Mutagenesis vol. 26 no. 5 pp. 643–650, 2011
Advance Access Publication 13 June 2011
doi:10.1093/mutage/ger027
Micronucleus frequencies in peripheral blood lymphocytes of children with chronic
kidney disease
Gonca Cakmak Demircigil*, Banu Aykanat,
Kibriya Fidan1, Kaan Gulleroglu2, Umut Selda Bayrakci2,
Aylin Sepici3, Bahar Buyukkaragoz1, Hamdi Karakayali4,
Mehmet Haberal4, Esra Baskin2, Necla Buyan1 and
Sema Burgaz
*
To whom correspondence should be addressed. Tel: þ90 312 2023089; Fax:
þ90 312 222 2326; Email: goncacd@gmail.com
Received on February 06, 2011; revised on March 16, 2011;
accepted on March 16, 2011
One of the crucial adverse effects of chronic kidney disease
(CKD) and its treatment is an elevated cancer risk. There
are no data on cytogenetic effects in children with CKD or
children undergoing dialysis or those who have received
a transplant. In this study, cytogenetic effects in children
with CKD in pre-dialysis (PreD) stage, on regular
haemodialysis (HD) and transplanted (Tx) compared with
a control group of healthy children has been investigated
using the cytokinesis-blocked micronucleus (CBMN) assay
and fluorescence in situ hybridisation (FISH) combined
with CBMN (CBMN-FISH) in peripheral blood lymphocytes. The results revealed a significant increase (P < 0.001)
in micronucleus (MN) frequencies [mean 6 SD (n)] in the
PreD, HD and Tx groups versus the control group [CBMN
assay; 9.19 6 2.61 (16), 9.07 6 4.86 (15), 6.12 6 5.33 (17)
versus 1.60 6 0.99 (20), respectively]. Moreover, centromere negative micronucleus (C2 MN) and centromere
positive micronucleus (C1 MN) frequencies were significantly higher in each subgroup children (PreD, HD and
Tx) than in the control group (P < 0.01) although children
in Tx group had lower C2 MN frequencies than PreD and
lower C1 MN frequencies than PreD and HD groups (P <
0.05). Additionally, MN frequencies in mononuclear cells,
nucleoplasmic bridges and nuclear buds in binucleated
cells were increased in children with CKD (P < 0.001, P <
0.001, P > 0.05, respectively). The nuclear division index
significantly decreased in Tx group relative to the control,
PreD and HD groups (P < 0.001). Associations between
cytogenetic parameters and creatinine or blood urea
nitrogen were found (P < 0.05). To provide longer and
better life expectancy of children with CKD and treatment
modes, further research is needed to better understand and
avoid these effects.
Chronic kidney disease (CKD) is a major public health concern
(1). A high burden of cardiovascular morbidity and mortality
(2), hepatitis B virus and hepatitis C virus infection-mediated
liver damage (3), diabetes (4), chronic and low-grade inflammation (5), neuromuscular disease (6), oxidative stress (7)
and increased incidence of cancer (8,9) are among the
outcomes of the CKD and/or end-stage renal disease.
The adverse health-related outcomes of CKD, and treatment
modes such as haemodialysis (HD) and transplantation, are
subjects of research to clarify the underlying mechanisms. In
paediatric patients, the causes, development and the treatment
strategies of CKD differ from adult patients (10,11). It is well
known that long periods of HD treatment are linked to DNA
damage due to oxidative stress (8), cancer incidence and
cytogenetic effects in peripheral lymphocytes are elevated in
patients with disease (12) and, in addition, susceptibility to
neoplastic disorders is increased as a consequence of prolonged
immunusuppression following transplantation (13).
It is advantageous to use some biomarkers of DNA damage
due to genetic instability to predict cancer risk as well as for
determining the effects of the potential genotoxic chemicals.
Cytogenetic biomarkers are the most frequently used end
points in human population studies (14).
Previous cytogenetic effect studies on CKD and treatment
modes have compared patients with moderate to severe chronic
kidney insufficiency, those on HD, peritoneal dialysis,
haemodiafiltration therapy, patients undergoing daily versus
standard HD (8,12,15–25) and kidney transplant patients (26)
with groups of healthy individuals or those receiving other
treatment modes. Micronuclei in peripheral blood lymphocytes
(PBL) (12,15–18,26) and buccal epithelial cells (8), chromosome aberrations, sister chromatid exchanges (19) and DNA
strand breaks (comet assay) (17,19–25) were the biomarkers
used in these studies, all of which were all carried out on
adults.
To our knowledge, there are no data on genetic damage in
children with CKD. Therefore, our aim was to evaluate the
level of cytogenetic damage of children with CKD including
children in pre-dialysis (PreD), children on regular (HD) and
transplanted (Tx) children using the cytokinesis-blocked
micronucleus (CBMN) assay and CBMN combined with the
fluorescence in situ hybridisation assay (CBMN-FISH) in PBL.
