full blood count, which indicated significantly lower hemoglobin levels in patients than in controls (P < 0.05), and they were low in patients with DNA damage compared with patients with no DNA damage. Leukocytic counts were significantly higher in patients than in controls (P < 0.05). There was no statistically significant difference in platelet count between patients and controls. Serum Ca levels were significantly low in patients when compared with controls (P < 0.05) and significantly low in patients with DNA damage compared with patients with no DNA damage (P < 0.05). The pathogenetic mechanism by which birth asphyxia causes hypocalcemia is poorly understood. However, it has been speculated that delayed introduction of feeds, increased calcitonin production, increased endogenous phosphate load, renal insufficiency, and reduced parathyroid hormone secretion may all contribute toward hypocalcemia [14]. In our study, there was a significant increase in blood urea and creatinine in the patients when compared with the controls (P < 0.05) and it was significantly high in patients with DNA damage compared with patients with no DNA damage (P < 0.05). These results are in agreement with those of Risso and colleagues, who found that kidney function parameters such as blood creatinine and urea concentrations and urine gravity were significantly higher (P < 0.01, for all) in the perinatal asphyxia group [9].


  Conclusion Top


Perinatal asphyxia results from compromised placental or pulmonary gas exchange, which may result in neonatal hypoxia and tissue/organ injury.

A variety of maternal, obstetric, and neonatal conditions predispose the fetus and newborn to asphyxia that can occur before, during, or after birth.

Perinatal asphyxia causes significant oxidative stress, which leads to DNA damage.

This study is another proof of occurrence of DNA damage in asphyxiated newborns and the positive correlation of DNA damage with the severity and clinical complications of perinatal asphyxia, which may be considered as a cofactor for DNA damage rather than the sequelae of events [Figure 1].
Figure 1:

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Recommendation

  1. Perinatal asphyxia can be prevented by good prenatal care and management of medical conditions such as maternal diabetes.
  2. Early identification of high-risk infants should be performed and early interventions should be initiated as soon as possible after delivery.
  3. Study of DNA damage in asphyxiated newborns helps in evaluation of severity and prognosis of perinatal asphyxia.
  4. Study of DNA damage in asphyxiated newborns helps in establishment of new guidelines of management through interference before neurological sequelae occurs.



  Acknowledgements Top


Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.Volpe JJ. In: Volpe JJ, editor. Hypoxic-ischemic encephalopathy: biochemical and physiological aspects. Neurology of the newborn. Philadelphia, PA: Elsevier Saunders; 2008. 247-324.  Back to cited text no. 1
    
2. Kurinczuk JJ, White-Koning M, Badawi N. Epidemiology of neonatal encephalopathy and hypoxic-ischaemic encephalopathy. Early Hum Dev 2010; 86:329-338.  Back to cited text no. 2
    
3. Halliwell B, Arouma OI. DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems. FEBS Lett 1991; 281:9-19.  Back to cited text no. 3
    
4. Dypbukt JM1, Thor H, Nicotera P. Intracellular Ca2+ chelators prevent DNA damage and protect hepatoma 1C1C7 cells from quinone-induced cell killing. Free Radic Res Commun. 1990; 8:347-54.  Back to cited text no. 4
    
5. Higgins RD, Raju T, Edwards AD, et al. Hypothermia and other treatment options for neonatal encephalopathy: an executive summary of the Eunice Kennedy Shriver NICHD workshop. J Pediatr 2011; 159:851.  Back to cited text no. 5
    
6. Klinger G, Beyene J, Shah P, Perlman M. Do hyperoxemia and hypocapnia add to the risk of brain injury after intrapartum asphyxia? Arch Dis Child Fetal Neonatal Ed 2005; 90:49-52.  Back to cited text no. 6
    
7. American Academy of Pediatrics (AAP). The Apgar score. Adv Neo Care 2006; 6:220.  Back to cited text no. 7
    
8. Zanelli SA, Naylor M, Dobbins N. Hypoxic ischemic encephalopathy. Philadelphia, PA: Elsevier Saunders; 2008. 1-9.  Back to cited text no. 8
    
9. Risso FM, Serpero LD, Zimmermann LJ, Gavilanes AW, Frulio R, Michetti F, et al. Perinatal asphyxia: kidney failure does not affect S100B urine concentrations. Chin Chim Acta 2012; 413:150-153.  Back to cited text no. 9
    
10.1Berseth CL, McCoy HH. Birth asphyxia alters neonatal intestinal motility in term neonates. Pediatrics 1992; 90:669-673.  Back to cited text no. 10
    
11.1Madan A, Hamrick SEG, Ferriero DM. In: Taeusch HW, Ballard RA, Gleason CA, editors. Central nervous system injury and neuroprotection. Avery′s diseases of the newborn. 8th ed. Philadelphia, PA: Elsevier Saunders; 2005. 965-992.  Back to cited text no. 11
    
12.1Manoj A, Rao RK, Bhat VB, Venkatesh C, Bobby Z. Oxidative stress induced DNA damage in perinatal asphyxia. Curr Ped Res 2011; 15:19-23.  Back to cited text no. 12
    
13.1Sarnat HB, Sarnat MS. Neonatal encephalopathy following fetal distress: a clinical and electroencephalographic study. Arch Neuro 1976; 33:696-705.  Back to cited text no. 13
    
14.1Gowen CW Jr. In: Kliegman RM, Marcdante KJ, Jenson HB, Behrman RE, editors. Fetal and neonatal medicine. Nelson essentials of pediatrics. 5 th ed. New Delhi: Elsevier Publishers Limited; 2006. 271-335.  Back to cited text no. 14
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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Abstract
Introduction
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