1. Healthcare

Mutation Research 491 (2001) 25–30
Cytogenetic analysis of children under long-term antibacterial
therapy with nitroheterocyclic compound furagin
G. Slapšyt˙e a,∗ , A. Jankauskien˙e b , J. Mierauskien˙e a , J.R. Lazutka a
a
b
Department of Botany and Genetics, Vilnius University,
ˇ
21 Ciurlionis
St., 2009 Vilnius, Lithuania
Department of Pediatrics, Vilnius University Children’s Hospital,
7 Santariški˛u St., 2600 Vilnius, Lithuania
Received 6 June 2000; received in revised form 14 November 2000; accepted 19 November 2000
Abstract
Cytogenetic analysis of chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) was performed in 109
blood samples from 95 pediatric patients with urinary tract infections (UTIs). Children were exposed to diagnostic levels
of X-rays during voiding cystourethrography and subsequently treated for one to 12 months with low doses of furagin —
N-(5-nitro-2-furyl)-allylidene-1-aminohydantoin. Furagin is 2-substituted 5-nitrofuran, chemically and structurally similar to
well-known antibacterial compound nitrofurantoin. Increased frequencies of CAs were found in children undergoing voiding
cystourethrography as compared with the unexposed, acentric fragments being the most frequent alteration (2.03 versus 0.88
per 100 cells, P = 0.006). However, a significant decrease in the frequency of acentric fragments was determined with the time
elapsed since X-ray examination was performed. A time-independent increase in SCE frequency was found in lymphocytes of
children treated with furagin. Total CA frequency did not differ significantly between groups of children with various duration
of furagin treatment. However, frequency of chromatid exchanges (triradials and quadriradials) increased significantly with
duration of treatment. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: Furagin; Chromosome aberration; Sister chromatid exchange; Human lymphocytes; Ionizing radiation
1. Introduction
Many nitrofurans are known for their excellent
antibacterial action and, therefore, are widely used in
clinical [1] and veterinary [2] practice. Furagin (furazidine) — N-(5-nitro-2-furyl)-allylidene)-1-aminohydantoin, CAS No. 1672-88-4 — is 2-substituted
5-nitrofuran chemically and structurally similar to the
well-known antibacterial compound nitrofurantoin.
∗ Corresponding author. Tel.: +370-2-332-864;
fax: +370-2-333-844.
E-mail address: [email protected] (G. Slapšyt˙e).
As nitrofurantoin, furagin is used in the therapy of
urinary tract infections (UTIs). It is used, however,
mainly in states of the former Soviet Union [3] and
some eastern European countries [4]. Clinical trials
in some western European countries were performed,
too [5].
Data on genotoxicity of furagin are scarce. In some
papers, mutagenicity of furagin in E. coli has been
described [6–8]. In one study, genotoxicity of nitrofurantoin and furagin at equimolar concentrations
was compared using the Ames test, chromosome
aberrations (CAs) and sister chromatid exchanges
(SCEs) in cultured human lymphocytes, sex-linked
1383-5718/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 3 - 5 7 1 8 ( 0 1 ) 0 0 1 3 1 - 0
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G. Slapšyt˙e et al. / Mutation Research 491 (2001) 25–30
recessive lethals and dominant lethals in Drosophila
melanogaster [9]. It appeared that furagin and nitrofurantoin had similar genotoxic potencies. No studies of patients receiving furagin therapy have been
performed until now.
Contrary to furagin, there are numbers of studies
concerning genotoxicity of its congener nitrofurantoin
in vitro and in vivo. Nitrofurantoin was clearly mutagenic in vitro for bacteria [10] and mammalian cells
[11]. Nitrofurantoin induced chromosome damage in
rodent bone marrow in vivo [12], but no induction of
somatic gene mutations in mouse embryos [13] and
CA in spermatocytes [14] was detected. One study
performed with people showed that nitrofurantoin
does not cause detectable cytogenetic abnormalities
in lymphocytes of adult UTI patients [15].
Urinary tract infection is one of the most prevalent form of infectious diseases among children.
Recurrence of UTI is common in susceptible individuals, especially in those with vesicouretheral reflux.
Long-term low-dose antimicrobial prophylaxis often
is necessary in such patients. Nitrofurans, mostly
nitrofurantoin, are used effectively in preventing recurrences of UTI for many years [16]. Use of furagin
for these purposes have also been suggested [3]. Since
such low-dose antimicrobial therapy may last up to 12
months, it is quite important to know whether furagin
is able to induce cytogenetic damage in lymphocytes
of the treated patients.
