http://informahealthcare.com/hem ISSN: 0363-0269 (print), 1532-432X (electronic) Hemoglobin, 2014; 38(5): 359–364 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03630269.2014.951890
ORIGINAL ARTICLE
N-Acetylcysteine Supplementation Reduces Oxidative Stress and DNA Damage in Children with b-Thalassemia Zeynep Canan Ozdemir1, Ahmet Koc2, Ali Aycicek3, and Abdurrahim Kocyigit4 Hemoglobin Downloaded from informahealthcare.com by Memorial University of Newfoundland on 10/04/14 For personal use only.
1
Pediatric Hematology Department, Harran University, Medical Faculty, Sanlıurfa, Turkey, Pediatric Hematology/Oncology Department, Marmara University, Medical Faculty, Istanbul, Turkey, 3 Pediatric Hematology/Oncology Clinic, Eskisehir State Hospital, Eskisehir, Turkey, and 4 Department of Clinical Biochemistry, Bezmi Alem Vakif University, Medical Faculty, Istanbul, Turkey 2
Abstract
Keywords
There are several reports that increased oxidative stress and DNA damage were found in b-thalassemia major (b-TM) patients. In this study, we aimed to evaluate the effects of N-acetylcysteine (NAC) and vitamin E on total oxidative stress and DNA damage in children with b-TM. Seventy-five children with transfusion-dependent b-thalassemia (b-thal) were randomly chosen to receive 10 mg/kg/day of NAC or 10 IU/kg/day of vitamin E or no supplementation; 28 healthy controls were also included in the study. Serum total oxidant status (TOS) and total antioxidant capacity (TAC) were measured, oxidative stress index (OSI) was calculated, and mononuclear DNA damage was assessed by alkaline comet assay; they were determined before treatment and after 3 months of treatment. Total oxydent status, OSI, and DNA damage levels were significantly higher and TAC levels were significantly lower in the thalassemic children than in the healthy controls (p50.001). In both supplemented groups, mean TOS and OSI levels were decreased; TAC and pre transfusion hemoglobin (Hb) levels were significantly increased after 3 months (p 0.002). In the NAC group, DNA damage score decreased (p ¼ 0.001). N-acetylcysteine and vitamin E may be effective in reducing serum oxidative stress and increase pre transfusion Hb levels in children with b-thal. N-acetylcysteine also can reduce DNA damage.
b-Thalassemia (b-thal), child, DNA damage, N-acetylcysteine (NAC), oxidative stress, vitamin E
Introduction b-Thalassemias are characterized by the presence of mutations on the b-globin gene, resulting in the absence or reduced synthesis of b-globin chains of the hemoglobin (Hb) tetramer (1). Early and regular blood transfusion therapy decreases the complications of severe anemia and prolongs survival. In the long term, however, the beneficial effects of transfusions are limited by the organ damage resulting from cumulative iron burden (2). When body iron is excessive, increased non transferrin-bound iron induces free radicals (3). Reactive oxygen species (ROS) are capable of causing oxidative damage to macromolecules, leading to lipid peroxidation, oxidation of amino acid side chains, and oxidation of polypeptide chains resulting in protein fragmentation, DNA damage, and DNA strand breaks (3,4). Therefore, iron chelation is essential in the management of this otherwise fatal disease. The selection of effective and safe chelation therapy protocols that can reduce and maintain the iron load of patients at near normal physiological ranges is important (5).
History Received 25 January 2014 Accepted 12 March 2014 Published online 15 September 2014
Antioxidants are potentially protective agents that help to remove toxic oxygen radicals (6). Investigators found deficiency of antioxidants and vitamin E in these patients (7–9). Vitamin E is a radical scavenger as a lipid soluble antioxidant. Many reports have shown similar outcomes, namely that supplementation of vitamin E can improve biochemical indicators, but no clinical improvement in Hb levels was seen (7,10,11). Another antioxidant is N-acetylcysteine (NAC) that readily enters cells, and within the cytoplasm it is converted to l-cysteine, which is a precursor to the aminothiol glutathione (GSH) and a major intracellular antioxidant (12). It is reported that treatment with combinations of antioxidants such as NAC in addition to iron chelators could neutralize the deleterious effects of ROS (13). Moreover, antioxidant supplementation decreases oxidative DNA damage in human lymphocytes (14). The present study aimed to evaluate the effects of NAC and vitamin E on total oxidative stress and DNA damage and to determine whether there were any effects on Hb levels in children with b-thalassemia major (b-TM).
