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.