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Year : 2019  |  Volume : 32  |  Issue : 3  |  Page : 1078-1082

A study of DNA damage in epileptic children treated with valproic acid or carbamazepine

Pediatric Department, Faculty of Medicine, Menoufia University, Shebeen El-Kom, Egypt

Date of Submission23-Jan-2018
Date of Acceptance06-Mar-2018
Date of Web Publication17-Oct-2019

Correspondence Address:
Dina Abd El-Aziz Hammad
Pediatric Department, Faculty of Medicine, Menoufia University, Shebeen El-Kom 32511
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mmj.mmj_41_18

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The aim of this study was to determine DNA damage in epileptic children due to seizures and/or due to the most widely used antiepileptic drugs (valproic acid or carbamazepine).
Epilepsy is a common neurological disease that requires early diagnosis and treatment. However, long-term use of antiepileptic drugs has been shown to cause oxidative damage to protein and DNA.
Patients and methods
We enrolled 15 epileptic patients receiving valproic acid monotherapy, 15 epileptic patients receiving carbamazepine monotherapy, 10 epileptic children with no treatment, and 10 control healthy children. Blood samples were collected from a peripheral vein into heparinized tubes and estimation of DNA damage in peripheral leukocytes by DNA fragmentation assay was carried out by DNA extraction and then gel electrophoresis.
Epileptic children of both groups receiving carbamazepine or valproic acid monotherapy had significantly higher more DNA damaged cells than that of the control group P = 0.002 and 0.04, respectively. This indicate a significant DNA damaging effect of both CBZ and valproic acid monotherapy on human lymphocytes. No significant correlations were detected as regards the duration of treatment, dose, or serum level of drugs. No significant differences were found between epileptic patients' group not receiving antiepileptic drugs and the control group.
It can be concluded that patients on either valproic acid or carbamazepine monotherapy are at risk to develop significant DNA damage effects more than those without treatment, while epilepsy itself does not cause DNA damage.

Keywords: carbamazepine, child, DNA damage, epilepsy, valproic acid

How to cite this article:
Mahmoud AT, Tawfik MA, Abd-El-Naby SA, El-Aziz Hammad DA. A study of DNA damage in epileptic children treated with valproic acid or carbamazepine. Menoufia Med J 2019;32:1078-82

How to cite this URL:
Mahmoud AT, Tawfik MA, Abd-El-Naby SA, El-Aziz Hammad DA. A study of DNA damage in epileptic children treated with valproic acid or carbamazepine. Menoufia Med J [serial online] 2019 [cited 2020 Apr 4];32:1078-82. Available from: http://www.mmj.eg.net/text.asp?2019/32/3/1078/268826

  Introduction Top

Epilepsy is defined as the occurrence of two unprovoked seizures more than 24 h apart or a single unprovoked seizure with high risk of recurrence [1]. In some epileptic patients, complete relief is experienced when treated with antiepileptic drugs. Carbamazepine and valproic acid are among the most commonly prescribed antiepileptic drugs in epilepsy [2]. There are several studies showing that seizure results in free radical production and oxidative damage to proteins and DNA [3]. Oxidative stress usually results from either increasing oxidant enzymes or decreasing antioxidant enzymes and results in excessive levels of free radical intermediates [4]. The oxidant/antioxidant balance in epilepsy is not only modulated by seizures per se, but also by antiepileptic drugs [2]. The use of drugs such as valproic acid or carbamazepine for a long term has been shown to increase free radical formation and cause oxidative damage within neuronal cells, the free radicals generated mediated decomposition of vital molecules, such as DNA, proteins, and lipids and cause a cascade of neurochemical events leading to neurodegeneration and cell death [5]. The metabolism of valproic acid may trigger oxygen-dependent tissue injury and elevate the free radicals in the body. Furthermore, valproic acid enhances the clearance of zinc, copper, and selenium, subsequently leading to decreased synthesis of free radical scavenging enzymes such as glutathione peroxidase and glutathione reductase activities [6]. More recently, valproic acid has been described as an HDAC inhibitor. Inhibition of HDAC promotes decondensed chromatin formation, thereby promoting the expression of genes. The corresponding proteins were described to play important roles in cellular activity and could influence several important pathways such as cell cycle control, differentiation, DNA repair, and apoptosis [7]. Antiepileptic drugs (AEDs) may interfere with homocysteine (which is an intermediate product in one-carbon metabolism), by affecting the levels of folate and vitamin B12 in patients with epilepsy. Folate is a mediator for the transfer of one-carbon units and is involved in DNA synthesis and methylation [8]. Valproic acid monotherapy may induce specific regions of DNA hypomethylation through their effects on one-carbon metabolism [9]. The role of carbamazepine in generating free radicals that cause neuronal damage is not so evident. Carbamazepine decreases the levels of antioxidant enzymes especially glutathione peroxidase and catalase in erythrocytes and decreases glutathione level which is a cofactor of several enzymes against oxidative stress [10]. On the other hand, valproic acid was shown to have a protective effect against oxidative stress in both, in-vitro and in-vivo models of epilepsy; it was suggested that valproic acid increase levels of glutathione. Thus, AEDs have been shown to contribute to both oxidant and antioxidant effects [11]. In the present study, we investigated the effect of valproic acid and carbamazepine monotherapy on DNA in epileptic children in relation to clinical and laboratory findings.