Materials and methods Address correspondence to Ali Aycicek, M.D., Pediatric Hematology/ Oncology Clinic, Eskisehir State Hospital, Yenidogan Street, 26010 Eskisehir, Turkey. Tel.: +90-222-237-48-00. Fax: +90-222-237-61-34. E-mail: [email protected]
A total of 98 consecutive children with b-thalassemia (b-thal) (48 females, 50 males) who attended the Pediatric Hematology Clinic and Outpatient Clinic at Harran
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University Research Hospital, Sanlıurfa, Turkey, between April and July 2010 were enrolled in this open-label randomized controlled trial. Their diagnoses were confirmed through molecular genetic analyses. The patients were regularly interviewed and examined by a staff of physicians at intervals of 15 days to 1 month. Serum ferritin, iron and liver enzymes were measured monthly. The patients received approximately 15 mL of packed red blood cells per kilogram of their body weight at each transfusion to maintain Hb levels above 9.5 g/dL. Deferasirox (DFX), deferiprone (DFP), or deferoxamine (DFO) plus DFP was applied as chelation therapy. Deferoxamine therapy did not involve intake of ascorbate. The study protocol was approved by the ethics committee and written informed consent was obtained from the parents of the patients. Twenty-five healthy children (12 females, 13 males) were enrolled as controls. Exclusion criteria Three children under 2 years old were not receiving iron chelators, three had exhibited endocrine complications, two had massive splenomegaly and hypersplenism, five had chronic hepatitis B or were hepatitis B carriers, two had hepatitis C, two had autoimmune hemolytic anemia, one had brucellosis, and five did not take antioxidant drugs regularly. In total, 23 children were excluded from the study. Study groups and treatment protocol Children with b-thal were randomly allocated into three groups. The duration of the study was 3 months. The first group (NAC) included 25 children (12 females, 13 males) who were supplemented with NAC at a dose of 10 mg/kg (Oxxa susp/ Toprak, maximum dose of 600 mg) orally, once daily for three months. The second group (vitamin E) included 25 children (12 females, 13 males) who were supplemented with vitamin E at a dose of 10 U/kg (Evicap caps 100 IU or 200 IU/Kocak, maximum dose 400 IU) orally, once daily. The third group included 25 children with b-thal (14 females, 11 males) who were not supplemented with any antioxidant drug (NS) treatment or dietary supplements. The patients maintained chelation therapy of DFX 20–30 mg/kg/day, orally once daily or DFP 75 mg/kg/day, orally three times daily or DFO 35– 40 mg/kg/day, subcutaneous infusion. Chelation therapy was with a single agent or a combination of two agents. Blood sample collection Blood samples were collected from a peripheral vein into vacutainers containing EDTA (ethylenediaminetetraacetic acid), jelled serum tubes, and heparinized tubes. The samples were collected before packed red blood cell transfusions were administered. Serum samples were separated from the cells by centrifugation at 1500 g for 10 min. and were stored at 80 C without preservative until assayed. All analyses were performed at a single laboratory. Measurement of total oxidant and antioxidant status Serum total oxidant status (TOS) and total antioxidant capacity (TAC) levels were determined using a commercially
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available kit (Rel Assay Diagnostics, Gaziantep, Turkey) developed by Erel (15,16). The results are expressed as mmol Trolox Eqv./L and mmol H2O2 Eqv./L, respectively. Percent ratio of the TOS level to the TAC level gave the oxidative stress index (OSI). The OSI value was calculated according to the following formula: OSI (arbitrary unit) ¼ TOS (mmol H2O2 Eqv./L)/TAC (mmol Trolox Eqv./L) 100 (17). Determination of DNA damage by alkaline comet assay Peripheral blood mononuclear leukocyte isolation for the comet assay was performed by density gradient separation (Histopaque 1077; Sigma-Aldrich, Inc., St. Louis, MO, USA). Heparinized blood of 1 mL was carefully layered over 1 mL of Histopaque and centrifuged for 35 min. at 500 g and 25 C. The interface band containing mononuclear leukocyte was washed with phosphate buffered saline (PBS) and then collected by 15 min. centrifugation at 400 g. The resulting pellets were resuspended in PBS to obtain 20,000 cells in 10 mL. Membrane integrity was assessed by trypan blue exclusion. Mononuclear DNA damage was analyzed by the alkaline comet assay as described by Faust et al. (18) with minor modifications (19). All of the analysis steps were conducted to prevent additional DNA damage under red light or without direct light. The images of 100 randomly chosen nuclei (50 nuclei from each of two duplicated slides) were analyzed visually for each subject. Each image was classified according to the intensity of the fluorescence in the comet tail and was given a value of 0, 1, 2, 3 or 4 (from undamaged, class 0, to maximally damaged, class 4) (20). These samples were randomly chosen from the microscopic slides of patients, and so the total score of a slide was between 0 and 400 arbitrary units (20,21). The same biochemistry staff performed all the procedures and a single observer unaware of the subject’s group determined the DNA damage score. Other parameters Ferritin, iron binding capacity, and iron levels were analyzed using a hormone analyzer and chemiluminescent immunoassay (Elecsys E170; Roche Diagnostics GmbH, Penzberg, Germany). The hemogram was analyzed by an analyzer with an automated cell counter for complete blood cell count (Celldyn 3700; Abbott Laboratories, Abbott Park, North Chicago, IL, USA). Statistical analyses Normality of variances was accomplished using Levene’s statistics. The data were expressed as mean ± SD (standard deviation) and were compared using the 2 test, paired t-test, Student’s t-test, and analysis of variance (one-way ANOVA) followed by Tukey’s honestly significant difference (HSD) posthoc test. Bivariate associations between variables were assessed with Pearson’s correlation test. The differences were considered statistically significant at p50.05. Statistical analyses were conducted using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA) SPSS for Windows 11.5.
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Table 1. Clinical parameters in the study and control groups.
