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Evaluation of DNA damage in leukocytes of G6PD-deficient Iranian newborns (Mediterranean variant) using comet assay
Mutation Research 568 (2004) 179–185
Evaluation of DNA damage in leukocytes of G6PD-deficient Iranian newborns (Mediterranean variant) using comet assay Seyed A. Mesbah-Namina,∗ , Alireza Nematia , Taki Tiraihib a
Department of Clinical Biochemistry, School of Medical Sciences, Tarbiat Modarres University, P.O. Box 14115-331, Tehran, Iran b Department of Anatomy, School of Medical Sciences, Tarbiat Modarres University, P.O. Box 14115-331, Tehran, Iran Received 5 May 2004; received in revised form 14 August 2004; accepted 18 August 2004 Available online 30 September 2004
Abstract Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited disease, which causes neonatal hemolytic anemia and jaundice. Recent studies of our group showed that the Mediterranean variant of this enzyme (Gd-Md) is the predominant G6PD in Iranian male infants suffering from jaundice; this variant is classified as severe G6PD deficiency. Considering the importance of G6PD reaction and its products NADPH and glutathione (GSH) against oxidative stress, we hypothesized the failure of detoxification of H2 O2 in G6PD-deficient white blood cells that could probably induce primary DNA damage. For the evaluation of DNA damage, we analyzed mononuclear leukocytes of 36 males suffering from the Gd-Md deficiency using alkaline single cell gel electrophoresis (SCGE) or comet assay. The level of DNA damage was compared with the level of basal DNA damage in control group represented by healthy male infant donors (of the same age group). Visual scoring was used for the evaluation of DNA damages. The results showed that the mean level of the DNA strand breakage in mononuclear leukocytes of 36 male G6PD-deficient (Gd-Md) infants was significantly higher (P < 0.001) than those observed in the normal lymphocytes. In conclusion, this investigation indicates that the mononuclear leukocytes of the Gd-Md samples may be exposed to DNA damage due to oxidative stress. This is the first report using comet assay for evaluation of DNA damage in severe G6PD deficiency samples. © 2004 Elsevier B.V. All rights reserved. Keywords: G6PD deficiency; Iranian infants; G6PD Mediterranean; DNA damage; Comet assay
1. Introduction
∗ Corresponding author. Tel.: +98 21 8011001; fax: +98 21 8006544. E-mail addresses: [email protected], [email protected] (S.A. Mesbah-Namin).
0027-5107/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2004.08.010
Glucose-6-phosphate dehydrogenase (G6PD) is a cytosolic enzyme present in all cells. An important function of G6PD is to produce NADPH required for the reductive biosynthetic reactions as well as for the stability of catalase, and the preservation of
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the reduced form of glutathione (GSH). Catalase and GSH are essential for the detoxification of H2 O2 . The defense of cells against this compound depends on G6PD activity. This is especially true in red blood cells (RBCs), which are sensitive to oxidative damage and in which, other NADPH-producing enzymes are lacking [1,2]. In humans, G6PD deficiency is the most common enzymopathy affecting over 400 million people throughout the world. The most common clinical manifestations are neonatal jaundice and acute hemolytic anemia,which can be triggered by a number of drugs, infection or the ingestion of fava beans. G6PD deficiency is genetically heterogenous and has proved to be an X-linked trait. Over 400 genetically determined variants of the G6PD have been reported but actually only 176 of them are recognized from which, 122 variants are mutants [3,4]. The Mediterranean variant of the G6PD (Gd-Md, 563 C > T) is one of the most common G6PD deficiency observed in several Middle-East countries such as Iran [5,6]. This variant is characterized by possessing less than 10% of normal enzyme activity, which makes it one of the most severe forms of G6PD deficiency. G6PD deficiency is also characterized by increased susceptibility of erythrocytes to H2 O2 , a well-known oxidant. Moreover, H2 O2 can readily transverse lipid bilayers to sites that contain reactive substrates, such as nucleus and react with ions of iron to form highly reactive hydroxyl radicals. This kind of radical could be able to attack and damage DNA [7]. For evaluating possible DNA damage in leukocytes of male G6PD-deficient neonates, we investigated the probable induction of DNA strand breaks in the alkaline single cell gel electrophoresis (SCGE) or comet assay. Comet assay is the potential tool to estimate DNA damage at the single-cell level and it provides information on the presence of DNA damage among individual cells [8]. This is the first report using the comet assay for evaluating DNA damage in G6PD-deficient subjects.
