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Molecular cytogenetic evaluation of the aneugenic effects of teniposide in

somatic and germinal cells of male mice

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 Mutagenesis Advance Access published August 6, 2011
Mutagenesis pp. 1–9, 2011
Molecular cytogenetic evaluation of the aneugenic effects of teniposide in somatic and
germinal cells of male mice
Sabry M. Attia*
Department of Pharmacology, College of Pharmacy, King Saud University, PO
Box 11451 Riyadh, Saudi Arabia.
*
To whom correspondence should be addressed. Tel: þ966 542927708; Fax:
þ966 14677200; Email: attiasm@yahoo.com or attiasm@ksu.edu.sa
Received on April 25, 2011; revised on June 18, 2011;
accepted on July 2, 2011
The ability of topoisomerase II inhibitor, teniposide, to
induce aneuploidy and meiotic delay in somatic and
germinal cells of male mice was investigated by fluores-
cence in situ hybridisation (FISH) assay using labelled
DNA probes and 5-bromo-2#-deoxyuridine (BrdU) in-
corporation assay, respectively. Colchicine and mitomycin
C were used as a positive control aneugen and clastogen,
respectively, and these compounds produced the expected
responses. Using FISH assay with centromeric and
telomeric DNA probes for erythrocyte, micronuclei (MN)
showed that teniposide is not only clastogenic but also
aneugenic in somatic cells in vivo. The assay also showed
that chromosomes can be enclosed in the MN before and
after centromere separation. By using the BrdU incorpo-
ration assay, it could be shown that the meiotic delay
caused by teniposide in germ cells was
48 h. Disomic and
diploid sperms were shown in epididymal sperm hybri-
dised with DNA probes specific for chromosomes 8, X and
Y after teniposide treatment. The prevalence of autodiploid
(XX88 and YY88) sperm and disomic XX8 or YY8 sperm
indicates that the second meiotic division was more
sensitive to teniposide than the first meiotic division. The
results also suggest that earlier prophase stages contribute
relatively less to teniposide-induced aneuploidy. Both the
clastogenic and the aneugenic potential of teniposide can
give rise to the development of secondary tumours and
abnormal reproductive outcomes in cured cancer patients
and medical personnel exposing to drug regimens that
include teniposide. Thus, genetic counselling of these
patients should take place before the start of chemotherapy
and should take the present results into consideration.
Introduction
Type II topoisomerases are essential nuclear enzymes found in
all living organisms (1). Their basic role in cells is to catalyse
the transport of one DNA double helix through a transient
double-strand break in another DNA molecule. This activity
helps relieve tensions built up in DNA during various DNA
metabolic processes, such as DNA replication, chromosome
condensation and decondensation, chromosome segregation
and transcription (2). Because of its essential role in cell growth,
cell cycle, as well as the high expression in proliferating cells,
Ó The Author 2011. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
doi:10.1093/mutage/ger051
this enzyme is an ideal target for cancer chemotherapeutic
drug (3). Virtually, every form of cancers that can be cured by
systemic chemotherapy were always treated with regimens that
focus on (or at least contain) drugs targeted to topoisomerase
II (4). Indeed, drugs targeted to topoisomerase II are now
indispensable components of systemic chemotherapy schedule,
which could effectively cure several known types of human
cancers.
Teniposide is a podophyllotoxin derivative and was found to
be able to increase the concentration of topoisomerase II–DNA
cleavage complexes (3). This action converts topoisomerase
II to a potent cellular toxin that fragments the genome.
Consequently, teniposide is referred to as a topoisomerase II
poison. It has been known for more than a decade that
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teniposide stabilises topoisomerase II-associated double-
stranded DNA breaks by inhibiting the ability of the enzyme
to ligate cleaved nucleic acid molecules (4). A pervious study
that examined drug effects on human topoisomerase IIa
indicated that podophyllotoxins action at both scissile bonds
are required to stabilise double-stranded breaks in the genetic
material (5). The presence of podophyllotoxins at either scissile
bond inhibits DNA ligation only on that strand and hence
stabilises a single-stranded break in the double helix (5). It has
been shown that in the treatment against small cell lung cancer,
malignant lymphoma, breast cancer and ovarian cancer etc.
through both clinical findings from chemotherapy results and in
vitro chemosensitivity test of tumour specimen, anti-tumour
effect on teniposide was stronger than other chemotherapeutic
drugs (6,7). However, podophyllotoxins utilisation is associ-
ated with an increased risk of secondary acute myeloid
leukaemia and increased sperm frequencies with chromosomal
abnormalities (8,9).
As with many other anticancer drugs, high doses of
podophyllotoxins as etoposide and teniposide induce apoptosis
and disruption of inner mitochondrial membrane potential
(10,11). However, several lines of evidence indicate that low
doses of various chemotherapeutic drugs are capable of inducing
mitotic catastrophe, resulting from abnormal mitotic events that
produce improper chromosomal segregation and cell division,