Parameters
N-acetylcysteine Group (n ¼ 25)
Vitamin E Group (n ¼ 25)
Non Supplemented Group (n ¼ 25)
p Valuea
Control Group (n ¼ 25)
9.65 ± 4.15 M: 13; F: 12 28.5 ± 11.7 2617.0 ± 865.0 190.0 ± 64.0 90.0 ± 67.0 10 (40.0) 17 (68.0) 4 (16.0) 4 (16.0)
10.30 ± 4.09 M: 13; F: 12 18.2 ± 9.0 3157.0 ± 1156.0 217.0 ± 67.0 125.0 ± 85.0 6 (24.0) 22 (88.0) 1 (4.0) 2 (8.0)
8.56 ± 4.10 M: 14; F: 11 24.0 ± 9.7 2934.0 ± 1104.0 188.0 ± 70.0 74.0 ± 51.0 5 (20.0) 22 (88.0) 1 (4.0) 2 (8.0)
0.274 0.923b 0.223 0.277 0.493 0.292 0.249b 0.359b – –
8.00 ± 3.78 M: 13; F: 12 29.0 ± 5.0 55.0 ± 25.0 61.0 ± 21.0 350.0 ± 58.0 NA NA NA NA
Mean age (years) Gender Body weight (kg) Ferritin (ng/mL) Iron (mg/dL) TIBC (mg/dL) Splenectomy, n (%) DFX, n (%) DFP, n (%) DFO + DFP, n (%)
TIBC: total iron binding capacity; NA: not applicable; DFX: deferasirox; DFP: deferiprone; DFO: deferoxamine. One-way ANOVA. b 2 test. Hemoglobin Downloaded from informahealthcare.com by Memorial University of Newfoundland on 10/04/14 For personal use only.
a
Table 2. Mean levels of hemoglobin, total oxidant status, total antioxidant capacity, oxidative stress index, and DNA damage in the thalassemic groups before treatment and in the control group.
Parameters Hb (g/dL) TOS (mmol; H2O2 Eqv./L) TAC (mmol; Trolox Eqv./L) OSI (AU) DNA damage (AU)
N-acetylcysteine Group (n ¼ 25)
Vitamin E Group (n ¼ 25)
Non Supplemented Group (n ¼ 25)
p Valuea
Control Group (n ¼ 25)
8.4 ± 1.3 16.3 ± 4.1b 0.72 ± 0.09 2.29 ± 0.75b 8.9 ± 6.0a
8.7 ± 1.1 11.9 ± 2.8 0.74 ± 0.12 1.65 ± 0.36 4.2 ± 5.0
8.0 ± 1.1 8.9 ± 2.1 0.76 ± 0.11 0.19 ± 0.36 3.2 ± 3.9
0.145 50.001 0.515 50.001 0.001
12.1 ± 0.9 6.4 ± 1.5 1.03 ± 1.19 6.80 ± 0.21 1.2 ± 1.7
Hb: hemoglobin; TOS: total oxidant status; TAC: total antioxidant capacity; OSI: oxidative stress index; AU: arbitrary unit. One-way ANOVA with the Tukey’s HSD multiple comparison test. b Different than the other groups according to the posthoc test. a
Results The demographic and clinical characteristics of the subjects are listed in Table 1. There were no differences between the study and control groups with respect to mean age, body weight, or sex distribution. Mean ferritin, iron, iron binding capacity levels, splenectomy, and chelator distribution were similar in the study groups (p40.05). Mean DFX dose was 24 mg/kg/day, mean DFP dose was 75 mg/kg/day, and mean DFO dose was 36 mg/kg/day. At the beginning of the study (before supplementation), mean levels of DNA damage, and TOS and OSI levels were significantly higher and TAC was significantly lower in the thalassemia groups than in the control group (Table 2). Mean levels of DNA damage, and TOS and OSI levels were significantly higher in the NAC group than in the other groups before supplementation (Table 2). The supplemented groups’ mean pre tranfusional Hb and TAC levels were significantly higher after 3 months of treatment compared to before treatment (p 0.002), and mean levels of TOS and OSI were significantly reduced after 3 months of treatment compared to before treatment (p50.001) (Table 3). Both supplemented groups’ OSI levels significantly decreased after supplementation (Figure 1). In the NAC group, mean levels of DNA damage were significantly reduced to levels similar to those in the other groups after 3 months (p ¼ 0.001); there were no important changes in the vitamin E and NS groups (Figure 2). Positive significant correlations were found between serum OSI and DNA damage score r ¼ 0.429, p50.001 (Figure 3);
this correlation disappeared after antioxidant supplementation (p40.05). Moreover, in the NAC group, before treatment, a positive significant correlation was detected between DNA damage and ferritin levels (r ¼ 690, p50.001). There was no correlation between splenectomy and DNA damage score (p ¼ 0.367).
Discussion In our study, we found that antioxidant supplementation such as vitamin E and NAC was effective in reducing oxidative status and increasing pre transfusion Hb levels. We also found that NAC decreased mononuclear DNA damage. To the best of our knowledge, there has been no report published regarding DNA damage in children with b-thal. It is well known that oxidative damage may play an important role in erythrocyte hemolysis in b-thal (22,23). The increased oxidative damage results from the generation of free radicals by an excess of denaturated a- or b-globin chains and intracellular iron overload (11,24–26). Previous studies showed that thalassemia patients have decreased antioxidant defense mechanisms but supplementation of antioxidants does not affect Hb level or transfusion requirement in patients with b-TM (27–31). Oxidative stress is caused by prolonged imbalance and antioxidant depletion as well as hyperproduction of ROS (11,22,25). Antioxidant capacity is the result of the overall effect of water-soluble antioxidants, lipid-soluble antioxidants, and antioxidant enzymes. Various studies have reported that antioxidant enzymes such as superoxide dismutase and
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Table 3. Mean levels of hemoglobin, total oxidant status, total antioxidant capacity, oxidative stress index, and DNA damage in the thalassemic groups before and after treatment. N-acetylcysteine Group (n ¼ 25)
Vitamin E Group (n ¼ 25)
Non Supplemented Group (n ¼ 25)
Parameters
Before Treatment
Third Month
p Valuea
Before Treatment
Third Month
p Valuea
Before Treatment
Third Month
p Valuea
Hb (g/dL) TOS (mmol; H2O2 Eqv./L) TAC (mmol; Trolox Eqv./L) OSI (AU) DNA damage (AU)
8.4 ± 1.3 16.3 ± 4.1 0.72 ± 0.09 2.29 ± 0.75 8.9 ± 6.0
9.6 ± 0.8 7.8 ± 2.3 0.84 ± 0.13 0.94 ± 0.23 2.5 ± 2.0
50.001 50.001 0.001 50.001 0.001
8.7 ± 1.1 11.9 ± 2.8 0.74 ± 0.12 1.65 ± 0.05 4.2 ± 5.0
9.7 ± 1.2 7.8 ± 2.1 0.84 ± 0.21 0.92 ± 0.30 4.4 ± 3.6
0.001 50.001 0.002 50.001 0.510
8.0 ± 1.1 8.9 ± 2.1 0.76 ± 0.11 1.19 ± 0.36 3.2 ± 3.9
8.1 ± 0.9 8.2 ± 2.1 0.81 ± 0.13 1.10 ± 0.27 4.0 ± 3.4
0.669 0.062 0.230 0.189 0.918
Hemoglobin Downloaded from informahealthcare.com by Memorial University of Newfoundland on 10/04/14 For personal use only.