Associations of genetic damage with routine biochemical
parameters such as blood urea nitrogen (BUN), blood
creatinine levels and ferritin were shown in some previous
studies (8,15,16,18,27). Thus, it was also of interest to
determine whether the micronucleus (MN) frequencies are
associated with these biochemical markers in children.
The CBMN assay has been applied to examining the effect
of lifestyle, environmental, dietary and genetic factors on
chromosomal stability and mitotic function in several studies
(14,28). Recent data from a prospective approach showed that
Ó The Author 2011. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
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Department of Toxicology, Faculty of Pharmacy, Gazi University, 06330
Ankara, Turkey, 1Department of Pediatric Nephrology, Faculty of Medicine,
Gazi University, 06560 Ankara, Turkey, 2Department of Pediatric Nephrology,
Faculty of Medicine, Baskent University, 06500 Ankara, Turkey, 3Department
of Biochemistry, Faculty of Medicine, Gazi University, 06560 Ankara, Turkey
and 4Department of General Surgery, Faculty of Medicine, Baskent University,
06500 Ankara, Turkey.
Introduction
G. Cakmak Demircigil et al.
background MN frequencies have predictive value for cancer
risk in healthy subjects (28), affirming the use of this
cytogenetic biomarker in human biomonitoring studies. Thus,
our study will provide information on the implications of both
genomic instability of the disease itself as well as treatmentrelated genotoxic effects in children with CKD.
Materials and methods
Biological sampling
Biological sampling was completed between July 16, 2008 and July 29, 2009
both for children with disease and the control group successively. Venous
blood samples were collected in various sterile tubes for the analysis of
biochemical parameters and cytogenetic end points. The coded blood
specimens for cytogenetic assays were delivered to the laboratory within 2 h
and incubation started immediately. The tests (blood cultures) were performed
under identical conditions. One scorer who was unaware of subjects’ enrolment
status performed all microscopic analysis blindly on the coded slides.
Biochemical parameters
The routine diagnostic and follow-up parameters in kidney disease such as
BUN, creatinine, uric acid, total cholesterol, triglycerides, total protein,
albumin, ferritin, parathyroid hormone, alkaline phosphatase, calcium,
phosphorus, serum iron, homocysteine and C-reactive protein (CRP) were
analysed for all subjects. Plasma Total Antioxidant Status (TAS) Assay
determinations were done by Randox commercial kit (Lot No: 115813; Randox
Laboratories Ltd., Crumlin, UK). Human interleukin-6 (IL-6) was determined
by the Invitrogen Hu IL-6 kit that is a solid-phase sandwich enzyme-linked
immunosorbent assay (ELISA, Lot No: 082702E; Invitrogen Corporation,
Camarillo, CA, USA).
MN analysis
CBMN in PBL. Incubation at 37°C for 72 h was started on the day of blood
sampling. Briefly, whole blood (0.5 ml) from the donors was added to 4.5 ml
culture medium composed of RPMI supplemented with 20% foetal calf serum,
2% phytohaemagglutinin and 0.4% L-glutamine (all provided by Biological
Industries, Kibbutz Beit Haemek, Israel). Binucleated cells were accumulated
by adding cytochalasin B (Sigma, St Louis, MO, USA) to a final concentration
of 6 lg/ml (cytochalasin B in dimethyl sulphoxide) at 44 h. At the end of 72 h,
samples were centrifuged and re-suspended in 0.075 M KCl (Merck, Darmstadt,
Germany) at 4°C for 3 min for hypotonic treatment. Cells were fixed by
methanol–acetic acid (3:1; v/v) four times, dropped onto cold slides, air-dried
and stained with Giemsa–May Grünwald (Merck). In the CBMN method,
micronuclei were scored in binucleated cells according to the scoring and
identification criteria of Fenech et al. (29). The scoring of MN was performed
blindly on the coded slides at 400 magnification (Axioscope 2 Microscope,
Carl Zeiss AG, Goettingen, Germany). MN and micronucleated cell
frequencies, nucleoplasmic bridges (NPB) and nuclear buds (NB) were scored
in 1000 binucleated cells and micronucleated mononuclear cells were scored in
1000 mononucleated cells (30,31). The nuclear division index (NDI), a cell
proliferation index, was calculated by scoring mono-, tri- and tetranucleated
cells while counting 250 binucleated cells and calculated according to
Eastmond and Tucker (32). MN and micronucleated cell frequencies, NPB
and NB were expressed as per thousand (&) binucleated PBL.