2. Materials and methods 2.1. Patients and blood sampling Blood samples were obtained from 36 unrelated male newborns (15–20 days old) diagnosed G6PD-deficient, suffering from jaundice and hyper-
bilirubinemia (total bilirubin was more than 18 mg/dl), and 10 male newborns of the same age without G6PD deficiency or any other disease (as the control samples) were referred to the pediatric service of medical center, Mofid hospital, Ali-Asghar hospital and Shariyat Razavi hospital in Tehran. Only 1 ml of whole blood was necessary to be used for this purpose. All parents had given their informed consent to participate in this study. All of 36 male infants had severe G6PD deficiency (less than 10% G6PD activity) and were diagnosed as G6PD-Mediterranean (Gd-Md) using PCR-RFLP analysis as described elsewhere [9]. Briefly, genomic DNA was extracted from whole blood using standard procedure. A 583 bp fragment encompassing exons 6 and 7 was amplified from genomic DNA by PCR with primers 91 and 92. The PCR product was digested by Mbo II at 37 ◦ C for 4 h; digested products were separated on 2.5 Nusieve agarose, stained with ethidium bromide, lighted with UV light and photographed. After Mbo II digestion of PCR-amplified fragment of the normal samples, three bands of 379, 120 and 60 bp were obtained (Fig. 1, lanes 9 and 10). In contrast, Mbo II digestion of the fragment obtained from the infants carrying Gd-Md mutation were 276, 120, 103 and 60 bp fragments (Fig. 1, lanes 15–19). 2.2. Alkaline comet assay The protocol of the comet assay was based on the procedure described previously [10] with slight modifications as presented by Collins [11]. All stages of experiments have been done at 4 ◦ C. Volume of 30 l of whole blood cells of G6PD-deficient newborns was mixed with 1 ml of phosphate-buffered saline (PBS) and left on ice for 30 min. Volume of 100 l of ficoll hypaque solution was added carefully below the mixture. Following a centrifugation (eppendorf centrifuge, Model 5810) for a period of 3 min at 650 × g, 100 l of lymphocyte layer was then withdrawn from the boundary between PBS and ficoll solution. Lymphocytes were then resuspended in 1 ml of PBS and centrifuged again at 1250 × g. The supernatant was then removed using pipette pasture. The remaining lymphocytes were used for DNA extraction and subsequently, for detecting the Gd-Md variant by standard method discussed in the following section. The lymphocytes were then dispersed in a small volume of remaining supernatant by tapping microtubes; this
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Fig. 1. Gel electrophoresis pattern showing DNA fragments for the Mediterranean mutation using Mbo II digestion of the PCR fragments. It shows that 11 samples have the Mediterranean mutation on the G6PD gene. Lane 1, uncut PCR sample; lanes 2 and 11, 50 bp DNA ladder marker; lane 10, Gd-Md positive control.
was used for the assessment of the cell viability and the comet assay. Cell viability was measured by the trypan blue exclusion assay [12] at the beginning of the comet assay for the two groups of samples: G6PDdeficient and normal lymphocytes. The cell viability was more than 90% for each experiment throughout this study. For the comet assay, the cells were quickly added to 140 l of 1% low-melting agarose (MBI fermentas, Iranian distributor) in PBS at 37 ◦ C and mixed by tapping the tubes. The cell suspensions (70 l) were placed on specially prepared frosted slides (made in our university, which contained one or two unfrosted rectangular spaces on the frosted surface; one slide is suitable for only one layer of agarose gel to perform comet assay). Coverslips (18 m × 18 m) were placed on the unfrosted windows of slides to allow uniform spreading. Agarose gel formation is performed at a cold temperature of 4 ◦ C for 5 min. After the removal of the coverslips, the slides were dipped into the lysing solution (2.5 M NaCl, 0.1 M Na2 EDTA, 10 mM Tris–HCl; pH 10, 1% Triton X-100) for 40 min. The slides were then placed on a horizontal electrophoresis unit. The reservoir was filled with freshly prepared buffer (1 mM Na2 EDTA, 0.3 NaOH, pH > 13). DNA unwinding was allowed to proceed for 40 min. SCGE was performed for 30 min using a field strength of 0.7 V/cm (300 mA; 25 V), then the slides were rinsed three times (each time
for 5 min) with neutralizing buffer (0.4 M Tris–HCl, pH 7.5). All steps from the lysis until the end of neutralization were performed under yellow light or in the dark. The dried microscope slides were stained with ethidium bromide (20 g/ml; 20 l/slide) and examined for scoring and analysis at ×400 magnification in a fluorescent microscope (Zeiss, Axiophot, Germany) equipped with excitation filter 516–546 nm; barrier filter 590 nm and plan-Neoflour UV objective. 2.3. Image analysis and scoring Visual scoring of the cellular DNA on each slide was based on characterization of 100 randomly selected nucleoids. Two independent observers evaluated the extent of DNA damage. The comet-like DNA formations were visually graded into four classes depending on DNA damage level: undamaged (class 1), slightly damaged (class 2), damaged (class 3), and highly damaged (class 4) (Figs. 2 and 3). The classification was carried out on the basis of an appearance of comets (i.e. tail length, head diameter and intensity) as previously described [13]. The objects with bright heads and no apparent tails were assigned to class 1 (Fig. 2) and comets with little heads and long tails fell in the class 4 (Fig. 3). Each comet was assigned a value according to its class and the overall score for 100 comets ranged
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Fig. 2. A typical photographic comet image of a normal lymphocyte with bright head of nucleoid, undamaged DNA, represented by class 1 of comet assay.