leading to the formation of cells bearing mutations (12). These
cells can develop resistance to the therapeutic agents or may lead
to the development of secondary tumours and abnormal
reproductive outcomes. Chromosomal imbalance (aneuploidy)
induced by aneugens is a phenomenon observed in .90% of all
solid tumours. Even haematological cancers, known to have
a rather stable chromosome number, frequently display loss or
gain of few chromosomes (13). Diploidy and aneuploidy in
germ cells may cause abortions or children with congenital
abnormalities such as trisomy 21, Klinefelter’s or Turner
syndromes, respectively (14).
Teniposide has been reported to exert strong mutagenic
effects in mammalian cells in vitro but not in bacteria (15–17).
It is a potent clastogenic agent in mouse lymphoma cell lines
1
S. M. Attia
(17) and it has been reported to induce sister chromatid
exchanges in CHO cells (18). Exposure of V79 cells to dif-
ferent concentrations of teniposide resulted in a concentration-
related elevation in the frequency of micronucleated binucleate
cells (19). Teniposide induced chromosomal aberrations,
aneuploidy and micronucleus formation in CHO cells (20,21).
The drug also induced sister chromatid exchange in V79 cells
and mutation and somatic recombination in Drosophila
melanogaster in the wing spot test (22,23). Despite podo-
phyllotoxin’s increasing use in malignancies, scarce data are
available in literature on its potential aneugenicity in vivo. In
an effort to predict patients’ reaction to tumour therapy, the
present study was designed to evaluate the aneugenicity of
teniposide in somatic and germinal cells of male mice in vivo.
The bone marrow micronucleus test complemented by
fluorescence in situ hybridisation (FISH) assay was performed
in mouse bone marrow cells to determine the aneugenic and
clastogenic origin of induced micronuclei (MN). The 5-bromo-
2#-deoxyuridine (BrdU) incorporation assay was used to test if
teniposide treatment altered the duration of the meiotic
divisions and the sperm-FISH assay was performed for
aneugenicity induction during male meiosis. In order to
determine the reliability of the methods, two model mutagens,
colchicine and mitomycin C, known to be predominantly
aneugenic and clastogenic, respectively, were used as positive
control substances.
Materials and methods
Animals
Adult male white Swiss albino mice weighing 23–27 g (10–12 weeks old) were
obtained from Experimental Animal Care Center at our university. The animals
were maintained in an air-conditioned animal house at a temperature of 25–
28°C, relative humidity at
50% and photocycle of 12:12 h light and dark
periods. The animals were provided with standard diet pellets and water ad
libitum. All experiments were carried out according to the Guidelines of the
Animal Care and Use Committee at our university. Each treatment group and
vehicle control group consisted of five randomly assigned animals. The total
number of animals in the somatic cell study was 15 treated mice and 5 vehicle
control whereas the total number of animals in the germ cell studies was 60
treated and 35 vehicle control mice.
Drugs and treatment
Teniposide (Developmental Therapeutics Program, National Cancer Institute,
Bethesda, MD, USA) was dissolved in dimethyl sulfoxide (DMSO) in sterile
dH2O, mixed on a magnetic stirrer for at least 30 min prior to administration
and administered by intraperitonial injection within 1 h following preparation.
Colchicine and mitomycin C (Sigma Chemical St Louis, MO, USA) were
dissolved in sterile water and were used as a positive control aneugen and
clastogen, respectively. Control mice receiving equal amount of DMSO (15%
DMSO in single treatment or 2% in multiple treatment) was included in order
to code the slides and avoid scoring biases. The injected volume was 0.1 ml/10 g
body weight. All other chemicals were of the finest analytical grade.
Bone marrow conventional micronucleus test
Animals were treated with 0.5 mg/kg teniposide and bone marrow was sampled
24 h after treatment. The dose of teniposide was selected based on the data of
Bakheet et al. (24). Colchicine and mitomycin C were used as a positive control
aneugen and clastogen, respectively, at the dose of 2 mg/kg each (25). Bone
marrow smears were prepared and stained with May-Gruenwald/Giemsa
solutions (26). At least four slides were made for each animal and allowed to
dry overnight. One slide per animal was stained with May-Gruenwald/Giemsa
solutions for conventional assessment of the MN frequencies in polychromatic
erythrocytes (PCEs) and normochromatic erythrocytes (NCEs). The remaining
unstained slides were stored at 20°C for the distinction between the
clastogenic and aneugenic effects by identifying the origin of MN with the
mouse DNA probes. Per animal, 1000 PCE of coded slides were scored for the
presence of MN. In addition, the number of PCEs among 1000 NCE per animal
was recorded to evaluate bone marrow suppression and mitotic activity was
calculated as %PCE 5 [PCE/(PCE þ NCE)]  100.
2
Bone marrow FISH analysis of MN using centromeric and telomeric DNA probes
Two DNA probes were used for tandem labelling: a murine minor satellite
DNA probe specific for the centromeric region (27) and a murine hexamers
DNA probe specific for the telomeric region (28). The centromeric and
telomeric probes were labelled with the fluorescent dyes Cy3 and fluorescein-