CBMN-FISH in PBL. CBMN was carried out as mentioned above. The slides
were washed in 2 saline-sodium citrate buffer (SSC) for 5 min at 37°C and
Table I. Demographic characteristics of the children with CKD and control group
Children with CKD
Control group
n
Age (mean SD)
Gender
Male, n (%)
Female, n (%)
BMI (mean SD)a
ETS exposure, n (%)b
Use of vitamin, n (%)b
Viral infections, n (%)
Vaccination, n (%)b
X-ray exposure (yes/no), n (%)b
TS
PreD
HD
Tx
49
14.29 4.03
17
12.71 5.15
15
15.20 3.17
17
15.06 3.05
20
12.80 3.87
26 (53.1)
23 (46.9)
20.99 8.27
26 (53.1)
26 (53.1)
4 (8.2)
12 (24.5)
20 (40.8)
9 (52.9)
8 (47.1)
20.99 7.56
9 (52.9)
5 (29.4)
1 (5.9)
4 (23.5)
6 (35.3)
7 (46.7)
8 (53.3)
17.61 3.29
11 (73.3)
15 (100.0)
1 (6.7)
4 (26.7)
7 (46.7)
10 (58.8)
7 (41.2)
23.98 10.87
6 (35.3)
6 (35.3)
2 (11.8)
4 (23.5)
7 (42.1)
11 (55.0)
9 (45.0)
17.79 3.27
19 (95.0)
1 (5.0)
0 (0.0)
0 (0.0)
1 (5.0)
For comparisons, one-way ANOVA and Pearson chi-square tests were used. Differences were accepted as statistically significant at a P-value ,0.05. TS, total study
group children with CKD; PreD, children in predialysis; HD, children in haemodialysis; Tx, children transplanted.
a
P , 0.05 control group versus Tx.
b
P , 0.05 control group versus TS, PreD, HD and Tx.
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Subjects
The study group consisted of 49 children with CKD between 2 and 19 years
old. Among them, 17 (9 males and 8 females) were in PreD stage (without
dialysis treatment; PreD), 15 (7 males and 8 females) were on regular HD and
17 (10 males and 7 females) were Tx. The inclusion criteria for the PreD
children were based on a blood creatinine level higher than 1 mg/dL (.1 mg/
dL) and glomerular filtration rate in the range of 15–90 ml/min. The HD group
was chosen from children on regular HD for 4 h three times weekly. All the
children were non-smokers. Children without alcohol intake and malignancy
were included in the study. The control group consisted of 20 healthy children
(11 males and 9 females), between 4 and 18 years old. Age and sex-matched
healthy children free from medication and infection were screened for study
eligibility in face-to-face interviews with the children’s parents. Only one child
had vitamin supplementation in the control group. The parents of the children
were informed previously about the study and all gave their informed consent
prior to their children’s involvement. Detailed questionnaire forms including
age, height, weight, education, cigarette smoking and environmental tobacco
smoke (ETS) exposure, recent diagnostic X-ray examination (yes/no) (3
months prior to the sampling), recent vaccination and sports activity of the
children were completed by the parents.
The information in the questionnaire regarding kidney disease type,
parameters, grade, treatment type, medication and viral infection of the study
group was completed by their physicians. Accordingly, the causes of primary
kidney disease among the children (n 5 49) were neurogenic bladder and
vesicoureteral reflux (n 5 20), congenital urogenital anomalies (n 5 6),
glomerulonephritis (n 5 7), nephrolithiasis (n 5 6), familial Mediterranean
fever (n 5 2), cystic kidney disease (n 5 3) and unknown (n 5 5). The duration
of being in PreD, HD and Tx groups [months standard deviation (SD)] were
41.35 38.83, 29.75 27.68 and 22.35 19.43 months, respectively. Tx
group was under immunosuppressant treatment as follows: ciclosporinmycophenolate mofetil (MMF)–steroid combination (n 5 4), tacrolimus–
MMF–steroid combination (n 5 8), rapamycin–MMF–steroid combination (n
5 1), ciclosporin–MMF combination (n 5 1), tacrolimus–steroid combination
(n 5 1), tacrolimus (n 5 1) and rapamycin (n 5 1). Other than
immunosuppression treatment and vitamin supplementation, 41% of the
children in PreD, HD and Tx groups had been administering antihypertensives,
25% erythropoietin, 31% osteoporosis drugs, 10% iron preparates, 16%
calcium acetate preparates, 16% Shohl’s solution (citric acid and sodium
citrate), 18% calcium citrate, 20% antibiotics and 84% other various drugs such
as analgesics. The demographic data are summarised in Table I. This study was
approved by the local ethical committee of Baskent University, Faculty of
Medicine (07.03.2007 - 07/39).
MN frequencies of children with disease
Statistical analysis
Data analysis was performed by using SPSS for Windows,
version 11.5 (SPSS Inc., Chicago, IL, USA). Whether the
distributions of continuous variables were normal or not was
determined by using Shapiro Wilk test. Levene test was used
for the evaluation of homogeneity of variances. Data expressed
as mean SD for continuous data and nominal variables have
been given as number of cases and/or as percentage (%).
Mean differences were compared by Student’s t-test or oneway analysis of variance (ANOVA), while for independent
groups, the median values were compared using the Mann–
Whitney U-test or Kruskal–Wallis test. When the P-values
from one-way ANOVA or Kruskal–Wallis test statistics were
statistically significant (P , 0.05), post-hoc Tukey or
Conover’s multiple comparison test were used to determine
which groups differ from which others. Whether the differences between C MN and Cþ MN proportions were
statistically significant (P , 0.05) or not was evaluated by
Wilcoxon signed-ranks test. Nominal data were evaluated by
Pearson chi-square or Fisher’s exact test, where applicable.