from 100 (100% of comets being in class 1) to 400 (100% of comets in class 4). The number of comets in each class was counted and an average DNA damage (DD) was calculated as follows: DD = (n1 + 2n2 + 3n3 + 4n4 )/(/100), n1 –n4 = number of comets in class 1–4 and = sum of all counted comets. The DD values were expressed in arbitrary units (au) [14]. In this experiment, one hundred of comets counted on each of slide and the average DD could be therefore calculated just as follows: DD = (n1 + 2n2 + 3n3 + 4n4 ).
Fig. 4. The percentage of comet classes of DNA damage distributed in two groups of samples. () Control samples with normal activity of G6PD, most of cells (92.4%) are considered in the class 1 of comet assay or undamaged state. () Patients, most of cells are damaged and distributed in all of comet classes (21.5% ± 1.45 S.E.M. for class 1, .42.03% ± 1.98 S.E.M. for class 2, 33.7% ± 2.05% S.E.M. for class 3 and 2.75% ± 0.8 S.E.M. for class 4).
all possible differences between the DD values in the study groups.
2.4. Statistical analysis 3. Results For each slide, distributions of cell damage population of 100 randomly selected cells in two groups of this study (normal and Gd-Md samples) were analyzed by parametric and non-parametric tests. We used the χ2 -test for comparing the distribution of DNA damage in all the samples. The non-parametric statistical methods (Mann–Whitney tests) were also used for verifying
3.1. The comet assay Before reporting the results obtained in the comet assay, it must be pointed out that the viability of the cells, normal infant lymphocytes (control) and the lymphocytes of the Gd-Md samples, were more
Fig. 3. Typical digitized comet images of various types of DNA damage (in four classes) of Gd-Md lymphocytes.
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G6PD deficiency, encouraged us to investigate the use of the comet assay for evaluation of presence of DNA damage in these patients. 4.2. DNA damage in the G6PD-deficient cells
Fig. 5. The Average distribution of DNA damage expressed by arbitrary unit (100–400 for class 1–4 in 100 cells scored) among two groups of samples. () C, control samples with normal G6PD activity with 108.3 ± 1.9 S.E.M. au score indicated ‘undamaged DNA’. () Gd-Md samples with 217.72 ± 4.03 S.E.M. au score indicated ‘damaged DNA’.
than 90%.Typical comet images of the migration of lymphocytes of normal and patient samples in different classes are shown in Figs. 2 and 3, respectively. Fig. 4 shows and summarizes the data results of the comet assays obtained from two groups of samples. As this figure indicates, the normal cells were mostly undamaged (92.4% for class 1) when used as a control, while the lymphocytes of patients with G6PD deficiency (Gd-Md) were mostly damaged cells (42.03% for class 2 and 33.72% for class 3). Fig. 5 shows the distribution of DNA damage classes by DD values among two groups of samples.