Precise diagnosis of UTI in children is unavoidably associated with cystourethrography. Although
X-ray doses received during medical examinations
are usually low, exposures to even such low levels
of ionizing radiation have been reported to produce
chromosome damage in lymphocytes of the exposed
persons. Significant increase of chromosome damage
was reported in patients undergoing gastro-intestinal
studies [17], mammography [18], cardiac catheterization and computed tomography [19], and in patients monitored with multiple chest fluoroscopies
[20].
In the present study, two cytogenetic endpoints
(CA and SCE) were used as indicators of the chromosome damage in peripheral blood lymphocytes
of pediatric UTI patients exposed to diagnostic levels of X-rays during voiding cystourethrography and
subsequently treated with low doses of furagin for one
— 12 months.
2. Materials and methods
2.1. Patients
One hundred and nine blood samples were taken
from 95 children suffering from UTI (mainly pyelonephritis and chronic cystitis) and hospitalised in
Vilnius University Children’s Hospital. With parental
consent an extra 2-ml blood samples from children
were obtained when blood was collected for routine
biochemical monitoring. Their ages ranged from 0.2
to 13 years, with a mean of 5.9 ± 0.3 years. The blood
urea nitrogen and creatinine levels of all patients were
within the normal range: blood urea nitrogen levels
ranged from 3.0 to 6.9 mmol/l; creatinine from 0.03
to 0.08 mmol/l.
Twenty-three children were studied at the diagnosis
and had no previous medication. Others had received
antibiotics, mainly ampicillin (50–100 mg/kg per day)
or gentamicin (3 mg/kg per day) for periods up to 7
days.
All patients had undergone voiding cystourethrography (effective dose equivalent to the procedure involving two film exposures was 0.4–2.4 mSv) before
the long-term antimicrobial therapy with furagin.
The treatment with furagin consisted of oral administration in doses of 5–8 mg/kg per day for the first 7
days of treatment, and in doses of 1–2 mg/kg per day
for rest of the days.
Blood sampling was done before cystourethrography (25 samples), just after cystourethrography
(32 samples), after one (17 samples), three (7 samples), six (8 samples) and 12 months (20 samples) of
the therapy.
2.2. Cytogenetic procedures
Blood samples were obtained by venipuncture and
collected into heparinized syringes. For each subject,
three lymphocyte cultures were usually set up according to conventional techniques. Cultures were made
in RPMI 1640 medium supplemented with 12% newborn calf serum, 7.8 ␮g/ml PHA, 100 U/ml penicillin,
100 ␮g/ml streptomycin. All reagents were purchased
from Sigma (St. Louis, MO, USA). The cells were
grown at 37◦ C for 72 h in complete darkness. Cultures
assigned to sister chromatid exchange assay were
treated with 10 ␮g/ml 5-bromo-20 -deoxyuridine for
G. Slapšyt˙e et al. / Mutation Research 491 (2001) 25–30
the entire culture period and colchicine (0.6 ␮g/ml)
for the last 3 h of incubation. In the cultures assigned
to chromosome aberration analysis colchicine at a
final concentration of 0.25 ␮g/ml was present during
the entire culture period. In such cultures even cultivated for 72 h, the majority (95–97%) of metaphases
were in the first-mitotic division. This methodology
was recommended and described by Chen and Zhang
[21]. The authors showed that colchicine or colcemide
added for the entire culture period may be used to obtain pure populations of first-division cells. Moreover,
in such cultures mitotically arrested first-division
cells represent lymphocytes with fast and slow proliferation rate, and thus a non-random selection of cell
populations may be avoided. The suitability of the
method in cytogenetic studies of human populations
was supported by other authors [22,23]. Cultures
were harvested by centrifugation, followed by 20 min
hypotonic treatment (0.075 M KCl) and three periods
of fixation in ethanol–glacial acetic acid (3:1). Flame
dried chromosome preparations were made, and a
modification of the fluorescence plus Giemsa method
was applied to obtain the sister chromatid differential
staining [24].
27
aberrations, including at least one exchange (polycentric, ring chromosome, translocation or inversion)
were defined as rogue cells. Chromosome damage
present in rogue cells was not included in the analysis. SCEs were scored in 25–50 second-mitotic
division cells per individual. Analysis of variance
(ANOVA) and Student’s t-test was performed using
SPSS/PC + statistical package. Chromosome aberration data were transformed using average square-root
transformation Y = 0.5[(X)1/2 + (X + 1)1/2 ], where
X is the number of chromosome damage per 100
cells, Y the transformed variable. SCE data were
log-transformed before statistical analysis.