isothiocyanate (FITC) using the biotin or digoxygenin systems according to the
manufacturer’s instructions (GIBCO Invitrogen, Carlsbad, CA, USA). In situ
hybridisation assay was performed as described elsewhere (29–32) with slight
modifications. Briefly, frozen slides were brought to room temperature and
rehydrated in 2 saline sodium citrate buffer (SSC) buffer for 5 min prior to
treatment with 0.1% Triton X-100 in 2 SSC for 3 min. After washing the
slides in 2 SSC for 2 min, they were fixed in 4% paraformaldehyde for 10
min at room temperature and washed again in 2 SSC for 3 min. Then, the
slides were denaturated for 10 min in 70% formamide at 78°C and dehydrated
in an ice-cold ethanol series of 70, 90 and 100% for 2 min each. After air-
drying, the slides were brought to 37°C for ,3 min on a slide warming plate
prior to mixing with the denaturated hybridisation mix. The hybridisation mix
contained 20 parts of the master mix 1.0 (50% formamide and 10% dextran
sulphate), 2 parts of centromeric probe (
20 ng per slide), 2 parts of telomeric
probe (
12 ng per slide), 3 parts of herring sperm DNA (500 lg/ml) and 3
parts of distilled water. The hybridisation mix was denatured for 5 min at 78°C
and chilled on ice. Aliquots of 30 ll of the hybridisation mix were added to
each slide, covered with coverslip and sealed with rubber cement. Hybridisation
was carried out overnight in a moist chamber at 37°C.
Post-hybridisation washing consisted of two steps: 40 min in 50%
formamide at 45°C and two washes for 15 min at 37°C in phosphate-nonidet
P-40 (PN) buffer (0.1 M sodium phosphate, 0.1% Nonidet P40, pH 8.0). After
incubation with 40 ll PNBR (PN buffer plus 5% blocking reagent and 0.02%
Downloaded from mutage.oxfordjournals.org at King Saud University on September 3, 2011
sodium azide) for 10 min, the hybridised probes were detected by streptavidin-
Cy3 for the biotin-labelled centromeric DNA probe and anti-digoxigenin–FITC
for the digoxigenin-labelled telomeric DNA probe. The hybridisation signals
were amplified by two rounds of incubation with biotinylated anti-streptavidin
followed by streptavidin-Cy3 and FITC-labelled anti-sheep IgG antibody made
in rabbit. The cells were counterstained with 4,6-diamidino-2-phenylindole
[DAPI; 0.1–0.5 lg/ml in phosphate-buffered saline (PBS)] for 10 min at room
temperature and coverslipped in Vectashield mounting medium. The slides
were scored immediately or following storage for several days at 4°C in the
dark. The two applied DNA probes are identified by the regular appearance of
a red domain for centromeric DNA probe (labelled with Cy3) and a green
domain for the telomeric DNA probe (labelled with FITC). Strict scoring
criteria were applied to avoid misinterpretation of signal numbers. MN were
scored as having two or more domains of the same colour if the signals were
(i) nearly similar in size and intensity, (ii) separated by at least the distance of
half the diameter of the domain and (iii) regular in appearance, not diffuse and
clearly positioned within the MN. For signal detection, 50–150 MN per group
were examined for the presence or absence of DNA probes and the numbers of
signals per MN were counted under an Axioplan Fluorescence Microscope.
Sperm BrdU incorporation assay
The time of development from meiotic divisions in spermatocytes to epididymal
sperm was assessed by labelling cells with BrdU. Mice were injected with 100 mg/
kg BrdU in order to label spermatocytes at S-phase during preleptotene of meiosis.
During meiosis I and II, 13 days later, the mice were treated with 24 mg/kg
teniposide. For human chemotherapy, teniposide is administered typically at doses
of 30–50 mg/m2/day for 5 days or three doses of
200 mg/m2 over 7 days. The
mouse has a body weight/surface area ratio of
3 kg/m2. Thus, the dose of 24 mg/
kg used in the current study corresponds to
72 mg/m2 and is within the dose range
used for human chemotherapy. Five treated and five solvent control mice were
sacrificed per day 33–37 after BrdU injection (20–24 days after drug treatment).
Both Caudae epididymes from each male were isolated and several incisions
were made. The epididymes were then placed in eppendorf cups with 300 ll of
foetal calf serum and the sperms were allowed to actively leave the epididymes for
60 min at 37°C. Sperm suspensions (7 ll) were pipetted onto one end of a clean
dry glass slide and spread across the slide (33). Decondensation of the sperm
heads was performed by incubation of the coded slides in 10 mM dithiothreitol
for 30 min on ice followed by incubation in 4 mM lithium 3,5-diiodosalicylic acid
for 60 min at room temperature. The slides were denatured in 2N HCl at room
temperature for 60 min and washed two times for 10 min in a Coplin jar with PBS
at room temperature to neutralise the acid. Immunodetection was carried out in
two steps in a humidified chamber: slides were incubated with 40 ll per slide of
a rat anti-BrdU (Seralab, 1/100) antibody for 30 min at room temperature. After
washing for 10 min with PN buffer at room temperature, the slides were
incubated with 40 ll per slide of a FITC-conjugated anti-rat IgG antibody
(Dianova, 1/100) for 30 min at room temperature and washed again for 10 min in
PN buffer at room temperature. Propidium iodide (2 lg/ml) was used as
counterstaining. Averages of 10 000 BrdU-positive and BrdU-negative cells per
animal were blind scored under a Fluorescence Microscope.
 Aneugenic effects of teniposide