Hb: hemoglobin; TOS: total oxidant status; TAC: total antioxidant capacity; OSI: oxidative stress index; AU: arbitrary unit. a Paired t-test within group.
Figure 1. Boxplot graph of serum OSI levels before treatment and after 3 months of treatment in the groups. NAC: N-acetylcysteine group; VITE: vitamin E group; NS: non supplemented thalassemic group. *Student’s t-test.
glutathione peroxidase were increased in thalassemic patients (8,11,23). However, it was shown that such increased enzyme activities were insufficient to prevent oxidative damage and antioxidant imbalance occurs (9,26,27,32). Many studies have shown that decreased level of vitamin E in b-TM and supplementation with vitamin E can improve biochemical markers, but not provide clinical improvement in Hb level or transfusion requirement. Tesoriere et al. (28) demonstrated that low level of vitamin E and a high level of malondialdehyde in serum tended to normalize after 6 months of treatment with 600 mg/day vitamin E in b-thal intermedia (b-TI) patients, and also erythrocyte count and hematocrit value increased, but Hb values did not change after 9 months of treatment. Livrea et al. (22) showed significantly decreased lipid peroxidation, significantly increased erythrocyte catalase
Figure 2. Boxplot graph of DNA damage levels before treatment and after 3 months of treatment in the groups. NAC: N-acetylcysteine group; VITE: vitamin E group; NS: received no antioxidant drug group.
activity, and a return to near normal levels of superoxide dismutase in b-thalassemic children supplemented with 10 mg/kg/day vitamin E for 4 weeks. Dissayabutra et al. (27) reported no change in Hb values with vitamin E supplementation in thalassemic children. In contrast, we showed that pre transfusion Hb levels were significantly higher after 3 months of treatment than before treatment in both supplemented groups. On the other hand, we found that serum TOS and OSI levels were significantly reduced after 3 months of treatment but TAC levels did not change. The observed increase in Hb levels may be associated with reduced oxidative stress and this may contribute to the prolongation of life expectancy of erythrocytes with vitamin E supplementation. Increased oxidative stress as well as iron overload in the liver leads to use of glutathione and might contribute to
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other hand, DNA damage did not change with vitamin E supplementation. The limitations of this research were that the parameters studied were not homogeneous before supplementation and that it was not a double-blind, placebo-controlled, crossover study. However, patients were randomly allocated into the groups and there was no intervention regarding patient distribution. In general, if researchers describe a trial as double-blind, readers can assume that they have avoided bias (39). A comparison of drugs is most readily accepted if the results are from randomized controlled trials. Open-label studies are frequently incorporated in the design of randomized controlled trials. We concluded that increased oxidant status and increased DNA damage in children with transfusion-dependent b-thal can be effectively reduced by antioxidant supplementation, especially with NAC.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. Figure 3. Correlation graphic of serum OSI and mononuclear DNA damage score (r ¼ 0.429, p50.001) before antioxidant supplementation.
References
decreased levels of glutathione in thalassemic patients. Therefore, NAC use in these patients, by increasing intracellular glutathione, may be expected to have a positive influence on oxidative effects. In an experimental study, Amer et al. (33) examined the effects of various oxidants and antioxidants on lipid peroxidation in normal and thalassemic eythrocytes, and they found lipid peroxidation was higher in the thalassemic erythrocytes and these effects were reversed by NAC. We found that the levels of TOS and OSI were decreased, and TAC levels were increased after supplementation. Our findings suggest that NAC supplementation reduces oxidative stress in thalassemic children. We also showed a significant increase in Hb level with a decrease in the amount of blood transfused during the study period. Oxidant damage to erythroid precursors leads to accelerated apoptosis and ineffective erythropoiesis (25). Therefore, there is a possible mechanism of NAC to reduce the level of oxidant status and increased antioxidant capacity as a result of protection of erythrocytes from oxidative damage, thus extending the life-span of erythrocytes. Oxidative DNA damage is one of the pathological consequences of iron. Shires et al. (34) reported that incubation of isolated rat liver cells with iron resulted in fragmentation of single-stranded DNA; iron-mediated DNA damage was required in the formation of reactive forms of oxygen but was not evidence for direct interaction of reactive oxygen with DNA (35). Experimental studies showed that iron plays an important role in hydrogen peroxide associated DNA damage and this damage can be reduced with iron chelation therapies (36,37). Our study showed that DNA damage is higher in thalassemic children than in healthy controls. N-acetylcysteine has an antioxidant effect, an anticarcinogenic effect, and a protective effect towards DNA damage (38). Thus, NAC has proven to be effective in treating increased DNA damage in b-thalassemic children. On the
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ORIGINAL ARTICLE
N-Acetylcysteine Supplementation Reduces Oxidative Stress and DNA Damage in Children with b-Thalassemia Zeynep Canan Ozdemir1, Ahmet Koc2, Ali Aycicek3, and Abdurrahim Kocyigit4 Hemoglobin Downloaded from informahealthcare.com by Memorial University of Newfoundland on 10/04/14 For personal use only.