4. Discussion 4.1. G6PD Mediterranean (Gd-Md) Gd-Md is polymorphic in all countries surrounding the Mediterranean sea including north African countries, but it is also widespread in the Middle-East including Arab countries and Iran. In our pervious study on the molecular characterization of G6PD deficiency in northern Iran [6], the prevalence of Gd-Md showed to be 66.7% (49/74). In the second study done by another group in southern Iran [5], it showed to be at an even higher rate, 83.8% (62/74). The high incidence of Gd-Md in Iran, which is the most severe variant of
Although two studies relating to DNA damage in G6PD-deficient cells have been previously published, this is the first report in which the damage is investigated in Gd-Md patients and the comet assay is used. Tian et al. [15] examined the role of G6PD in the regulation of cell death using two cell lines (PC12 cells and BALB/c cells). The cells were incubated with 50 M or 100 M H2 O2 in the presence and absence of known inhibitors of G6PD. They found that alterations in G6PD activity can significantly affect oxidative stress-induced cell death and apoptosis. The authors concluded that G6PD plays an important role in cell death by regulating intracellular redox status. Efferth et al. [16] investigated DNA damage and apoptosis in mononuclear cells (MNCs) of G6PD-deficient individuals after exposure of the cells to UV irradiation. Seven of the individuals, three males and four females, were Germans suffering from the rare G6PD Aachen variant and one was an Iranian whose G6PD variant was not reported. The inhibition of PCR amplification technique was used to measure DNA damage induced by UV. The authors found minimal–intermediate rates of DNA damage and apoptosis in MNCs of females and increased DNA damage and apoptosis in MNCs of male G6PD Aachen and Iranian male G6PD-deficient individuals. They concluded that increased DNA damage may occur as a result of deficient detoxification of reactive oxygen species (ROS) by GSH, and this may ultimately account for the higher rate of apoptosis in G6PD-deficient MNCs. Although the hemoglobin damage in erythrocytes of G6PD-deficient subjects is well known, damage to nucleic acids in their nucleated cells is poorly understood. Erythrocytes of newborns consume more O2 and produce more H2 O2 than erythrocytes of adults [17]. Detoxification of H2 O2 depends on availability of NADPH and the presence of GSH. Maintenance of GSH in a reduced state has been thought to be the most important function of G6PD [18]. Accordingly, reduced capacity of detoxification of H2 O2 by GSH in G6PD-deficient neonates would render their DNA more vulnerable to oxidative damage. The aim of the present study was to investigate
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DNA damage in the peripheral lymphocytes of Gd-Md Iranian male infants using the alkaline comet assay. This assay is suitable for evaluation of single strand DNA breakages. For this purpose, samples were examined as described in Section 2.2. Visual scoring of one hundred cells under a fluorescent microscope was used for evaluation of damages. Visual scoring was time-efficient and its sensitivity was equal to that of computerized image analysis [19]. We were able to obtain only 10 blood samples from the normal infants. The results of the comet assay showed a highly significant increase in DNA damage in Gd-Md patients as compared to normal controls (P < 0.001). The comet assay revealed that class 2 and class 3 of DNA damage are predominant in the Gd-Md samples (Fig. 4). The result of the DD distribution for the patients and control samples also confirmed the significantly different values between the two groups, 108.3 and 217.72 arbitrary units for normal and patient samples, respectively (Fig 5). In conclusion, the results of this study suggest that increased DNA damages in the lymphocytes of the Gd-Md newborns may occur because of reduction in GSH production. To ascertain that the observed increase in DNA strand breakage is in fact due to decreased GSH production resulting from G6PD deficiency rather than other features associated with jaundice, it would be best to investigate degree of damage in newborns with neonatal jaundice who do not have a deficiency in G6PD. It would also be useful to examine newborns with G6PD deficiency who do not have jaundice, if one can be found. This is the first report of an attempt to use comet assay for evaluating DNA damage in the severe G6PD deficiency samples. Clinical consequences of this DNA damage in the Gd-Md patients need further investigations.
Acknowledgements We would like to thank Dr. E. Hajizadeh for helping in the statistical calculations and Dr. G. Sarrafi, Prof. E. Elahi and Prof, B. Farzami for editing the MS.
References [1] P.J. Mason, T.J. Vulliamy, Genetic Variation of Human Erythrocyte G6PD, Imperial College Press, London, 2000.