Degrees of association between continuous variables were
evaluated by Spearman’s rank correlation test. In order to
define the risk factors of MN assay, stepwise linear regression
analyses were used. Any variable whose univariate tests had
statistical significance (P , 0.05) and variables with clinical
importance were accepted as candidate for the multivariable
model. Differences between study groups were evaluated by
multiple linear regression (MLR) analysis, after being adjusted
for factors such as age, gender, body mass index (BMI),
smoking, X-ray exposure, drug usage and sports activity.
Coefficients of regression and 95% confidence intervals for
each independent variable were also calculated. For all
analyses, differences were accepted as statistically significant
at a P-value of ,0.05.
Results
Some general characteristics of the study population are shown
in Table I. Age and gender were consistent among the groups
(P . 0.05). Although children in the study were all nonsmokers, ETS exposure in the control group was higher than in
the PreD, HD and Tx and total study (TS) groups (P , 0.05).
BMI was found to be the highest in the Tx group and
significantly different from the control group (P , 0.05). The
groups were similar according to viral infections over the
previous 2 years. Children in each of the CKD groups had
higher frequency of use of vitamins (multivitamin, folate,
vitamin B and vitamin C), vaccination in the previous year and
X-ray exposure in the previous 3 months compared with the
control group (P , 0.05).
With regard to biochemical parameters, the children with
CKD (TS) and the subgroups of PreD, HD and Tx had
statistically different BUN, creatinine, uric acid, cholesterol,
triglycerides, homocysteine, ferritin and parathyroid hormone
levels from the control group (P , 0.05). The subgroups of
PreD and HD had statistically different albumin and CRP
levels from the control group (P , 0.05). All these parameters
were clinically out of the normal range in PreD, HD and Tx
groups but within the normal range for control group children.
TAS of the control group found to be higher than in the PreD,
HD and Tx groups (not significant, P . 0.05) but the increase
was significant in comparison to TS (P , 0.05). Alkaline
phosphatase in the control group was found to be lower than
the PreD, HD and Tx groups (not significant, P . 0.05) but the
decrease was significant in comparison to TS (P , 0.05).
BUN, creatinine and parathyroid hormone levels were
significantly higher and albumin level was significantly lower
in Tx group than in the HD and PreD groups (P , 0.05).
Ferritin and CRP levels were significantly increased in HD
group relative to the Tx (P , 0.05). Among the groups, there
were no significant differences for IL-6, total protein, calcium,
phosphorus and serum iron values (P . 0.05).
In the CBMN assay, MN and micronucleated cell frequencies in binucleated PBL were significantly higher in PreD, HD,
Tx and TS groups when compared with that of the control
group (P , 0.001, Table II). The statistical results of
micronucleated cells were not shown because of similar results
with MN frequencies. Among the children with CKD, MN
frequencies in HD and PreD groups were significantly higher
than in the Tx group (P , 0.05). In addition, as a measure of
cytotoxicity, NDI of children in the Tx group was found to be
significantly lower than in the other subgroups and control
group (P , 0.001, Table II). According to the NB data, there
was an increase for the subgroups versus the control group
which was not significant (P . 0.05, Table II). MN frequencies
in mononuclear cells were significantly higher in PreD, HD, Tx
and TS groups than in the control group (P , 0.001, Table II).
NPB in the binucleated cells were significantly increased in
HD, Tx and TS groups relative to the control group (P , 0.05,
Table II).
In the CBMN-FISH assay, the total MN count was 344 in
the TS group, of which 202 were Cþ MN (59%), while 142
were C MN (41%). In the control group, out of 35 MN, 22
were Cþ MN (63%) and 13 were C MN (37%). Cþ MN to
C MN percentages (Cþ MN%/C MN%) in the total MN
counts for PreD, HD and Tx groups were 55/45, 65/35 and 56/
44, respectively. The total counted binucleated cells and the
total MN, Cþ MN and C MN counts in CBMN-FISH assay
are shown in Table III. The significant differences among the
groups with regard to MN frequencies (mean SD) are also
shown in Table III. Both Cþ MN and C MN frequencies of
the subgroups of PreD, HD and Tx were significantly higher
than those of the control frequencies (P , 0.05, Table III).