Sperm multicolour FISH assay
Random groups of five mice each were treated with single doses of 6, 12 and 24
mg/kg teniposide. Colchicine was used as a positive control aneugen at the dose
of 3 mg/kg (34). After drug administration, the animals were maintained with
food and water ad libitum until being sacrificed. Mice were sacrificed by
cervical dislocation 23 days after drug treatment. In order to determine if
subacute treatment would have an effect because prophase stages would be
included in teniposide treatment, doses of 0.5, 1 and 2 mg/kg of teniposide in
2% DMSO were injected on 12 consecutive days and sperms were sampled 23
days after the last treatment. Immediately after animal sacrificing, the sperm
were collected from the C. epididymis. The time of sampling was chosen on the
basis of the BrdU incorporation study.
The frequencies of disomic and diploid sperm were determined by FISH
with DNA probes specific for mouse chromosomes 8 (35), X (36) and Y (37).
The probes for chromosomes 8 and Y were labelled with the fluorescent dyes
Cy3 and FITC using the biotin or digoxygenin systems, respectively, while the
chromosome X probe was labelled with a combination of Cy3 and FITC using
the biotin and digoxygenin systems according to the manufacturer’s
instructions (GIBCO Invitrogen, Carlsbad, CA, USA). The probe mixture
[4.5 lg probes in 2.1. master mix (55% formamide, 10% dextran sulfate) 2
SSC] was denatured at 78°C for 10 min and chilled on ice. Hybridisations with
fluorochrome-labelled DNA probes were performed as previously described by
Attia et al. (34) with some modifications. Briefly, the sperm heads were
decondensed as described above in BrdU incorporation assay and then the
slides were denaturated for 10 min in 70% formamide at 78°C and dehydrated
in ethanol series. Following the application of 30 ll of probe mixture,
hybridisation was performed at 37°C for 24–48 h in a moist chamber. Post-
hybridisation washing and detection of the hybridised probes were performed
of bone marrow was noted (P , 0.05). In NCEs, the MN
frequencies were between 0.02 and 0.06/100 NCE in all
groups. Thus, no discrimination between MN induced in PCE
and NCE was required for the fluorescent analysis of MN with
in situ hybridisation since the NCE only contributed minimally
to the total number of MN.
Bone marrow FISH analysis of MN using centromeric and
telomeric DNA probes
The results for double labelling with centromeric and telomeric
probes in both PCE and NCE are shown in Table II. The
telomere-positive MN without a centromere signal represents
MN containing an acentric fragment with pericentric hetero-
chromatin. All MN with centromeric signals also had telomeric
signals and these MN contained entire chromatids or entire
chromosomes. After treatment of mice with the positive control
aneugen colchicine, the majority of induced MN were
telomere- as well as centromere positive, reflecting the
expected aneugenic effects of colchicine. After treatment of
mice with the positive control clastogen mitomycin C, only
19.27% of induced MN were double-labelled with the telomere
and the centromere probes, confirming the predominantly
clastogenic effects of mitomycin C. As shown in Figure 1 and