[2] L. Luzzatto, A. Mehta, Glucose-6-phosphate dehydrogenase deficiency, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic Basis of Inherited Disease, McGraw-Hill, London, 1995, pp. 3367–3398. [3] A. Mehta, P.J. Mason, T.J. Vulliamy, Glucose-6-phosphate dehydrogenase deficiency, Baillieres Clin. Haematol. 13 (2000) 21–38. [4] E. Beutler, T.J. Vulliamy, Hematologically important mutations, Glucose-6-phosphate dehydrogenase, Blood Cells Mol. Dis. 28 (2002) 93–103. [5] M. Karimi, F.M. di Montemuros, M.G. Danielli, S. Farjadian, A. Afrasiabi, G. Fiorelli, M.D. Cappellini, Molecular characterization of glucose-6-phosphate dehydrogenase deficiency in the Fars province of Iran, Haematologica 88 (2003) 346– 347. [6] S.A. Mesbah-Namin, M.H. Sanati, A. Mowjoodi, P.J. Mason, T.J. Vulliamy, M.R. Noori-Dalooii, Three major G6PDdeficient polymorphic variants identified in Mazandaran state of Iran, Br. J. Hematol. 177 (2002) 763–764. [7] M. Kruszewski, M.H. Green, J.E. Lowe, I. Szumiel, DNA strand breakage, cytotoxicity and mutagenicity of H2 O2 treatment at 4 ◦ C and 37◦ C in L5178Y sublines, Mutat. Res. 308 (1994) 233–241. [8] A.R. Collins, V.L. Dobson, M. Dusinska, G. Kennedy, R. Stetina, The comet assay: what can it really tell us? Mutat. Res. 375 (1997) 183–193. [9] V. Poggi, M. Town, N.S. Foulkes, L. Luzzatto, Identification of a single base change in a new human mutant glucose-6phosphate dehydrogenase gene by polymerase chain reaction amplification of the entire coding region from genomic DNA, Biochem. J. 271 (1990) 157–160. [10] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1998) 184–191. [11] A.R. Collins, Measurement of oxidative DNA damage using the comet assay, in: J. Lunec, H.R. Griffiths (Eds.), Measuring In Vivo Oxidative Damage: A Practical Approach, John Wiley and Sons, 2000, pp. 83–94. [12] A. Boyem, Separation of leukocytes from blood and bone marrow, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97) (1968) 77–89. [13] P. Lebailly, C. Vigreux, T. Godard, F. Sichel, E. Bar, J.Y. LeTalaer, M. Henry-Amar, P. Gauduchon, Assessment of DNA damage induced in vitro by etoposide and two fungicides (carbendazim and chlorothalonil) in human lymphocytes with the comet assay, Mutat. Res. 375 (1997) 205–217. [14] A.R. Collins, M. Ai-guo, S.G. Duthie, The kinetics of repair of oxidative DNA damage (strand breaks and oxidized pyrimidines) in human cells, Mutat. Res. 336 (1995) 69–77. [15] W.Ni. Tian, L.D. Braunstein, K. Apse, J. Pang, M. Rose, X. Tain, R.C. Stanton, Importance of Glucose-6-phosphate dehydrogenase activity in cell death, Am. J. Physiol. 276 (1999) C1121–C1131. [16] T. Efferth, U. Fabry, R. Osieka, DNA damage and apoptosis in mononuclear cells from G6PD-deficient patients (G6PD Aachen variant) after UV irradiation, J. Leukoc. Biol. 69 (2001) 340–342.
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[19] H. Kobayashi, C. Sugiyama, Y. Morikawa, M. Hayashi, T. Sofuni, A comparison between manual microscopic analysis and computerized image analysis in the single cell gel electrophoresis assay, MMS Commun. 3 (1995) 103– 111.
Evaluation of DNA damage in leukocytes of G6PD-deficient Iranian newborns (Mediterranean variant) using comet assay Seyed A. Mesbah-Namina,∗ , Alireza Nematia , Taki Tiraihib a
Department of Clinical Biochemistry, School of Medical Sciences, Tarbiat Modarres University, P.O. Box 14115-331, Tehran, Iran b Department of Anatomy, School of Medical Sciences, Tarbiat Modarres University, P.O. Box 14115-331, Tehran, Iran Received 5 May 2004; received in revised form 14 August 2004; accepted 18 August 2004 Available online 30 September 2004
Abstract Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited disease, which causes neonatal hemolytic anemia and jaundice. Recent studies of our group showed that the Mediterranean variant of this enzyme (Gd-Md) is the predominant G6PD in Iranian male infants suffering from jaundice; this variant is classified as severe G6PD deficiency. Considering the importance of G6PD reaction and its products NADPH and glutathione (GSH) against oxidative stress, we hypothesized the failure of detoxification of H2 O2 in G6PD-deficient white blood cells that could probably induce primary DNA damage. For the evaluation of DNA damage, we analyzed mononuclear leukocytes of 36 males suffering from the Gd-Md deficiency using alkaline single cell gel electrophoresis (SCGE) or comet assay. The level of DNA damage was compared with the level of basal DNA damage in control group represented by healthy male infant donors (of the same age group). Visual scoring was used for the evaluation of DNA damages. The results showed that the mean level of the DNA strand breakage in mononuclear leukocytes of 36 male G6PD-deficient (Gd-Md) infants was significantly higher (P < 0.001) than those observed in the normal lymphocytes. In conclusion, this investigation indicates that the mononuclear leukocytes of the Gd-Md samples may be exposed to DNA damage due to oxidative stress. This is the first report using comet assay for evaluation of DNA damage in severe G6PD deficiency samples. © 2004 Elsevier B.V. All rights reserved. Keywords: G6PD deficiency; Iranian infants; G6PD Mediterranean; DNA damage; Comet assay
1. Introduction
∗ Corresponding author. Tel.: +98 21 8011001; fax: +98 21 8006544. E-mail addresses: [email protected], [email protected] (S.A. Mesbah-Namin).