Among the subgroups, Tx group showed the lowest Cþ MN
and C MN frequencies (Table III). In the TS and HD groups,
Cþ MN frequencies were significantly higher than the C MN
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then air-dried at room temperature. Then, they were incubated in pepsin
solution for 7 min at 37°C. The slides were washed in 2 SSC for 5 min at
37°C and then were rinsed in distilled water at 37°C twice. The slides were
dehydrated by washing in 70% ethanol (3 min), 90% ethanol (5 min) and then
air-dried at room temperature. Fifteen microlitre fluorescein isothiocyanatelabelled human chromosome pan-centromeric probe (Cat No.1695-F-01;
Cambio, Cambridge, UK) was applied to slide and covered with a glass
coverslip. Coverslip edges were sealed with a rubber cement. Probes/slides
were denatured at 70°C for 7 min in a hot water bath and were incubated at
37°C overnight. After hybridisation, rubber cement and coverslips were
removed. Then slides were washed in 2 SSC for 5 min at 37°C twice and in
4 SSC for 5 min at 37°C twice. The slides were stained with a counterstain
medium containing 15 ll 4#-6-diamidino-2-phenylindole (Cambio) and were
covered with a glass coverslip. The slides were scored on the coded slides
blindly with fluorescence-equipped microscope (Axioscope 2 Microscope, Carl
Zeiss AG, Goettingen, Germany) at 400 magnification (33,34). The MN
present in the binucleated cells with intact cytoplasm were examined for the
presence of centromeric spots and were classified as centromere positive
micronucleus (Cþ MN) or centromere negative micronucleus (C MN). A
total of 1000 binucleated lymphocytes were scored for each subject. MN
frequencies in CBMN-FISH Assay were expressed as MN per thousand (&)
binucleated PBL and each of the MN has been determined as either C or Cþ.
G. Cakmak Demircigil et al.
Table II. MN, NPB and NB frequencies and NDI levels in children with CKD and control group children in CBMN assay
Children with CKD
Control group
MN frequency in binucleated PBL (&)
Mean SD
Micronucleated cell frequency in binucleated PBL (&)
Mean SD
NPB frequency in binucleated PBL (&)
Mean SD
NB frequency in binucleated PBL (&)
Mean SD
MN frequency in mononucleated PBL (&)
PreD
HD
Tx
8.06 4.58
9.19 2.61
9.07 4.86
6.12 5.33a
1.60 0.99b
7.94 4.48
9.13 2.55
8.87 4.67
6.00 5.24a
1.60 0.99b
1.75 2.96
1.06 2.17
1.27 1.87
2.82 4.05
0.05 0.22c
0.75 1.67
0.94 1.18
0.87 2.64
0.47 0.80
0.40 0.75
6.21 5.12
5.19 3.06
5.40 5.40
7.88 6.18
1.10 0.85b
1.96 0.27
48
2.02 0.19
16
2.16 0.20
15
1.72 0.22a
17
2.01 0.13
20
For comparisons, Kruskal–Wallis and Mann–Whitney U-tests were used. Differences were accepted as statistically significant at a P-value ,0.05. TS, total study
group children with CKD; PreD, children in predialysis; HD, children in haemodialysis; Tx, children transplanted; NDI, (mononucleated cells þ 2 binucleated
cells þ 3 trinucleated cells þ 4 tetranucleated cells)/total number of cells.
a
P , 0.001; Tx versus HD, PreD.
b
P , 0.001; control group versus TS, PreD, HD and Tx.
c
P , 0.01; control group versus TS, HD and Tx.
d
The MN data of one children in PreD were not available due to technical reasons.
Table III. MN, Cþ MN and C MN frequencies in children with CKD and control group children in CBMN-FISH assay
MN frequencies in binucleated PBL (&) (mean SD)
Groupa
Total binucleated cell
TS
PreD
HD
Tx
Control
46
16
15
15
20
103
103
103
103
103
Total MN
Cþ MN
C MN
Total MN
Cþ MN
344
147
119
78
35
202
81
77
44
22
142
66
42
34
13
7.48
9.19
7.93
5.20
1.75
4.39
5.06
5.13
2.93
1.10
3.78
3.69
3.45
3.21c
1.12e
2.38b
1.44
2.45b
2.58c
0.97e
C MN
3.09
4.13
2.80
2.27
0.65
2.47
2.90
2.11
2.02d
0.67e
For comparisons Wilcoxon signed ranks test was used. Differences were accepted as statistically significant at a P-value ,0.05. TS, total study group children with
CKD; PreD, children in predialysis; HD, children in haemodialysis; Tx, children transplanted.
a
Cþ MN and C MN data of three children in CKD were not evaluated due to technical problems. For one child from CKD group and two children from control
group, there were no MN induction.
b
P , 0.02; Cþ MN versus C MN.
c
P , 0.05; Tx versus PreD and HD.
d
P , 0.05; Tx versus PreD.
e
P , 0.02; control group versus PreD, HD and Tx groups.
frequencies (P , 0.05, Table III). MN frequency in PBL was
significantly correlated with Cþ MN frequency (r 5 0.852; P
, 0.001) and C MN frequency (r 5 0.678; P , 0.001) in the
whole study population (disease and control groups).
Univariate analysis indicated that CBMN assay MN frequency
and CBMN-FISH assay Cþ MN and C MN frequencies in
children with diagnostic X-ray exposure (mean SD; 8.65
4.998, 4.53 2.37 and 3.79 2.90, respectively) were not
significantly higher than those in children without diagnostic Xray exposure (mean SD; 7.64 4.31, 4.30 2.43 and 2.59
2.02, respectively) (P . 0.05).