1
Pediatric Hematology Department, Harran University, Medical Faculty, Sanlıurfa, Turkey, Pediatric Hematology/Oncology Department, Marmara University, Medical Faculty, Istanbul, Turkey, 3 Pediatric Hematology/Oncology Clinic, Eskisehir State Hospital, Eskisehir, Turkey, and 4 Department of Clinical Biochemistry, Bezmi Alem Vakif University, Medical Faculty, Istanbul, Turkey 2
Abstract
Keywords
There are several reports that increased oxidative stress and DNA damage were found in b-thalassemia major (b-TM) patients. In this study, we aimed to evaluate the effects of N-acetylcysteine (NAC) and vitamin E on total oxidative stress and DNA damage in children with b-TM. Seventy-five children with transfusion-dependent b-thalassemia (b-thal) were randomly chosen to receive 10 mg/kg/day of NAC or 10 IU/kg/day of vitamin E or no supplementation; 28 healthy controls were also included in the study. Serum total oxidant status (TOS) and total antioxidant capacity (TAC) were measured, oxidative stress index (OSI) was calculated, and mononuclear DNA damage was assessed by alkaline comet assay; they were determined before treatment and after 3 months of treatment. Total oxydent status, OSI, and DNA damage levels were significantly higher and TAC levels were significantly lower in the thalassemic children than in the healthy controls (p50.001). In both supplemented groups, mean TOS and OSI levels were decreased; TAC and pre transfusion hemoglobin (Hb) levels were significantly increased after 3 months (p 0.002). In the NAC group, DNA damage score decreased (p ¼ 0.001). N-acetylcysteine and vitamin E may be effective in reducing serum oxidative stress and increase pre transfusion Hb levels in children with b-thal. N-acetylcysteine also can reduce DNA damage.
b-Thalassemia (b-thal), child, DNA damage, N-acetylcysteine (NAC), oxidative stress, vitamin E
Introduction b-Thalassemias are characterized by the presence of mutations on the b-globin gene, resulting in the absence or reduced synthesis of b-globin chains of the hemoglobin (Hb) tetramer (1). Early and regular blood transfusion therapy decreases the complications of severe anemia and prolongs survival. In the long term, however, the beneficial effects of transfusions are limited by the organ damage resulting from cumulative iron burden (2). When body iron is excessive, increased non transferrin-bound iron induces free radicals (3). Reactive oxygen species (ROS) are capable of causing oxidative damage to macromolecules, leading to lipid peroxidation, oxidation of amino acid side chains, and oxidation of polypeptide chains resulting in protein fragmentation, DNA damage, and DNA strand breaks (3,4). Therefore, iron chelation is essential in the management of this otherwise fatal disease. The selection of effective and safe chelation therapy protocols that can reduce and maintain the iron load of patients at near normal physiological ranges is important (5).
History Received 25 January 2014 Accepted 12 March 2014 Published online 15 September 2014
Antioxidants are potentially protective agents that help to remove toxic oxygen radicals (6). Investigators found deficiency of antioxidants and vitamin E in these patients (7–9). Vitamin E is a radical scavenger as a lipid soluble antioxidant. Many reports have shown similar outcomes, namely that supplementation of vitamin E can improve biochemical indicators, but no clinical improvement in Hb levels was seen (7,10,11). Another antioxidant is N-acetylcysteine (NAC) that readily enters cells, and within the cytoplasm it is converted to l-cysteine, which is a precursor to the aminothiol glutathione (GSH) and a major intracellular antioxidant (12). It is reported that treatment with combinations of antioxidants such as NAC in addition to iron chelators could neutralize the deleterious effects of ROS (13). Moreover, antioxidant supplementation decreases oxidative DNA damage in human lymphocytes (14). The present study aimed to evaluate the effects of NAC and vitamin E on total oxidative stress and DNA damage and to determine whether there were any effects on Hb levels in children with b-thalassemia major (b-TM).