0027-5107/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2004.08.010
Glucose-6-phosphate dehydrogenase (G6PD) is a cytosolic enzyme present in all cells. An important function of G6PD is to produce NADPH required for the reductive biosynthetic reactions as well as for the stability of catalase, and the preservation of
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the reduced form of glutathione (GSH). Catalase and GSH are essential for the detoxification of H2 O2 . The defense of cells against this compound depends on G6PD activity. This is especially true in red blood cells (RBCs), which are sensitive to oxidative damage and in which, other NADPH-producing enzymes are lacking [1,2]. In humans, G6PD deficiency is the most common enzymopathy affecting over 400 million people throughout the world. The most common clinical manifestations are neonatal jaundice and acute hemolytic anemia,which can be triggered by a number of drugs, infection or the ingestion of fava beans. G6PD deficiency is genetically heterogenous and has proved to be an X-linked trait. Over 400 genetically determined variants of the G6PD have been reported but actually only 176 of them are recognized from which, 122 variants are mutants [3,4]. The Mediterranean variant of the G6PD (Gd-Md, 563 C > T) is one of the most common G6PD deficiency observed in several Middle-East countries such as Iran [5,6]. This variant is characterized by possessing less than 10% of normal enzyme activity, which makes it one of the most severe forms of G6PD deficiency. G6PD deficiency is also characterized by increased susceptibility of erythrocytes to H2 O2 , a well-known oxidant. Moreover, H2 O2 can readily transverse lipid bilayers to sites that contain reactive substrates, such as nucleus and react with ions of iron to form highly reactive hydroxyl radicals. This kind of radical could be able to attack and damage DNA [7]. For evaluating possible DNA damage in leukocytes of male G6PD-deficient neonates, we investigated the probable induction of DNA strand breaks in the alkaline single cell gel electrophoresis (SCGE) or comet assay. Comet assay is the potential tool to estimate DNA damage at the single-cell level and it provides information on the presence of DNA damage among individual cells [8]. This is the first report using the comet assay for evaluating DNA damage in G6PD-deficient subjects.
2. Materials and methods 2.1. Patients and blood sampling Blood samples were obtained from 36 unrelated male newborns (15–20 days old) diagnosed G6PD-deficient, suffering from jaundice and hyper-
bilirubinemia (total bilirubin was more than 18 mg/dl), and 10 male newborns of the same age without G6PD deficiency or any other disease (as the control samples) were referred to the pediatric service of medical center, Mofid hospital, Ali-Asghar hospital and Shariyat Razavi hospital in Tehran. Only 1 ml of whole blood was necessary to be used for this purpose. All parents had given their informed consent to participate in this study. All of 36 male infants had severe G6PD deficiency (less than 10% G6PD activity) and were diagnosed as G6PD-Mediterranean (Gd-Md) using PCR-RFLP analysis as described elsewhere [9]. Briefly, genomic DNA was extracted from whole blood using standard procedure. A 583 bp fragment encompassing exons 6 and 7 was amplified from genomic DNA by PCR with primers 91 and 92. The PCR product was digested by Mbo II at 37 ◦ C for 4 h; digested products were separated on 2.5 Nusieve agarose, stained with ethidium bromide, lighted with UV light and photographed. After Mbo II digestion of PCR-amplified fragment of the normal samples, three bands of 379, 120 and 60 bp were obtained (Fig. 1, lanes 9 and 10). In contrast, Mbo II digestion of the fragment obtained from the infants carrying Gd-Md mutation were 276, 120, 103 and 60 bp fragments (Fig. 1, lanes 15–19). 2.2. Alkaline comet assay The protocol of the comet assay was based on the procedure described previously [10] with slight modifications as presented by Collins [11]. All stages of experiments have been done at 4 ◦ C. Volume of 30 l of whole blood cells of G6PD-deficient newborns was mixed with 1 ml of phosphate-buffered saline (PBS) and left on ice for 30 min. Volume of 100 l of ficoll hypaque solution was added carefully below the mixture. Following a centrifugation (eppendorf centrifuge, Model 5810) for a period of 3 min at 650 × g, 100 l of lymphocyte layer was then withdrawn from the boundary between PBS and ficoll solution. Lymphocytes were then resuspended in 1 ml of PBS and centrifuged again at 1250 × g. The supernatant was then removed using pipette pasture. The remaining lymphocytes were used for DNA extraction and subsequently, for detecting the Gd-Md variant by standard method discussed in the following section. The lymphocytes were then dispersed in a small volume of remaining supernatant by tapping microtubes; this
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Fig. 1. Gel electrophoresis pattern showing DNA fragments for the Mediterranean mutation using Mbo II digestion of the PCR fragments. It shows that 11 samples have the Mediterranean mutation on the G6PD gene. Lane 1, uncut PCR sample; lanes 2 and 11, 50 bp DNA ladder marker; lane 10, Gd-Md positive control.