There was no correlation between duration of dialysis and MN
frequencies and between the time since transplantation and MN
frequencies (r 5 0.249, P . 0.05; r 5 0.256, P . 0.05,
respectively). Significant correlations were found between
both BUN and creatinine levels and MN frequencies in the
CBMN assay (r 5 0.379, P 5 0.008; r 5 0.298, P 5 0.040,
respectively) and between BUN or creatinine levels and Cþ MN
frequencies in the CBMN-FISH assay (r 5 0.374, P 5 0.010 and
r 5 0.337, P 5 0.022, respectively) for the TS group (P , 0.05).
646
Multiple regression analysis showed that PreD (P , 0.001),
HD (P , 0.001) and Tx (P , 0.001) groups had significantly
influenced MN frequency in PBL (Table IV). Similarly,
statistically significant parameters affecting both Cþ and C
MN frequencies were detected as PreD, HD and Tx groups.
Additionally diagnostic X-ray examination had a negative
significant effect on C MN frequency (Table V).
Discussion
Among the wide range of health effects associated with CKD, the
risk of cancer is under investigation in order to elucidate the
underlying mechanisms and to prevent or reduce the risk of the
disease from treatment modes, such as dialysis and transplantation.
It is well known that kidney transplantation and dialysis are
the most common treatments for people with CKD (35). In case
of kidney transplantation, immunosuppressives are used to
prevent immunologic rejection. However, patients undergoing
immunosuppressive therapy are at risk of developing cancer
(26, 36). There is a trend towards increasing incidence and
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NDI
Mean SD
nd
TS
MN frequencies of children with disease
earlier occurrence of post-transplant lymphoproliferative disorder in the paediatric kidney transplant population (37).
Similarly, patients undergoing dialysis treatment carry an
increased cancer risk (38).
The aim of our study was to evaluate cytogenetic effects as
one of the signalling or triggering parameters of likely cancer
development for paediatric patients. The 10-fold increased
cancer risk compared to the general population among children
with end-stage renal disease (38), causative differences from
adult patients (10), lower quality of life scores (39), possibility
of growth retardation and post-transplant malignancy (40) as
well as dialysis and immunosuppression treatment for
relatively long periods were the reasons to carry out such
a study on children.
PreD
HD
Tx
Age
Gender
BMI
ETS exposure
Sports (h)
Vitamin (yes/no)
X-ray exposure
Medication 1
Medication 2
Medication 3
Medication 4
BUN
TAS
MN frequency in PBL (&), R2 5 0.577
B (95% CIs)
P
1.243
1.187
0.790
0.008
0.218
0.016
0.201
0.021
0.175
0.125
0.164
0.014
0.005
0.126
0.004
0.253
,0.001
,0.001
,0.001
0.655
0.107
0.112
0.215
0.669
0.405
0.415
0.283
0.956
0.979
0.649
0.192
0.214
(0.756 to 1.731)
(0.483 to 1.890)
(0.320 to 1.260)
(0.046 to 0.029)
(0.049 to 0.485)
(0.004 to 0.037)
(0.523 to 0.120)
(0.079 to 0.122)
(0.243 to 0.593)
(0.432 to 0.181)
(0.468 to 0.140)
(0.497 to 0.526)
(0.362 to 0.371)
(0.425 to 0.676)
(0.002 to 0.011)
(0.655 to 0.150)
PreD, children in predialysis; HD, children in haemodialysis; Tx, children
transplanted; BMI, body mass index; medication 1, antihypertensives,
medication 2, erythropoietin; medication 3, osteoporosis drugs; medication 4,
iron preparates; B, regression coefficient; CIs, confidence intervals. A P-value
,0.05 accepted as statistically significant.
Table V. MLR analysis of Cþ MN and C MN frequencies in children with CKD and control group children in the CBMN-FISH assay
Cþ MN frequency in PBL (&), R2 5 0.449
PreD
HD
Tx
Age
Gender
BMI
ETS exposure
Sports (h)
Vitamin
X-ray exposure
Medication 1
Medication 2
Medication 3
Medication 5
BUN
C MN frequency in PBL (&), R2 5 0.402
B (95% CIs)
P
1.091
1.053
0.649
0.031
0.050
0.019
0.156
0.070
0.292
0.003
0.078
0.442
0.091
0.178
0.004
,0.001
0.005
0.008
0.119
0.729
0.245
0.355
0.186
0.185
0.984
0.642
0.145
0.641
0.547
0.279
(0.570 to 1.612)
(0.337 to 1.769)
(0.174 to 1.123)
(0.071 to 0.008)
(0.337 to 0.238)
(0.014 to 0.052)
(0.492 to 0.180)
(0.035 to 0.176)
(0.144 to 0.728)
(0.340 to 0.347)
(0.411 to 0.256)
(1.041 to 0.157)
(0.480 to 0.298)
(0.412 to 0.768)
(0.004 to 0.012)
PreD
HD
Tx
Age
Gender
BMI
ETS exposure
Sports (h)
Vitamin
X-ray exposure
Medication 1
Medication 2
Medication 3
Medication 4
BUN
B (95% CIs)
P
1.125
1.037
0.496
0.029
0.241
0.014
0.002
0.049
0.155
0.432
0.101
0.465
0.250
0.243
0.004
,0.001
0.006
0.043
0.151
0.094
0.393
0.989
0.342
0.486
0.012
0.544
0.095
0.211
0.403
0.233
(0.605 to 1.646)
(0.307 to 1.767)
(0.017 to 0.975)
(0.011 to 0.070)
(0.043 to 0.526)
(0.048 to 0.019)
(0.336 to 0.341)
(0.152 to 0.054)
(0.289 to 0.600)
(0.765 to 0.100)
(0.231 to 0.433)
(0.084 to 1.013)
(0.648 to 0.147)
(0.823 to 0.336)
(0.012 to 0.003)
PreD, children in predialysis; HD, children in haemodialysis; Tx, children transplanted; BMI, body mass index; Medication 1, antihypertensives; Medication 2,
erythropoietin; Medication 3, osteoporosis drugs; Medication 4, iron preparates; Medication 5, calcium acetate. B, regression coefficient; CIs, confidence intervals. A
P-value ,0.05 accepted as statistically significant.
