Statistical Analysis
SPSS 20.0 was a program utilized for statistical analysis of collected data. Categorical measurements were determined as number and percentage and numerical computations as mean and standard deviation. The Shapiro-Wilk test was used for the analysis of compliance with the normal distribution. Continuous variables were summarized by mean±standard deviations whereas non-parametric variables were shown as mean value ±standard deviation median (interquartile range). The Wilcoxon signed-rank test was used to compare two related samples. Variance analysis was performed for comparing the three times repeated measurements with normal distribution and Friedman analysis was done for parameters with non-normal distribution. In these tests, as there are three different groups, p<0,0167 is considered as statistically significant. Wilcoxon signed ranks test was also used for the comparisons of the subgroups. According to these comparisons, p values were done as follows: P1, group 1 vs. group 2, P2, group 1 vs. group 3, P3, group 2 vs. group 3. All statistical tests were two-sided. Results were evaluated at 95% confidence interval, and a p<0,05 was considered statistically significant except in comparison of three subgroups.
Results
Our study included 22 girls and 5 boys between 3 months and 15 years of age (mean age 6.9 ± 3.6 years) The mean MNL DNA damage levels were analyzed in 24 children, and the results of the first group, second group, and third group were; 2,125 arbitrary units (AU), 4,222 AU, and, 1,885 AU, respectively (Table 1). There was a statistically significant difference between the first (before scan) and second groups (immediately after scan) (p<0.05) and the second and third group (one week after the scan) (p<0.05) (Figure 1, 2). Overall, the mean DNA damage increased just after the scan and decreased one week after the scan. Power analysis was done by the Gpower statistical program to detect the power of the study by investigating groups with the statistically significant difference in DNA damage levels, between the first and second group and between the second and third group. The effect size was 5.94 for the difference between first and second group; 95% confidence level, power was 1. The effect size was 6.54 for the difference between the first and second group; 95% confidence level was 1.
There were no statistically significant differences between groups in terms of serum TAS, TOS, and OSI values (p>0.05) (Table 1). Due to a storage problem in laboratory, serum TAS levels were evaluated in 18 children and TOS in 15 children.
TAS/Cr, TOS/Cr, NAG/Cr ratios, and OSI levels were performed to all children, and results are illustrated in Table 2. There were no statistically significant differences between the groups (before and after scan) in terms of urine TAS/Cr, TOS/Cr, NAG/Cr ratios, and OSI levels (Table 2).
Discussion
To the best of our knowledge, this is the first study investigating the possible harmful effects of Tc-99m DMSA administration in children in terms of oxidative stress, DNA damage and renal tubular injury. We evaluated the levels of ROS in both urine and serum samples of pediatric patients, serum MNL DNA damage undergoing Tc-99m DMSA scintigraphy and, a urinary biomarker of acute tubular damage (NAG). Our results showed that MNL DNA damage immediately increased after the scan, but measured values decreased to baseline after one week. There were no differences between serum and urine oxidative stress levels before and after the DMSA scan and also, urinary NAG levels did not change.
Our study showed that radiopharmaceuticals might exhibit adverse effects on DNA through the direct impact of ionizing radiation, not by oxidative damage. The effect of Tc-99m DMSA radiopharmaceutical is not sufficient enough to create oxidative damage. Although DNA damage has been repaired, there is no information on the impact of repetitive Tc-99m DMSA or other scintigraphic images on DNA in children. Also we are not sure about some subtle permanent changes in DNA occurred.
DMSA scans are frequently used in the evaluation of recurrent febrile urinary tract infections in the pediatric population [7]. The pediatric nephrologists and urologists carefully follow up the children with renal scars and recurrent urinary tract infections in terms of chronic kidney disease, hypertension and investigate the possible underlying vesicoureteral reflux and try to preserve long term renal function [10]. DMSA scintigraphy administer the considerable amount of radiation. The radiation dose in children aged 1-15 years varies between 0.68 to 1.22 mSV (depending on guidelines used). The dose is approximately 1 mSv/examination regardless of the age of the child, adapted according to body surface compared to a pediatric chest CT which is 2-5mSV. [20-22]. Cellular exposure to ionizing radiation leads to oxidizing events, chronic inflammation and has long term effects on genomic instability [23]. It is well known that radiation has carcinogenic effects and pediatric exposure to radiation causes increased lifelong risk for the solid cancers and leukemia [22].