was used for the assessment of the cell viability and the comet assay. Cell viability was measured by the trypan blue exclusion assay [12] at the beginning of the comet assay for the two groups of samples: G6PDdeficient and normal lymphocytes. The cell viability was more than 90% for each experiment throughout this study. For the comet assay, the cells were quickly added to 140 l of 1% low-melting agarose (MBI fermentas, Iranian distributor) in PBS at 37 ◦ C and mixed by tapping the tubes. The cell suspensions (70 l) were placed on specially prepared frosted slides (made in our university, which contained one or two unfrosted rectangular spaces on the frosted surface; one slide is suitable for only one layer of agarose gel to perform comet assay). Coverslips (18 m × 18 m) were placed on the unfrosted windows of slides to allow uniform spreading. Agarose gel formation is performed at a cold temperature of 4 ◦ C for 5 min. After the removal of the coverslips, the slides were dipped into the lysing solution (2.5 M NaCl, 0.1 M Na2 EDTA, 10 mM Tris–HCl; pH 10, 1% Triton X-100) for 40 min. The slides were then placed on a horizontal electrophoresis unit. The reservoir was filled with freshly prepared buffer (1 mM Na2 EDTA, 0.3 NaOH, pH > 13). DNA unwinding was allowed to proceed for 40 min. SCGE was performed for 30 min using a field strength of 0.7 V/cm (300 mA; 25 V), then the slides were rinsed three times (each time
for 5 min) with neutralizing buffer (0.4 M Tris–HCl, pH 7.5). All steps from the lysis until the end of neutralization were performed under yellow light or in the dark. The dried microscope slides were stained with ethidium bromide (20 g/ml; 20 l/slide) and examined for scoring and analysis at ×400 magnification in a fluorescent microscope (Zeiss, Axiophot, Germany) equipped with excitation filter 516–546 nm; barrier filter 590 nm and plan-Neoflour UV objective. 2.3. Image analysis and scoring Visual scoring of the cellular DNA on each slide was based on characterization of 100 randomly selected nucleoids. Two independent observers evaluated the extent of DNA damage. The comet-like DNA formations were visually graded into four classes depending on DNA damage level: undamaged (class 1), slightly damaged (class 2), damaged (class 3), and highly damaged (class 4) (Figs. 2 and 3). The classification was carried out on the basis of an appearance of comets (i.e. tail length, head diameter and intensity) as previously described [13]. The objects with bright heads and no apparent tails were assigned to class 1 (Fig. 2) and comets with little heads and long tails fell in the class 4 (Fig. 3). Each comet was assigned a value according to its class and the overall score for 100 comets ranged
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Fig. 2. A typical photographic comet image of a normal lymphocyte with bright head of nucleoid, undamaged DNA, represented by class 1 of comet assay.
from 100 (100% of comets being in class 1) to 400 (100% of comets in class 4). The number of comets in each class was counted and an average DNA damage (DD) was calculated as follows: DD = (n1 + 2n2 + 3n3 + 4n4 )/(/100), n1 –n4 = number of comets in class 1–4 and = sum of all counted comets. The DD values were expressed in arbitrary units (au) [14]. In this experiment, one hundred of comets counted on each of slide and the average DD could be therefore calculated just as follows: DD = (n1 + 2n2 + 3n3 + 4n4 ).
Fig. 4. The percentage of comet classes of DNA damage distributed in two groups of samples. () Control samples with normal activity of G6PD, most of cells (92.4%) are considered in the class 1 of comet assay or undamaged state. () Patients, most of cells are damaged and distributed in all of comet classes (21.5% ± 1.45 S.E.M. for class 1, .42.03% ± 1.98 S.E.M. for class 2, 33.7% ± 2.05% S.E.M. for class 3 and 2.75% ± 0.8 S.E.M. for class 4).
all possible differences between the DD values in the study groups.
2.4. Statistical analysis 3. Results For each slide, distributions of cell damage population of 100 randomly selected cells in two groups of this study (normal and Gd-Md samples) were analyzed by parametric and non-parametric tests. We used the χ2 -test for comparing the distribution of DNA damage in all the samples. The non-parametric statistical methods (Mann–Whitney tests) were also used for verifying
3.1. The comet assay Before reporting the results obtained in the comet assay, it must be pointed out that the viability of the cells, normal infant lymphocytes (control) and the lymphocytes of the Gd-Md samples, were more
Fig. 3. Typical digitized comet images of various types of DNA damage (in four classes) of Gd-Md lymphocytes.
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G6PD deficiency, encouraged us to investigate the use of the comet assay for evaluation of presence of DNA damage in these patients. 4.2. DNA damage in the G6PD-deficient cells
Fig. 5. The Average distribution of DNA damage expressed by arbitrary unit (100–400 for class 1–4 in 100 cells scored) among two groups of samples. () C, control samples with normal G6PD activity with 108.3 ± 1.9 S.E.M. au score indicated ‘undamaged DNA’. () Gd-Md samples with 217.72 ± 4.03 S.E.M. au score indicated ‘damaged DNA’.
than 90%.Typical comet images of the migration of lymphocytes of normal and patient samples in different classes are shown in Figs. 2 and 3, respectively. Fig. 4 shows and summarizes the data results of the comet assays obtained from two groups of samples. As this figure indicates, the normal cells were mostly undamaged (92.4% for class 1) when used as a control, while the lymphocytes of patients with G6PD deficiency (Gd-Md) were mostly damaged cells (42.03% for class 2 and 33.72% for class 3). Fig. 5 shows the distribution of DNA damage classes by DD values among two groups of samples.