Downloaded from mutage.oxfordjournals.org at King Saud University on September 3, 2011

as described above in bone marrow FISH analysis. Sperm nuclei were
counterstained with 0.05–0.1 lg/ml DAPI. Slides were examined for disomic
and diploid sperm under an Axioplan Fluorescence Microscope. Per animal,
fluorescent signals from coded slides were counted in
10 000 sperms and
sperms were designated as normal (X8 and Y8), disomic (X88, Y88 and XY8)
or diploid (XY88, XX88 and YY88).
Statistical analysis
Data were expressed as the mean  standard deviation (SD) of the means. The
analysis parameters were tested for homogeneity of variance and normality and
were found normally distributed. The data were, therefore, analysed by
employing non-parametric tests, the Mann–Whitney U-test. Results were
considered significantly different if the P value was 0.05.
Table II, the ratios of MN arising from clastogenic (centromere
negative) and aneugenic (centromere positive) effects of the
vehicle and positive controls were found to be in the same
range as reported earlier (29–32). With teniposide, 18.24% of
the analysed MN had no signal, 35.14% showed telomere
signals and 46.62% were double labelled with the telomere and
the centromere probes. The distribution of the signal
frequencies in MN that were positive only with the telomere
probe is shown in Table III. The majority of analysed MN in

Table II. Results of double FISH labelling of colchicine (2 mg/kg),

Results mitomycin C (2 mg/kg) and teniposide (0.5 mg/kg)-induced MN with the
telomeric and the centromeric DNA probes
Bone marrow conventional MN test
The incidences of micronucleated polychromatic erythrocyte Chemicals In situ hybridisation analysis of MN
(MNPCE) in each treatment group as well as the PCE/NCEs Number MN MN with MN with
ratio are shown in Table I. After treatment with the positive of MN without any telomere telomere and
controls colchicine and mitomycin C, a statistically significant scored signal (%) signals centromere
increase in the incidence of MNPCE over the control value was only (%) signals (%)
observed (P , 0.01). Moreover, mitomycin C significantly Control 53 16 (30.19) 14 (26.42) 23 (43.39)
decreased the percent PCE (P , 0.05), indicating a reduction Colchicine 80 9 (11.25) 13 (16.25) 58 (72.5)
in erythroblast proliferation. Teniposide significantly increased Mitomycin C 83 14 (16.87) 53 (63.86) 16 (19.27)
MNPCE frequencies (P , 0.01) and the response was directly Teniposide 148 27 (18.24) 52 (35.14) 69 (46.62)
correlated with bone marrow toxicity as significant suppression

Table I. Frequencies of MNPCE, MNNCE and %PCE in bone marrow of

mice after treatment with colchicine (2 mg/kg), mitomycin C (2 mg/kg) and
teniposide (0.5 mg/kg)

Chemicals % MNPCE % MNNCE % PCE

(mean  (mean  (mean 
SD) SD) SD)

Control 0.30  0.07 0.02  0.04 48.6  2.60

Colchicine 1.06  0.16** 0.02  0.04 45.4  3.78
Mitomycin C 1.33  0.34** 0.04  0.05 41.6  3.43*
Teniposide 3.88  0.57** 0.06  0.05 42.8  4.32*
Fig. 1. Illustration of the contribution of clastogenicity and aneugenicity to the
*P , 0.05, **P , 0.01 compared with the solvent corresponding control induced MN frequencies in animals treated with colchicine (2 mg/kg),
(Mann–Whitney U-test). mitomycin C (2 mg/kg) and teniposide (0.5 mg/kg).