In recent years, interest in ROS and oxidative stress have increased. ROS has both mutagenic and carcinogenic consequences, affecting many cell components and causing oxidative stress and DNA damage [6]. Studies investigating the oxidative stress and ROS after DMSA scan are limited. Salmanoglu showed that oxidative and nitrosative stress was increased in adult patients who were administrated with 99mTc-DMSA compared to the control group [11]. Studies with adult patients who had undergone different scintigraphic techniques demonstrated that antioxidant enzymes decreased after the injection of radiopharmaceuticals [15, 24]. In our research, we showed that DMSA administration had no effect on the oxidative stress of children.
DNA is considered to be the primary target for the direct and indirect effects of ionizing radiation. The absorption of ionizing radiation can cause disruption of atomic structures, radiolysis of water and subsequently cauing damage to nucleic acids, proteins and lipids [23]. The majority of DNA damage is repaired within minutes or hours after induction. However, sometimes, repair cannot be performed or may be incorrect. Both conditions are considered to be a significant risk for carcinogenesis [25]. For these reasons, it is important to investigate possible DNA damage when examining the effects of nuclear imaging techniques using ionizing radiation. Some clinical studies have shown that a low dose of iodine-131 might cause chromosomal damages [26, 27]. Dantas et al. [28] also demonstrated an increased DNA damage in up to two hours following Technetium-99m-labelled radiopharmaceutical injection, which is subsequently reduced to zero after 24 hours, consisted with the results of our study. Researchers of the same study interpreted this as the effect of DNA repair mechanisms.  In our research, we evaluated DNA damage one week later and found that DNA damage have been reduced, which initially have increased just after radiopharmaceutical administration
In recent years NAG and similar proteins were commonly discussed in the literature. These proteins can be used as biomarkers for the early diagnosis of acute kidney injury [12, 29-30]. Several studies in the literature demonstrated that urinary NAG excretion is a very sensitive parameter in determining renal tubular damage, and levels can change when exposed to hypoxic, toxic, and even radioactive substances [12, 29-31]. It has been affirmed that increased ROS levels are associated with high urinary NAG excretion and renal tubular damage [32]. In our study, we analyzed the possibility of renal tubular influence in addition to oxidative damage in the early period by evaluating urinary NAG excretion. Urinary NAG, TAS, TOS, and OSI levels did not change after the administration of DMSA radiopharmaceutical. The absence of oxidative damage in the urine samples is a consistent finding along with no increase in urinary NAG levels. It also supports the absence of oxidative damage in serum examination. Based on these results, there was no short- or long-term renal tubular damage after the first DMSA scintigraphy. This might be due to the short-acting and low-dose radiation received from Tc-99m DMSA.
There are some limitations to the study. First, the study sample size is small and had a wide age range distribution. Another limitation, due to technical reasons, we were not able to analyze the serum levels of TAS, TOS, and OSI for all children. Also, for ethical concerns we did not include a control group without previous urinary tract infection. Our study group was free of infection for at least six months but the previous infection itself may have influenced renal tubular function and possible confounding if DMSA showed renal scarring, which may reflect kidney fibrosis and/or altered cellular structure/metabolism. Other limitations were; we did not directly quantify the radiation dosage from the DMSA scan and lack of study of other potential imaging studies that might cause similar oxidative stress injury or DNA damage, like CT scans.  Lastly, we did not determine the exact time of the recovery of DNA damage and study the effect of repetitive DMSA scans.
In conclusion, the mononuclear leukocyte DNA damage has increased just after the administration of Tc-99m DMSA radiopharmaceutical in children and normalized after one week. This finding suggest that DNA damage caused by radiopharmaceuticals are reversible. However, this does not exclude smaller permanent changes, which may be demonstrated by other techniques. DMSA scan was not associated with an increased oxidative stress or renal tubular damage in this study. Further researches with larger sample sizes are warranted in the future.