4. Discussion 4.1. G6PD Mediterranean (Gd-Md) Gd-Md is polymorphic in all countries surrounding the Mediterranean sea including north African countries, but it is also widespread in the Middle-East including Arab countries and Iran. In our pervious study on the molecular characterization of G6PD deficiency in northern Iran [6], the prevalence of Gd-Md showed to be 66.7% (49/74). In the second study done by another group in southern Iran [5], it showed to be at an even higher rate, 83.8% (62/74). The high incidence of Gd-Md in Iran, which is the most severe variant of
Although two studies relating to DNA damage in G6PD-deficient cells have been previously published, this is the first report in which the damage is investigated in Gd-Md patients and the comet assay is used. Tian et al. [15] examined the role of G6PD in the regulation of cell death using two cell lines (PC12 cells and BALB/c cells). The cells were incubated with 50 M or 100 M H2 O2 in the presence and absence of known inhibitors of G6PD. They found that alterations in G6PD activity can significantly affect oxidative stress-induced cell death and apoptosis. The authors concluded that G6PD plays an important role in cell death by regulating intracellular redox status. Efferth et al. [16] investigated DNA damage and apoptosis in mononuclear cells (MNCs) of G6PD-deficient individuals after exposure of the cells to UV irradiation. Seven of the individuals, three males and four females, were Germans suffering from the rare G6PD Aachen variant and one was an Iranian whose G6PD variant was not reported. The inhibition of PCR amplification technique was used to measure DNA damage induced by UV. The authors found minimal–intermediate rates of DNA damage and apoptosis in MNCs of females and increased DNA damage and apoptosis in MNCs of male G6PD Aachen and Iranian male G6PD-deficient individuals. They concluded that increased DNA damage may occur as a result of deficient detoxification of reactive oxygen species (ROS) by GSH, and this may ultimately account for the higher rate of apoptosis in G6PD-deficient MNCs. Although the hemoglobin damage in erythrocytes of G6PD-deficient subjects is well known, damage to nucleic acids in their nucleated cells is poorly understood. Erythrocytes of newborns consume more O2 and produce more H2 O2 than erythrocytes of adults [17]. Detoxification of H2 O2 depends on availability of NADPH and the presence of GSH. Maintenance of GSH in a reduced state has been thought to be the most important function of G6PD [18]. Accordingly, reduced capacity of detoxification of H2 O2 by GSH in G6PD-deficient neonates would render their DNA more vulnerable to oxidative damage. The aim of the present study was to investigate
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DNA damage in the peripheral lymphocytes of Gd-Md Iranian male infants using the alkaline comet assay. This assay is suitable for evaluation of single strand DNA breakages. For this purpose, samples were examined as described in Section 2.2. Visual scoring of one hundred cells under a fluorescent microscope was used for evaluation of damages. Visual scoring was time-efficient and its sensitivity was equal to that of computerized image analysis [19]. We were able to obtain only 10 blood samples from the normal infants. The results of the comet assay showed a highly significant increase in DNA damage in Gd-Md patients as compared to normal controls (P < 0.001). The comet assay revealed that class 2 and class 3 of DNA damage are predominant in the Gd-Md samples (Fig. 4). The result of the DD distribution for the patients and control samples also confirmed the significantly different values between the two groups, 108.3 and 217.72 arbitrary units for normal and patient samples, respectively (Fig 5). In conclusion, the results of this study suggest that increased DNA damages in the lymphocytes of the Gd-Md newborns may occur because of reduction in GSH production. To ascertain that the observed increase in DNA strand breakage is in fact due to decreased GSH production resulting from G6PD deficiency rather than other features associated with jaundice, it would be best to investigate degree of damage in newborns with neonatal jaundice who do not have a deficiency in G6PD. It would also be useful to examine newborns with G6PD deficiency who do not have jaundice, if one can be found. This is the first report of an attempt to use comet assay for evaluating DNA damage in the severe G6PD deficiency samples. Clinical consequences of this DNA damage in the Gd-Md patients need further investigations.
Acknowledgements We would like to thank Dr. E. Hajizadeh for helping in the statistical calculations and Dr. G. Sarrafi, Prof. E. Elahi and Prof, B. Farzami for editing the MS.
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