3. Results
The mean frequencies of CA were higher in the
group of children after cystourethrography than in
the group of children before the procedure with significant differences for chromatid breaks (1.29 versus
0.71, P = 0.0326) and acentric fragments (2.03
versus 0.88, P = 0.0060; Table 1). The rates of
dicentrics and chromatid exchanges were similar in
both groups. Increased frequency of CA was evident in groups of children either previously treated
or untreated with antibiotics (Table 2). ANOVA of
the CA data was conducted with subjects grouped by
exposure to X-rays and previous medication. Statistically significant influence of X-irradiation was found
(P = 0.0023), while previous treatment with antibiotics was found to be non-significant (P = 0.9450).
2.3. Scoring criteria and statistical methods
CA were scored on coded slides. As a rule, no less
than 100 first-mitotic division cells per individual were
analysed. Detailed scoring criteria were described in
our recent paper [24]. Gaps were not included into the
analysis. Cells having five or more chromosome-type
Table 1
Chromosome aberrations (CAs) in lymphocytes of children with urinary tract infection before treatment and treated with furagin
Treatment
duration (months)
Number
of patients
Chromosome aberrationsa per 100 cells ± S.E.M.
0
0
1
3
6
12
24b
0.71
1.29
1.41
1.43
1.57
1.29
31c
17
7
7
20
a
ctb
cte
±
±
±
±
±
±
0.14
0.19d
0.28
0.30
0.30
0.26
0.04
0.06
0.06
0.07
0.29
0.23
ace
±
±
±
±
±
±
0.04
0.04
0.06
0.07
0.18
0.09
0.88
2.03
1.65
1.43
1.36
1.13
dic
±
±
±
±
±
±
0.17
0.30e
0.32
0.43
0.58
0.33
0.04
0.06
0
0.14
0
0.10
ctb: Chromatid breaks; cte: chromatid exchanges; ace: acentric fragments; dic: dicentric chromosomes.
Children both before cystourethrography and treatment with furagin.
c Children after cystourethrography but before treatment with furagin.
d P = 0.0326.
e P = 0.006, Student’s t-test.
b
Total
± 0.04
± 0.04
± 0.09
± 0.10
1.67
3.45
3.12
3.07
3.21
2.75
±
±
±
±
±
±
0.20
0.40
0.55
0.66
0.74
0.46
G. Slapšyt˙e et al. / Mutation Research 491 (2001) 25–30
28
Table 2
Chromosome aberrations and sister chromatid exchanges (SCEs) in children with urinary tract infection before and after X-ray examination
Treatment with
antibiotics
CA per 100 cells ± S.E.M.
Before X-ray
After X-ray
Before X-ray
After X-ray
Not treated
Treated
1.72 ± 0.33
= 11)
1.61 ± 0.24 (N = 13)
3.40 ±
(N = 10)
3.47 ± 0.52b (N = 21)
6.45 ± 0.41 (N = 12)
6.86 ± 0.37 (N = 13)
6.40 ± 0.48 (N = 10)
6.91 ± 0.38 (N = 19)
a
b
(Na
SCE per cell ± S.E.M.
0.64b
N: number of cases.
P < 0.05 when compared to X-ray untreated children, Student’s t-test.
Table 3
Sister chromatid exchanges in lymphocytes of children with urinary tract infection before treatment and treated with furagin
Treatment duration (months)
Number of patients
SCE per cell ± S.E.M.
0
1
3
6
12
54a
6.70
8.26
7.67
7.37
8.37
15
2
5
12
a
b
±
±
±
±
±
0.20
0.45b
1.13
0.59
0.36b
Range
4.60–11.94
5.01–11.16
6.54–8.80
6.28–9.48
6.82–11.12
Children both after and before cystourethrography but before treatment with furagin.
P < 0.05, Student’s t-test.
No effects of X-ray examination and treatment with
antibiotics on the SCE frequency was found (Table 2).
Results of the analysis of CA and SCE in lymphocytes of children before treatment and treated with
furagin are shown in Tables 1 and 3. There was timeindependent increase of the frequency of SCE in lymphocytes of the treated children. Total CA frequency
did not differ significantly between various groups of
children. However, analysis of different types of CA
revealed two statistically significant trends. First, frequency of chromosome breaks and acentrics dropped
significantly with the time elapsed since X-ray examination (Fig. 1). This dependency may be described by
the equation Y = 1.793 − 0.062X, (r 2 = 0.77), where
Y is the number of chromosome breaks per 100 cells, X
the time (in months) elapsed after X-ray examination.
Second, frequency of chromatid exchanges (triradials
and quadriradials) increased significantly with duration of treatment with furagin (Y = 0.066 + 0.019X,
r 2 = 0.83, where Y is the number of chromatid exchanges per 100 cells, X the duration of treatment with
furagin in months; Fig. 2).