3
S. M. Attia

colchicine group contained one telomere signal, whereas the
majority of analysed MN in mitomycin C contained four
telomere signals. Of the 52 MN analysed following treatment
of mice with the teniposide, one telomere signal per MN was
observed in 38.46% MN, two signals were seen in 32.69%
MN, three signals were seen in 17.3% MN and four signals
were seen in 11.53% MN.
The distribution of the signal frequencies in the MN that were
double labelled with both the centromere and the telomere
probes is shown in Table IV. The majority of analysed MN in
colchicine group contained one centromere and two telomere
signals or two centromere and four telomere signals. After
treatment with mitomycin C, only 25.0% double-labelled MN
had one centromere and one or two telomere signals, 31.25%
showed one centromere and three or four telomere signals,
18.75% showed two centromere and one or two telomere signals
and 25.0% showed two centromere and three or four telomere
signals. With teniposide, 40.5% of induced MN had one
centromere and one or two telomere signals, 23.09% showed
one centromere and three or four telomere signals, 23.09%
showed two centromere and one or two telomere signals and
17.38% showed two centromere and three or four telomere
signals. To correlate the FISH data with the conventional MN
groups were significantly below the control values (P , 0.05
and P , 0.01, respectively), while on days 23 and 24, there were
no significant differences between the teniposide-treated and
control animals. According to Oakberg (38), the development
from meiotic spermatocytes of mice to epididymal sperm takes
22 days. Thus, the meiotic delay caused by teniposide was
48
h. Therefore, the optimum day for sperm sampling in the sperm-
FISH assays was concluded to be 23 or 24 with teniposide.
Multicolour sperm-FISH assay
The results of the analysis of aneugenic effects in germ cells of
male mice after single exposure to teniposide are presented in
Table V, together with the negative and positive control data.
Sex ratios were found to be in the same range as the theoretical
ratio of 1:1 for X- versus Y-bearing sperm in all groups. A
significant increase in the frequency of diploid sperm was
caused by treatment with all doses of teniposide compared with
the control value. Treatment of mice with 6 mg/kg of
teniposide did not induce significant increases in disomic
sperm; however, treatment with 12 and 24 mg/kg of teniposide
significantly induced disomic sperm compared with the control
value. The dose–response curves for teniposide-induced
disomic and diploid sperm can be described by the linear

Downloaded from mutage.oxfordjournals.org at King Saud University on September 3, 2011

data, the expected percent of PCE with signals-positive and
-negative MN were calculated (Figure 1). For example, after
treatment with colchicine, 1.06% MNPCE were found in the
conventional MN test and 88.75% MN were signals positive in
the FISH analysis; thus, 0.94075% of the 1.06% MNPCE were
calculated to be signals positive and, correspondingly, 0.11925%
MN were calculated to be signals negative.
Sperm BrdU incorporation assay
The results of the BrdU incorporation assay are presented in
Figure 2. With 24 mg/kg of teniposide, prolongations of the
duration of the meiotic divisions were observed. On days 21 and
22, the frequencies of BrdU-labelled sperm in the teniposide
Table III. Distribution of the telomere frequencies among MN after treatment
with colchicine (2 mg/kg), mitomycin C (2 mg/kg) and teniposide (0.5 mg/
kg)
Chemicals MN with One Two Three Four
telomere telomere telomere telomere telomere
signals signal (%) signals (%) signals (%) signals (%)
only
equations y 5 0.004x þ 0.041 (r2 5 0.990) and y 5 0.002x þ
0.003 (r2 50.998), respectively (Figure 3). The frequency of
disomic sperm induced by colchicine was significantly in-
creased by a factor of 1.64 compared with the control value.
However, in contrast to teniposide, colchicine did not
significantly increase the frequency of diploid sperm, in-
dicating that no complete meiotic arrest occurred.
The results of the multiple exposures to teniposide are shown
in Table VI. There were no mortality or symptoms of toxicity
observed after repeated treatment with teniposide or the vehicle.
These results indicate that repeated treatment with teniposide and
the vehicle are well tolerated by experimental animals. Treatment
of mice with 0.5 and 1 mg/kg of teniposide on 12 consecutive
days did not induce any significant increases of disomic or
diploid sperm. However, the highest dose group (2 mg/kg/day),
which received a total of 24 mg/kg, induced significant increases
in disomic and diploid sperm compared with the corresponding
control values. The dose–response curves for teniposide-induced
disomic and diploid sperm can be described by the linear
equations y 5 0.017x þ 0.048 (r2 5 0.838) and y 5 0.011x þ
0.001 (r2 50.936), respectively (Figure 4).