Materials and methods Address correspondence to Ali Aycicek, M.D., Pediatric Hematology/ Oncology Clinic, Eskisehir State Hospital, Yenidogan Street, 26010 Eskisehir, Turkey. Tel.: +90-222-237-48-00. Fax: +90-222-237-61-34. E-mail: [email protected]
A total of 98 consecutive children with b-thalassemia (b-thal) (48 females, 50 males) who attended the Pediatric Hematology Clinic and Outpatient Clinic at Harran
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University Research Hospital, Sanlıurfa, Turkey, between April and July 2010 were enrolled in this open-label randomized controlled trial. Their diagnoses were confirmed through molecular genetic analyses. The patients were regularly interviewed and examined by a staff of physicians at intervals of 15 days to 1 month. Serum ferritin, iron and liver enzymes were measured monthly. The patients received approximately 15 mL of packed red blood cells per kilogram of their body weight at each transfusion to maintain Hb levels above 9.5 g/dL. Deferasirox (DFX), deferiprone (DFP), or deferoxamine (DFO) plus DFP was applied as chelation therapy. Deferoxamine therapy did not involve intake of ascorbate. The study protocol was approved by the ethics committee and written informed consent was obtained from the parents of the patients. Twenty-five healthy children (12 females, 13 males) were enrolled as controls. Exclusion criteria Three children under 2 years old were not receiving iron chelators, three had exhibited endocrine complications, two had massive splenomegaly and hypersplenism, five had chronic hepatitis B or were hepatitis B carriers, two had hepatitis C, two had autoimmune hemolytic anemia, one had brucellosis, and five did not take antioxidant drugs regularly. In total, 23 children were excluded from the study. Study groups and treatment protocol Children with b-thal were randomly allocated into three groups. The duration of the study was 3 months. The first group (NAC) included 25 children (12 females, 13 males) who were supplemented with NAC at a dose of 10 mg/kg (Oxxa susp/ Toprak, maximum dose of 600 mg) orally, once daily for three months. The second group (vitamin E) included 25 children (12 females, 13 males) who were supplemented with vitamin E at a dose of 10 U/kg (Evicap caps 100 IU or 200 IU/Kocak, maximum dose 400 IU) orally, once daily. The third group included 25 children with b-thal (14 females, 11 males) who were not supplemented with any antioxidant drug (NS) treatment or dietary supplements. The patients maintained chelation therapy of DFX 20–30 mg/kg/day, orally once daily or DFP 75 mg/kg/day, orally three times daily or DFO 35– 40 mg/kg/day, subcutaneous infusion. Chelation therapy was with a single agent or a combination of two agents. Blood sample collection Blood samples were collected from a peripheral vein into vacutainers containing EDTA (ethylenediaminetetraacetic acid), jelled serum tubes, and heparinized tubes. The samples were collected before packed red blood cell transfusions were administered. Serum samples were separated from the cells by centrifugation at 1500 g for 10 min. and were stored at 80 C without preservative until assayed. All analyses were performed at a single laboratory. Measurement of total oxidant and antioxidant status Serum total oxidant status (TOS) and total antioxidant capacity (TAC) levels were determined using a commercially
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available kit (Rel Assay Diagnostics, Gaziantep, Turkey) developed by Erel (15,16). The results are expressed as mmol Trolox Eqv./L and mmol H2O2 Eqv./L, respectively. Percent ratio of the TOS level to the TAC level gave the oxidative stress index (OSI). The OSI value was calculated according to the following formula: OSI (arbitrary unit) ¼ TOS (mmol H2O2 Eqv./L)/TAC (mmol Trolox Eqv./L) 100 (17). Determination of DNA damage by alkaline comet assay Peripheral blood mononuclear leukocyte isolation for the comet assay was performed by density gradient separation (Histopaque 1077; Sigma-Aldrich, Inc., St. Louis, MO, USA). Heparinized blood of 1 mL was carefully layered over 1 mL of Histopaque and centrifuged for 35 min. at 500 g and 25 C. The interface band containing mononuclear leukocyte was washed with phosphate buffered saline (PBS) and then collected by 15 min. centrifugation at 400 g. The resulting pellets were resuspended in PBS to obtain 20,000 cells in 10 mL. Membrane integrity was assessed by trypan blue exclusion. Mononuclear DNA damage was analyzed by the alkaline comet assay as described by Faust et al. (18) with minor modifications (19). All of the analysis steps were conducted to prevent additional DNA damage under red light or without direct light. The images of 100 randomly chosen nuclei (50 nuclei from each of two duplicated slides) were analyzed visually for each subject. Each image was classified according to the intensity of the fluorescence in the comet tail and was given a value of 0, 1, 2, 3 or 4 (from undamaged, class 0, to maximally damaged, class 4) (20). These samples were randomly chosen from the microscopic slides of patients, and so the total score of a slide was between 0 and 400 arbitrary units (20,21). The same biochemistry staff performed all the procedures and a single observer unaware of the subject’s group determined the DNA damage score. Other parameters Ferritin, iron binding capacity, and iron levels were analyzed using a hormone analyzer and chemiluminescent immunoassay (Elecsys E170; Roche Diagnostics GmbH, Penzberg, Germany). The hemogram was analyzed by an analyzer with an automated cell counter for complete blood cell count (Celldyn 3700; Abbott Laboratories, Abbott Park, North Chicago, IL, USA). Statistical analyses Normality of variances was accomplished using Levene’s statistics. The data were expressed as mean ± SD (standard deviation) and were compared using the 2 test, paired t-test, Student’s t-test, and analysis of variance (one-way ANOVA) followed by Tukey’s honestly significant difference (HSD) posthoc test. Bivariate associations between variables were assessed with Pearson’s correlation test. The differences were considered statistically significant at p50.05. Statistical analyses were conducted using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA) SPSS for Windows 11.5.
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Table 1. Clinical parameters in the study and control groups.