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Table IV. MLR analysis of MN frequencies in children with CKD and control
group children in the CBMN assay
Neri et al.. (41) have reviewed the effect biomarker studies
conducted on children for environmental exposures or disease
conditions up to 2006 (41). From 2006 to the present, a few
more studies have been published to our knowledge (42–52)
but none of these were studies of children with CKD.
In the present study, significantly increased MN frequencies in
children either in PreD or under treatment (HD and Tx) were
found. Furthermore, the highest and most significant effect on
MN frequencies was observed in PreD group of children
followed by HD and Tx groups, as evaluated with MLR analysis.
Our study concurs with increased cytogenetic effects with
regards to MN frequencies either in CKD or in treatment modes
of adult patients presented in previous studies (12,15–18,26).
Also, we have shown significant increases in the NPB and
MN frequencies in mononuclear cells, whereas there were nonsignificant increases in NB in study group children. NPB has
been accepted as the biomarker of DNA misrepair and/or
telomere end fusions and NB as the biomarker of elimination of
amplified DNA and/or DNA repair complexes (30). Gene
amplification is thought to play an important role in tumour
progression (53). Evaluation of MN frequency of mononuclear
cells in biomonitoring studies was suggested to give a more
comprehensive insight into the CBMN assay (31). Identifying
chromosomal rearrangements such as NPB and NB in the
CBMN assay with regards to its ‘cytome assay’ nature (54)
could be presumed to provide mechanistic insight which could
be investigated further in future studies. In this study, two
measures of MN frequency were used, i.e. the mean frequency
of MN per thousand cells and the mean frequency of cells
bearing at least one MN per thousand cells. The two indexes
have been considered to give similar performances; mean
frequency of MN is potentially more sensitive; however,
frequency of cells with MN is more stable (55). In our study,
both of these parameters were found to follow a similar trend.
The finding of cytotoxicity with significant reduction of NDI
only in the Tx group could be attributable to immunosuppression treatment in addition to disease-related effects. Likewise,
Oliviera et al. (26) showed decreased cytokinesis-block
proliferation index in kidney Tx patients undergoing immunosuppression therapy. Moreover, in the in vitro part of the study
G. Cakmak Demircigil et al.
648
In the present study, the time since diagnosis of the disease,
start of dialysis and transplantation until biological sampling
were relatively shorter than in adult studies of CKD
(8,20,21,24) and the correlation between these parameters
and MN frequencies were not significant. In the literature,
different results were reported according to the association of
dialysis duration with cytogenetic effects (8,20,21,24). Significant associations have been found with DNA damage by the
comet assay in the study of Stopper et al. (20) and with buccal
MN frequency in the study of Roth et al. (8), whereas in the
study of Kan et al. (24), there was no correlation results of the
comet assay. Conversely, in the study of Stoyanova et al. (21),
the correlation was negative between DNA damage (comet
assay) and dialysis duration. Differences with regards to the
size of the populations, age range and treatment factors could
be reasons for inconsistent associations.
The size of the patient groups versus control groups in the
CBMN assay studies were 35 versus 23 in the study of Stopper
et al. (15), 12 (without healthy control) in the study of Kobras
et al. (17), 25 versus 12 in the study of Fragedaki et al. (16), 14
versus 20 in the study of Oliviera et al. (26) and 201 versus 57
in the study of Sandoval et al. (18), whereas it was 48 versus
20 in our study on children. Roth et al. (8) assessed MN
frequencies in buccal epithelial cells in a group of 40 patient
versus 40 controls. One of the limitations of our study could be
the population size of the study even though it is not outside
the range of the aforementioned adult study populations.