Control 14 8 (57.14) 3 (21.42) 2 (14.28) 1 (7.14) Discussion

Colchicine 13 7 (53.84) 3 (23.07) 1 (7.692) 2 (15.38)
Mitomycin C 53 10 (18.86) 12 (22.64) 14 (26.41) 17 (32.07) The genotoxic activity of teniposide during mitosis in vivo was
Teniposide 52 20 (38.46) 17 (32.69) 9 (17.30) 6 (11.53) recently confirmed in our laboratory with the bone marrow MN

Table IV. Distribution of the signal frequencies in the double FISH labelled MN with the centromere and the telomere DNA probes after induction with colchicine
(2 mg/kg), mitomycin C (2 mg/kg) and teniposide (0.5 mg/kg)

Chemicals Number One One One One Two Two Two Two
of double- centromere centromere centromere centromere centromere centromere centromere centromere
labelled and one and two and three and four and one and two and three and four
MN telomere telomere telomere telomere telomere telomere telomere telomere
signals (%) signals (%) signals (%) signals (%) signals (%) signals (%) signals (%) signals (%)

Control 23 6 (26.08) 3 (13.04) 2 (8.69) 3 (13.04) 3 (13.04) 2 (8.69) 3 (13.04) 1 (4.34)

Colchicine 58 3 (5.17) 10 (17.2) 2 (3.44) 8 (13.79) 7 (12.06) 8 (13.79) 8 (13.79) 12 (20.6)
Mitomycin C 16 3 (18.75) 1 (6.25) 3 (18.75) 2 (12.5) 2 (12.5) 1 (6.25) 2 (12.5) 2 (12.5)
Teniposide 69 12 (17.4) 16 (23.1) 6 (8.69) 10 (14.4) 8 (11.59) 5 (7.24) 4 (5.79) 8 (11.59)

4
 Aneugenic effects of teniposide

test (24). So far the origin of the teniposide-induced MN is not
determined yet and there are no in vivo published aneuploidy
studies for teniposide in germ cells. Therefore, the current
study was designed to investigate the ability of teniposide to
induce aneuploidy in somatic and germinal cells of male mice.
The conventional bone marrow MN test complemented by
FISH with the mouse centromeric and telomeric DNA probes
was used to investigate the mechanisms of induction of MN in
mice treated with teniposide. This assessment is particularly
important for chemical clastogens, which may also have
aneugenic potential. Such chemicals may interact, on the
chromosomal level, with the respective targets for chromo-
somal missegregation. To determine the efficiency of the
method, two model mutagens, colchicine and mitomycin C,
known to have aneugenic and clastogenic actions, respectively,
were used. The observed results of the MNPCE and the
distribution of signals per MN in the solvent control and the
two positive mutagens in the current study correspond well
with the published data reported earlier (29,32). These data
confirmed the sensitivity of the experimental protocol followed
in the detection of genotoxic effects.
Teniposide at a dose of 0.5 mg/kg significantly increased the
frequency of MNPCE, as expected (24). Additionally, tenipo-
arrest. A total of 148 MN from teniposide-treatment group
were analysed by FISH and 69 (46.62%) MN of these were
double labelled with centromere and telomere probes, in-
dicating that they were formed by lagging chromosomes and
reflecting an aneugenic activity of teniposide. Similarly, 77
(53.38%) of the induced MN were centromere negative,
indicating that they were formed by acentric fragments and
reflecting the clastogenic activity of teniposide. Since the
centromere probe labelled mitotic chromosomes at both
chromatids, it can be concluded that equal proportions of
MN contained single chromatids (chromosome loss) and
biarmed chromosomes (non-disjunction).
These results indicate that teniposide is clastogenic and
aneugenic during mitotic phases. Both the clastogenic and
the aneugenic potential of teniposide in somatic cells can give
rise to the development of secondary tumours. Therefore, the
clinical use of this genotoxic drug must be weighed against
the risks of secondary malignancies in cured patients.
Importantly, experimental observations have shown that the
catalytic topoisomerase II inhibitor, dexrazoxane, is able to
enhance the cytotoxic action of teniposide (39) and modulates
teniposide-induced DNA damage and apoptosis in the bone
marrow cells in vivo (24). Thus, based on the results of these