  Patients and Methods Top

This study was carried out in Menoufia University Hospital during the period from May 2015 to May 2017 on 40 (16 boys, 24 girls) epileptic children, their mean age was 7.2 ± 4.5 and 10 clinically healthy nonepileptic children were taken as a control group (five boys, five women), the mean age was 7.5 ± 2.8. The 40 epileptic patients were classified into three groups: the first group (15) was treated with valporic acid monotherapy for more than 1 year; the second group (15) was treated with carbamazepine monotherapy for more than 1 year, and the third group (10) was recently diagnosed with epilepsy and has not started any treatment yet. The diagnosis was based on history, clinical examination, and was confirmed by investigations (electroencephalogram and imaging of the brain).

We excluded from this study patients with thyroid diseases, other endocrinopathies, liver, heart, or kidney diseases and diseases that could influence the level of oxidative stress (such as diabetes mellitus and arterial hypertension).

All the studied groups were subjected to: detailed history taking with special emphasis on age of onset of seizures, duration of attacks, types of seizures, any precipitating factors such as fever. A thorough neurological examination (cranial nerves examinations, gait, muscle tone, muscle power, reflexes and sensory system affection with stress on mental status) was done. Laboratory investigations including routine complete blood cell count and liver enzyme levels were done. 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 using GeneJet Genomic DNA Purification Mini Kit (Life Technologies Ltd, 3 Fountain Drive, Inchinnan Business Park, Paisley PA4 9RF, UK), and then did gel electrophoresis. Gels were prepared with 1% electrophoresis-grade agarose; the agarose was boiled with Tris-borate-EDTA buffer; and then 0.5 μg/ml ethidium bromide was added to the agarose mixture at 40°C. Gel was poured and allowed to solidify at room temperature for 1 h before the samples were loaded. After 30 min, electrophoresis was performed for 1 h at 50 V using 1× TBE buffer as the running buffer. Gel was photographed using a Europe GmbH Heesenstrasse 31 D-40549 Düsseldorf Germany while the DNA was visualized using a 312 nm light under a transilluminator. These photographs were analyzed in the genetics lab at Menoufia University Hospital, to detect apoptotic activity by measuring maximal optical density of apoptotic fragments of DNA.

Data were collected, tabulated and statistically analyzed using a personal computer with statistical package of the social sciences (SPSS) version 20 (SPSS Inc., Chicago, Illinois, USA). χ2 was used to find the association between two or more qualitative variables. Fischer's exact test was used for 2 × 2 tables when an expected cell count of more than 25% of cases was less than 5. Student's t-test is a test of significance used for comparison between two groups having quantitative variables and with independent parametric data. Mann–Whitney U-test was used for the comparison between two groups having quantitative variables with independent nonparametric data. A P value of less than 0.05 was considered significant.

  Results Top

The results of this study are shown in [Table 1], [Table 2], [Table 3], [Table 4], [Table 5].
Table 1: Demographic data of the four studied groups

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Table 2: Laboratory findings of the four studied groups

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Table 3: DNA result in the four studied groups

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Table 4: Comparison between patients with DNA damage and patients with no DNA damage in carbamazepine-treated group regarding drug characters

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Table 5: Comparison between patients with DNA damage and patients with no DNA damage in the valproic acid-treated group regarding drug characters

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In terms of demographic data, there were no significant differences between patients groups and control group as regards age, sex, mode of delivery, and residence [Table 1]. In this study, laboratory investigations showed that leukocyte counts were significantly higher in untreated epileptic patients than in the controls (P = 0.003). While alanine transferase levels were significantly higher in patients treated with carbamazepine than control children, other groups showed no significant difference in leukocyte counts or levels of alanine transferase enzyme. There were no statistically significant differences in platelet counts or hemoglobin levels between all studied groups and control [Table 2]. As regards DNA changes, our results showed that there were significant differences between the studied groups in gel results, with significantly higher DNA damage in patients on carbamazepine monotherapy (60.0%) (P = 0.002) and also in patients on valproic acid monotherapy (33.3%) (P = 0.04). No differences were found in gel result between untreated patients and healthy controls [Table 3]. [Table 4] and [Table 5] showed no relationships were observed between gel results in patients on antiepileptic drugs and the duration of treatment, its dose, or serum level of the drug.