Parameters
N-acetylcysteine Group (n ¼ 25)
Vitamin E Group (n ¼ 25)
Non Supplemented Group (n ¼ 25)
p Valuea
Control Group (n ¼ 25)
9.65 ± 4.15 M: 13; F: 12 28.5 ± 11.7 2617.0 ± 865.0 190.0 ± 64.0 90.0 ± 67.0 10 (40.0) 17 (68.0) 4 (16.0) 4 (16.0)
10.30 ± 4.09 M: 13; F: 12 18.2 ± 9.0 3157.0 ± 1156.0 217.0 ± 67.0 125.0 ± 85.0 6 (24.0) 22 (88.0) 1 (4.0) 2 (8.0)
8.56 ± 4.10 M: 14; F: 11 24.0 ± 9.7 2934.0 ± 1104.0 188.0 ± 70.0 74.0 ± 51.0 5 (20.0) 22 (88.0) 1 (4.0) 2 (8.0)
0.274 0.923b 0.223 0.277 0.493 0.292 0.249b 0.359b – –
8.00 ± 3.78 M: 13; F: 12 29.0 ± 5.0 55.0 ± 25.0 61.0 ± 21.0 350.0 ± 58.0 NA NA NA NA
Mean age (years) Gender Body weight (kg) Ferritin (ng/mL) Iron (mg/dL) TIBC (mg/dL) Splenectomy, n (%) DFX, n (%) DFP, n (%) DFO + DFP, n (%)
TIBC: total iron binding capacity; NA: not applicable; DFX: deferasirox; DFP: deferiprone; DFO: deferoxamine. One-way ANOVA. b 2 test. Hemoglobin Downloaded from informahealthcare.com by Memorial University of Newfoundland on 10/04/14 For personal use only.
a
Table 2. Mean levels of hemoglobin, total oxidant status, total antioxidant capacity, oxidative stress index, and DNA damage in the thalassemic groups before treatment and in the control group.
Parameters Hb (g/dL) TOS (mmol; H2O2 Eqv./L) TAC (mmol; Trolox Eqv./L) OSI (AU) DNA damage (AU)
N-acetylcysteine Group (n ¼ 25)
Vitamin E Group (n ¼ 25)
Non Supplemented Group (n ¼ 25)
p Valuea
Control Group (n ¼ 25)
8.4 ± 1.3 16.3 ± 4.1b 0.72 ± 0.09 2.29 ± 0.75b 8.9 ± 6.0a
8.7 ± 1.1 11.9 ± 2.8 0.74 ± 0.12 1.65 ± 0.36 4.2 ± 5.0
8.0 ± 1.1 8.9 ± 2.1 0.76 ± 0.11 0.19 ± 0.36 3.2 ± 3.9
0.145 50.001 0.515 50.001 0.001
12.1 ± 0.9 6.4 ± 1.5 1.03 ± 1.19 6.80 ± 0.21 1.2 ± 1.7
Hb: hemoglobin; TOS: total oxidant status; TAC: total antioxidant capacity; OSI: oxidative stress index; AU: arbitrary unit. One-way ANOVA with the Tukey’s HSD multiple comparison test. b Different than the other groups according to the posthoc test. a
Results The demographic and clinical characteristics of the subjects are listed in Table 1. There were no differences between the study and control groups with respect to mean age, body weight, or sex distribution. Mean ferritin, iron, iron binding capacity levels, splenectomy, and chelator distribution were similar in the study groups (p40.05). Mean DFX dose was 24 mg/kg/day, mean DFP dose was 75 mg/kg/day, and mean DFO dose was 36 mg/kg/day. At the beginning of the study (before supplementation), mean levels of DNA damage, and TOS and OSI levels were significantly higher and TAC was significantly lower in the thalassemia groups than in the control group (Table 2). Mean levels of DNA damage, and TOS and OSI levels were significantly higher in the NAC group than in the other groups before supplementation (Table 2). The supplemented groups’ mean pre tranfusional Hb and TAC levels were significantly higher after 3 months of treatment compared to before treatment (p 0.002), and mean levels of TOS and OSI were significantly reduced after 3 months of treatment compared to before treatment (p50.001) (Table 3). Both supplemented groups’ OSI levels significantly decreased after supplementation (Figure 1). In the NAC group, mean levels of DNA damage were significantly reduced to levels similar to those in the other groups after 3 months (p ¼ 0.001); there were no important changes in the vitamin E and NS groups (Figure 2). Positive significant correlations were found between serum OSI and DNA damage score r ¼ 0.429, p50.001 (Figure 3);
this correlation disappeared after antioxidant supplementation (p40.05). Moreover, in the NAC group, before treatment, a positive significant correlation was detected between DNA damage and ferritin levels (r ¼ 690, p50.001). There was no correlation between splenectomy and DNA damage score (p ¼ 0.367).
Discussion In our study, we found that antioxidant supplementation such as vitamin E and NAC was effective in reducing oxidative status and increasing pre transfusion Hb levels. We also found that NAC decreased mononuclear DNA damage. To the best of our knowledge, there has been no report published regarding DNA damage in children with b-thal. It is well known that oxidative damage may play an important role in erythrocyte hemolysis in b-thal (22,23). The increased oxidative damage results from the generation of free radicals by an excess of denaturated a- or b-globin chains and intracellular iron overload (11,24–26). Previous studies showed that thalassemia patients have decreased antioxidant defense mechanisms but supplementation of antioxidants does not affect Hb level or transfusion requirement in patients with b-TM (27–31). Oxidative stress is caused by prolonged imbalance and antioxidant depletion as well as hyperproduction of ROS (11,22,25). Antioxidant capacity is the result of the overall effect of water-soluble antioxidants, lipid-soluble antioxidants, and antioxidant enzymes. Various studies have reported that antioxidant enzymes such as superoxide dismutase and
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Table 3. Mean levels of hemoglobin, total oxidant status, total antioxidant capacity, oxidative stress index, and DNA damage in the thalassemic groups before and after treatment. N-acetylcysteine Group (n ¼ 25)
Vitamin E Group (n ¼ 25)
Non Supplemented Group (n ¼ 25)
Parameters
Before Treatment
Third Month
p Valuea
Before Treatment
Third Month
p Valuea
Before Treatment
Third Month
p Valuea
Hb (g/dL) TOS (mmol; H2O2 Eqv./L) TAC (mmol; Trolox Eqv./L) OSI (AU) DNA damage (AU)
8.4 ± 1.3 16.3 ± 4.1 0.72 ± 0.09 2.29 ± 0.75 8.9 ± 6.0
9.6 ± 0.8 7.8 ± 2.3 0.84 ± 0.13 0.94 ± 0.23 2.5 ± 2.0
50.001 50.001 0.001 50.001 0.001
8.7 ± 1.1 11.9 ± 2.8 0.74 ± 0.12 1.65 ± 0.05 4.2 ± 5.0
9.7 ± 1.2 7.8 ± 2.1 0.84 ± 0.21 0.92 ± 0.30 4.4 ± 3.6
0.001 50.001 0.002 50.001 0.510
8.0 ± 1.1 8.9 ± 2.1 0.76 ± 0.11 1.19 ± 0.36 3.2 ± 3.9
8.1 ± 0.9 8.2 ± 2.1 0.81 ± 0.13 1.10 ± 0.27 4.0 ± 3.4
0.669 0.062 0.230 0.189 0.918
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Hb: hemoglobin; TOS: total oxidant status; TAC: total antioxidant capacity; OSI: oxidative stress index; AU: arbitrary unit. a Paired t-test within group.