Six rogue cells (cells with multiple chromosometype aberrations) were detected in four children. Two
children with rogue cells were before any treatment,
one child was after 6 months and one child after 12
months of chemotherapy with furagin. The damage
consisted of multiple dicentric, tricentric and tetracentric chromosomes as well as of numerous acentric fragments, many of which with the appearance of
“double minutes”.
Fig. 1. Dependence of the frequency of chromosome breaks (csb)
on the time (in months) elapsed since X-ray examination. Mean
group values (black dots) and standard errors of the mean (bar
lines) are shown. Solid line shows linear regression fit, and dotted
lines — 95% confidence limit of the regression line.
G. Slapšyt˙e et al. / Mutation Research 491 (2001) 25–30
Fig. 2. Dependence of the frequency of chromatid exchanges (cte)
on the duration (in months) of furagin therapy. Mean group values
(black dots) and standard errors of the mean (bar lines) are shown.
Solid line shows linear regression fit, and dotted lines — 95%
confidence limit of the regression line.
4. Discussion
The results of the cytogenetic analysis indicate
that children undergoing voiding cystourethrography
and exposed to 0.4–2.4 mSv exhibit frequencies of
CA statistically higher than observed in unexposed
children, acentric fragments being the most frequent
alteration. Our results are in agreement with other
studies. Investigations on radiation workers exposed
to low doses between 0.25 and 3.3 mSv [25] and radiation exposed hospital workers — dose ranges from
1.6 to 42.7 mSv [26], revealed a significant increase
in acentric fragments as compared to the controls.
The differences, however, were not significant for
dicentric and ring chromosomes. More pronounced
increase in acentric fragments as compared to dicentrics has been assumed to be due to the very low
dose rates of exposure of the individuals in these studies, and acentric fragments were considered to be the
best indicators of irradiation at low doses (for doses
below 50 mSv) [26]. In general, our results confirm
this statement. However, we observed a significant
decrease in the frequency of acentric fragments with
the time elapsed since X-ray examination. Recently,
29
we have reported similar decrease of acentric fragments in lymphocytes of another group exposed to
ionizing radiation — Chernobyl clean-up workers
[23]. Thus, it seems that acentric fragments are good
indicators of low-dose irradiation only when effects
of relatively recent exposure are studied.
Demonstration of elevated levels of CA in children
undergoing voiding cystourethrography is a further
evidence that diagnostic irradiation involves exposures with consequential risk to health. Chromosome
damage is suggested as a relevant biomarker of cancer
risk [27]. Thus, cystourethrography in children may
be considered as cancer risk factor. However, current
epidemiological data on associations between diagnostic X-ray examination of the children and cancer
are quite contradictory [28,29].
We also found that long-term antibacterial therapy
with furagin induced chromosomal damage and SCEs
in lymphocytes of the treated children although this
increase is not so evident. Increase of the frequency
of SCE in lymphocytes of children treated with furagin was not time-dependent. Perhaps, the reason why
no increase of SCEs was found after 3 and 6 months
of therapy may be attributed to the fact that insufficient number of patients was analysed. Situation
with CA was even more complicated by two opposite trends: time-dependent decrease of chromosome
breaks induced by X-ray examination, and treatment
duration-dependent increase in the frequency of chromatid exchanges.
Our previous results [9] indicated that CA and SCEs
were induced in human lymphocytes in vitro after
continuous treatment with 5–40 ␮M of furagin. It also
induced CA when treated for the last 3 h of culture
period, however, much higher doses (150–250 ␮M)
were required. Consequently, furagin should be considered as S-independent genotoxic agent and its
genotoxicity in vivo might be expected. We also found
[9] that genotoxicity profile of furagin is comparable
with that one of nitrofurantoin (furagin congener).
One study in vivo with adult UTI patients treated
with nitrofurantoin showed no induction of chromosome damage [15]. Since both nitrofurantoin and
furagin are interchangeable in a sense of their antimicrobial efficiency [3], further use of nitrofurantoin in
treatment of UTIs might be recommended. It must be
noted, however, that treatment doses in this study and
in our present study were quite different: adult UTI
30
G. Slapšyt˙e et al. / Mutation Research 491 (2001) 25–30
patients were treated with 10 mg/kg per day of nitrofurantoin for 10 days, and children were treated with
1–2 mg/kg per day of furagin for 1–12 months. Thus,
further studies are needed to elucidate the genetic
safety of therapeutic use of furagin and nitrofurantoin.
[16]
[17]
[18]
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