  Discussion Top

Epilepsy is the most common neurological condition affecting individuals of all ages. Carbamazepine and valproic acid are the most commonly prescribed antiepileptic drugs.

In this study, we focused on studying DNA damage in patients who received carbamazepine or valproic acid drugs as monotherapies.

In our study, there were no significant differences between the four studied groups as regards demographic data such as age, sex, residence, family history of convulsions, and parents' consanguinity. Our result showed that the most common type of seizure in epileptic children was a generalized tonic-clonic (67.5%). These findings are concordant with Khan et al. [12] who showed that 71.0% of patients with epilepsy had generalized tonic-clonic seizures.

Our laboratory investigations of the studied groups included a complete blood count and levels of alanine transferase enzyme, which showed a significant higher white blood counts in untreated children than healthy controls (P = 0.004). Shah et al. [13] explained that leukocytosis may be due to demargination of leukocytes secondary to muscular activity during a generalized seizure or due to release of catecholamine or cortisol during a seizure. There were significantly higher alanine transferase levels in children on CBZ than healthy control children (P = 0.000). Similarly, Hussein et al. [14] reported that CBZ was associated with mild elevations of liver enzymes, which occurred in up to 50% of patients and the hepatotoxicity of carbamazepine was independent of the daily dose of the drug. There were no statistically significant differences in platelet counts or hemoglobin levels between studied groups.

As regards DNA changes, our results showed that there were significant differences between the four studied groups regarding gel results, with positive results being higher in epileptic patients on carbamazepine monotherapy (60.0%) when compared with those on valproic acid monotherapy (33.3%), while untreated epileptic patients and healthy controls showed no DNA damage (0%).

This could be explained by the effects of antiepileptic drugs such as carbamazepine and valproic acid which may lead to oxidative stress by decreasing the levels of cellular glutathione, glutathione peroxidase, and superoxide dismutase, increasing the levels of free radicals causing lipid peroxidation and strand breaks in DNA [15]. Our study showed that no relation between changes was found in gel result with antiepileptic drugs (both valproic acid (VPA) and Carbamazepine (CBZ)) and between dose, serum level of drug, or the duration of treatment.

Similar to our study, Varoglu et al. [4] showed that oxidative stress was present with VPA and CBZ monotherapy and found that oxidative stress started 2 months after the initiation of treatment with each AED. In contrast to the present results, Alshafei et al. [16] showed that patients treated with VAP and CBZ had significant elevation of total antioxidant capacity, and glutathione S transferase and significant decrease in lipid peroxidase versus controls.

Our results agree with Dundaroz et al. [17] who found the occurrence of DNA damage in epileptic patients receiving carbamazepine monotherapy with a statistically higher significant result than those of the controls using the single-cell gel electrophoresis method and no relationship was observed between the frequency of DNA damaged cells and the dose of CBZ or the duration of treatment. Our results are in accordance with Denli et al. [18] who studied the genotoxic effect of VPA monotherapy using the single-gel electrophoresis method and suggested that VPA increases carcinogenesis risk with long-term use and showed significant higher damaged cells in treated epileptic. Our results also agree with Schulpis et al. [5] who suggested that patients on VPA therapy may be at risk of developing oxidative stress to DNA and stated that the induction of oxidative stress by VPA contributes to the mechanism of hepatotoxicity associated with VPA therapy.