Figure 1. Boxplot graph of serum OSI levels before treatment and after 3 months of treatment in the groups. NAC: N-acetylcysteine group; VITE: vitamin E group; NS: non supplemented thalassemic group. *Student’s t-test.
glutathione peroxidase were increased in thalassemic patients (8,11,23). However, it was shown that such increased enzyme activities were insufficient to prevent oxidative damage and antioxidant imbalance occurs (9,26,27,32). Many studies have shown that decreased level of vitamin E in b-TM and supplementation with vitamin E can improve biochemical markers, but not provide clinical improvement in Hb level or transfusion requirement. Tesoriere et al. (28) demonstrated that low level of vitamin E and a high level of malondialdehyde in serum tended to normalize after 6 months of treatment with 600 mg/day vitamin E in b-thal intermedia (b-TI) patients, and also erythrocyte count and hematocrit value increased, but Hb values did not change after 9 months of treatment. Livrea et al. (22) showed significantly decreased lipid peroxidation, significantly increased erythrocyte catalase
Figure 2. Boxplot graph of DNA damage levels before treatment and after 3 months of treatment in the groups. NAC: N-acetylcysteine group; VITE: vitamin E group; NS: received no antioxidant drug group.
activity, and a return to near normal levels of superoxide dismutase in b-thalassemic children supplemented with 10 mg/kg/day vitamin E for 4 weeks. Dissayabutra et al. (27) reported no change in Hb values with vitamin E supplementation in thalassemic children. In contrast, we showed that pre transfusion Hb levels were significantly higher after 3 months of treatment than before treatment in both supplemented groups. On the other hand, we found that serum TOS and OSI levels were significantly reduced after 3 months of treatment but TAC levels did not change. The observed increase in Hb levels may be associated with reduced oxidative stress and this may contribute to the prolongation of life expectancy of erythrocytes with vitamin E supplementation. Increased oxidative stress as well as iron overload in the liver leads to use of glutathione and might contribute to
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other hand, DNA damage did not change with vitamin E supplementation. The limitations of this research were that the parameters studied were not homogeneous before supplementation and that it was not a double-blind, placebo-controlled, crossover study. However, patients were randomly allocated into the groups and there was no intervention regarding patient distribution. In general, if researchers describe a trial as double-blind, readers can assume that they have avoided bias (39). A comparison of drugs is most readily accepted if the results are from randomized controlled trials. Open-label studies are frequently incorporated in the design of randomized controlled trials. We concluded that increased oxidant status and increased DNA damage in children with transfusion-dependent b-thal can be effectively reduced by antioxidant supplementation, especially with NAC.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. Figure 3. Correlation graphic of serum OSI and mononuclear DNA damage score (r ¼ 0.429, p50.001) before antioxidant supplementation.
References
decreased levels of glutathione in thalassemic patients. Therefore, NAC use in these patients, by increasing intracellular glutathione, may be expected to have a positive influence on oxidative effects. In an experimental study, Amer et al. (33) examined the effects of various oxidants and antioxidants on lipid peroxidation in normal and thalassemic eythrocytes, and they found lipid peroxidation was higher in the thalassemic erythrocytes and these effects were reversed by NAC. We found that the levels of TOS and OSI were decreased, and TAC levels were increased after supplementation. Our findings suggest that NAC supplementation reduces oxidative stress in thalassemic children. We also showed a significant increase in Hb level with a decrease in the amount of blood transfused during the study period. Oxidant damage to erythroid precursors leads to accelerated apoptosis and ineffective erythropoiesis (25). Therefore, there is a possible mechanism of NAC to reduce the level of oxidant status and increased antioxidant capacity as a result of protection of erythrocytes from oxidative damage, thus extending the life-span of erythrocytes. Oxidative DNA damage is one of the pathological consequences of iron. Shires et al. (34) reported that incubation of isolated rat liver cells with iron resulted in fragmentation of single-stranded DNA; iron-mediated DNA damage was required in the formation of reactive forms of oxygen but was not evidence for direct interaction of reactive oxygen with DNA (35). Experimental studies showed that iron plays an important role in hydrogen peroxide associated DNA damage and this damage can be reduced with iron chelation therapies (36,37). Our study showed that DNA damage is higher in thalassemic children than in healthy controls. N-acetylcysteine has an antioxidant effect, an anticarcinogenic effect, and a protective effect towards DNA damage (38). Thus, NAC has proven to be effective in treating increased DNA damage in b-thalassemic children. On the
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