The consistency of the age and gender among the study
and control groups and the participation of non-smokers only
in the study eliminates the most important confounding
factors for cytogenetic effect end points especially for
CBMN assay (60). Although ETS exposure affects a higher
proportion of the control group, it did not affect the effect
biomarkers assessed. Conducting a children study could be
assumed to have advantage of removing the probable effects
of secondary diseases in the study population, such as
cardiovascular disease and cancer. For instance, a high
incidence of cardiovascular and cancer pathologies among
the CKD patients in the study of Sandoval et al. (18) was
reported. So the data from our study would reflect the
disease- and treatment-mediated cytogenetic effects. Vral
et al. (61) suggested the CBMN assay as a validated and
standardised assay to evaluate in vivo radiation exposure of
occupational, medical and accidentally exposed individuals
but because of the variable MN background frequency, only
exposures in excess of 0.2–0.3 Gy X-rays could be detected
(61). In our study, while 40% of the children in CKD group
had diagnostic X-ray exposure, univariate and multivariate
analyses demonstrated no effect of X-ray exposure. Overall,
X-ray exposure seemed to have no apparent confounding
effect on MN frequencies in the present study.
As a conclusion, we presented the first cytogenetic effect
findings on children with CKD in different treatment modes,
namely PreD, HD and Tx. The present study supplied
a comprehensive picture of cytogenetic effects in the study
group. From technical and mechanistic viewpoints, many
advantages of the MN assay, such as evaluating the nuclear
anomalies (NPB, NB), cytotoxicity (NDI), clastogenic (C
MN) and aneugenic (Cþ MN) outcomes, have been shown.
Assuming the kidney disease status, dialysis and posttransplantation immunosupression treatment are more crucial
in childhood, the observed clear cytogenetic effect in our study
could provide valuable insight into the likely cancer risk and
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of Oliviera et al. (26), increased MN frequencies for
mycophenolate mofetil (MMF)- and tacrolimus-treated cells
and a significant cytotoxic effect in MFF supplemented
cultures have been shown (26). Almost all Tx children in our
study had MFF and/or tacrolimus treatment, so the results
gained from the study of Oliviera et al. (26) supported our
findings.
As aneuploidy is a major cause of human reproductive
failure and an important contributor to cancer, any increase in
its frequency should be identified and controlled (56). In the
CBMN-FISH assay, labelling the centromeric region of the
chromosomes with anti-kinetochore antibody (56), pan-centromeric probe (45) or centromeric probe (34) allows distinction
between MN containing a whole chromosome (Cþ MN) and
an acentric chromosome fragment (C MN) (34). Thus, the
MN assay could give valuable mechanistic data for children
with disease status and treatment modes.
A further mechanistic step, according to our CBMN-FISH
assay data, is that individuals in each of the CKD groups
showed increases in both clastogenic and aneugenic events
relative to the control group. Moreover, the aneugenic effect in
the HD group has been found to be more significantly
contributing to the total MN frequency. None of the
cytogenetic studies on adults with CKD (12,15–18) used
centromeric probes to analyse the mechanism of MN
formation.
As with the CBMN assay, the MLR analysis revealed that
the most important factors shaping the aneugenic and
clastogenic effects were for children being in the disease or
treatment group. Recent diagnostic X-ray exposure of children
was also found to affect the C MN frequency but negatively.
Thus, children with CKD or children under treatment may
have an increased probability of cancer development according
to the cytogenetic effects measured by MN assay. This cancer
susceptibility would be more crucial than for similar cytogenetic effects in adults with CKD since children with the disease
have a longer period of life in front of them with the
expectancy of better health-related life quality.
In that respect, the presence of chronic infection, previous
treatment with immunosuppressive or cytotoxic drugs, nutritional deficiencies, viral-associated factors, increased levels of
oxidative stress, reduced antioxidant levels, accumulation of
uraemic toxins and altered DNA repair are suggested as
reasons for increased cancer risk (12,17,18,35) and these
factors could also be responsible for the cytogenetic effects
observed in our study.
With regard to the biochemical parameters, only BUN and
creatinine levels showed significant correlations with MN
frequencies in the children with CKD in our study. Sandoval
et al. (18) also found an association between creatinine levels
and MN frequencies in CKD in a large population size study
(201 patients), which has been interpreted as confirmation of
the association between the degree of renal failure, and its
progression, with genetic damage. Among these parameters,
TAS, ferritin and uraemic state have been associated with
oxidative stress-mediated genetic damage (27,57,58) and
consequently to an increase in MN formation (8,15,16).
Markers of inflammation such as IL-6 and CRP were found
to be significantly higher in children with CKD in the study of
Ece et al. (59). In our study, IL-6 levels were found to be nonsignificantly increased in children with disease. Our study
group deserves long-term follow-up programmes for determination of future health status and progression of the outcomes.
MN frequencies of children with disease
also into the prevention actions and improvements for the
treatment approaches for paediatric patients.
Recommendations from previous studies, such as controlling
the oxidative stress by vitamin supplementation, effective
removal of uraemic toxins, inhibiting the advanced glycation
end products, tailored medication regimes, daily HD and
controlling for HD conditions, antihypertensive therapy with
angiotensin blockade and enrichment of the enzyme polymorphism effects on the disease outcome and progression (16–
18,27,57,58), should also be considered for children. Our
findings point to the need for further studies in order to
eliminate or reduce the cytogenetic effects.
Funding
Acknowledgements
The authors are grateful to the all of the children for their participation in this
study. We would like to thank Mr Salih Ergocen for performing the statistical
analysis.
Conflict of interest statement: None declared.
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