  Conclusion Top

Prolonged therapy with carbamazepine and valproic acid causes significant oxidative stress, which leads to DNA damage as compared with controls or untreated patients. No statistically insignificant relations were found between the damaged effects of antiepileptic drugs on DNA and its dose, serum level of drug, or duration of treatment.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Fisher R, Acevedo C, Arzimanoglou A, Bogacz A, Cross JH, Elger CE, et al. A practical clinical definition of epilepsy. Epilepsia 2014; 55:475–482.  Back to cited text no. 1
Landmark JC, Larsson PG, Rytter E, Johannessen SI. Antiepileptic drugs in epilepsy and other disorders – a population-based study of prescriptions. Epilepsy Res 2009; 87:31–39.  Back to cited text no. 2
Ercegovac M, Jovic N, Simic T, Beslac-Bumbairevic L, Sokic D, Dukic T, et al. Byproducts of protein, lipid and DNA oxidative damage and antioxidant enzyme activities in seizure. Seizure 2010; 19:205–210.  Back to cited text no. 3
Varoglu AO, Yildirim A, Aygul R, Gundogdu OL, Sahin YN. Effects of valproate, carbamazepine and levetiracetam on the antioxidant and oxidant systems in epileptic patients and their clinical importance. Clin Neuropharmacol 2010; 33:155–157.  Back to cited text no. 4
Schulpis KH, Lazaropoulou C, Regoutas S, Karikas GA, Margeli A, Tsakiris S, et al. Valproic acid monotherapy induces DNA oxidative damage. Toxicology 2006; 217:228–232.  Back to cited text no. 5
Cengiz M, Yukcel A, Seven M. The effects of carbamazepine and valproic acid on the erythrocyte glutathione, glutathione peroxidase, superoxide dismutase and serum lipid peroxidation in epileptic children. Pharmacol Res 2000; 41:423–425.  Back to cited text no. 6
Chen Y, Pan RL, Zhang XL, Dong XJ, Zhang GR, et al. Induction of hepatic differentiation of mouse bone marrow stromal stem cells by the histone deacetylase inhibitor VPA. J Cell Mol Med 2009; 13:2582–2592.  Back to cited text no. 7
Belcastro V, Striano P, Gorgone G, Costa C, Ciampa C, et al. Hyperhomocysteinemia in epileptic patients on new antiepileptic drugs. Epilepsia 2010; 51:274–279.  Back to cited text no. 8
Ni G, Qin J, Li H, Chen Z, Zhou Y, et al. Effects of antiepileptic drug monotherapy on one-carbon metabolism and DNA methylation in patients with epilepsy. PLoS One 2015; 101–10.  Back to cited text no. 9
Alshafei MM, Seham S, Kassem SS, Abdel kader MM. Effect of long term treatment with antiepileptic drugs on oxidant status, zinc and magnesium in epileptic patients. World Appl Sci J 2013; 28:316–323.  Back to cited text no. 10
Cui J, Shao L, Young LT, Wang JF. Role of glutathione in neuro-protective effects of mood stabilizing drugs lithium and valproate. Neuroscience 2007; 144:1447–1453.  Back to cited text no. 11
Khan H, Mohamed A, Alsakini Z, Zulfiquar K, Sohail A, Shaikh R, et al. Consanguinity, family history and risk of epilepsy: a case–control study. Gulf Med J 2012; 1:32–36.  Back to cited text no. 12
Shah AK, Shein N, Fuerst D, Yangala R, Shah J, Watson C. Peripheral WBC count and serum prolactin level in various seizure types and nonepileptic events. Epilepsia 2001; 42:1472–1475.  Back to cited text no. 13
Hussein RR, Soliman RH, Abdelhaleem AM, Tawfeik MH, Abdelrahim ME. Effect of antiepileptic drugs on liver enzymes Beni-Suef University. J Basic Appl Sci 2013; 2:14–19.  Back to cited text no. 14
Sobaniec W, Solowiej E, Kulak W, Bockowski L, Smigielska-Kuzia J, Artemowicz B. Evaluation of the influence of antiepileptic therapy on antioxidant enzyme activity and lipid peroxidation in erythrocytes of children with epilepsy. J Child Neurol 2006; 21:558–562.  Back to cited text no. 15
Alshafei MM, Kassem SS, Abdel kader MM. Effect of long term treatment with antiepileptic drugs on oxidant status, zinc and magnesium in epileptic patients. World Appl Sci J 2013; 28:316–323.  Back to cited text no. 16
Dundaroz R, Aydin HI, Gungor T, Gok F, Denl M, Baltaci V. Assessment of dna damage induced by carbamazepine in epileptic Women. Med J Kocatepe 2001; 2:87–93.  Back to cited text no. 17
Denli M, Aydin HI, Dundaroz R, Ozisik T, Erdem E, Baltaci V. Genotoxicity evaluation in female patients on valproic acid monotherapy using alkaline single cell gel electrophoresis. East J Med 2000; 5:61–65.  Back to cited text no. 18


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


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