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side significantly decreased the percent PCE, indicating
a reduction in erythroblast proliferation most likely by mitotic
Fig. 2. Time course of appearance of BrdU-labelled sperm in the epididymis
after treatment with 24 mg/kg teniposide. *P , 0.05, **P , 0.01 compared
with the concurrent control (Mann–Whitney U-test).
previous in vivo studies, strategies can be developed to
decrease the teniposide-induced genotoxicity in non-tumour
cells using dexrazoxane. A total of 35.14% of the analysed
MN was telomere positive and must be interpreted as MN
harbouring terminal acentric fragments (Table III). Addition-
ally, 18.24% of the analysed MN had no FISH signals and
must be interpreted as MN harbouring interstitial acentric
fragments (Table II). Centromere plus telomere double-
labelled MN that gave one centromeric signal and two
telomere signals (Table IV) probably contained a chromosome
lagging after centromere separation. MN with two centromere
signals and four telomere signals most likely contained
a chromosome lagging before centromere separation or two
chromatids after centromere separation.
Studies in humans have shown that certain chemotherapy
regimens increase the frequencies of aneuploidy in germinal
cells (40–43), suggesting that such patients may be at higher
risk for the development of secondary tumours and abnormal
reproductive outcomes. Therefore, it is of general concern to

Table V. Results of the multi-colour FISH with epididymal sperm of mice treated with colchicine and teniposide

Control Colchicine (3 mg/kg) Teniposide (6 mg/kg) Teniposide (12 mg/kg) Teniposide (24 mg/kg)

Number of animals 5 5 5 5 5
Sperm scored 50011 50029 50081 50012 50041
X/Y-bearing sperm 0.9984 1.0011 1.0515 1.0311 1.0081
Disomic sperm
X-X-8 6 14 9 11 18
Y-Y-8 7 12 7 11 17
X-Y-8 2 2 3 4 7
X-8-8 4 6 7 13 13
Y-8-8 6 6 6 9 13
Total 25 40 32 48 68
% Disomies  SD 0.05  0.010 0.08** 0.012 0.064  0.015 0.096**  0.015 0.136**  0.019
Diploid sperm
X-Y-8-8 0 0 2 3 5
X-X-8-8 1 1 5 9 16
Y-Y-8-8 0 1 3 5 11
Total 1 2 10 17 32
% Diploidies  SD 0.002  0.004 0.004  0.005 0.02  0.007* 0.034**  0.015 0.064**  0.005

*P, 0.05, **P, 0.01 compared with the concurrent control (Mann–Whitney U-test).

5
S. M. Attia

decrease the risk of aneuploidy production, detection of germ
cell aneugens and understandings of the causal mechanisms are
essential. In the current study, aneuploidy was determined in
germinal cell by the sperm-FISH assay. In order to determine
the reliability of the methods, colchicine, known to be
predominantly aneugenic, was used as positive control sub-
stance and the results of the positive and negative control were
in the same range as those of the earlier studies (32,34,44,45).
These data confirmed the sensitivity of the experimental
protocol followed in the detection of aneuploidogenic effects.
It has been often discussed that chemicals with aneugenic
properties can alter the progression of cell division in both
meiotic and mitotic cells (46,47). In the present study, the time
of development from meiotic divisions in spermatocytes to
epididymal sperm was assessed by the BrdU incorporation
assay. The results clearly indicate that teniposide prolonged the
duration of the meiotic divisions in mouse spermatocytes for
48 h. These observations therefore confirm previous results
that inhibition of topoisomerase II function during different
phases of the cell cycle by teniposide slow down cell cycle
progression and causes cells to arrest at the G2/M phase
(11,48,49). Such G2/M arrest has been proposed to be due to
induction of G2 checkpoint machinery that allows damaged
In the present experiments, teniposide caused dose-dependent
significant increases in the frequencies of disomic and diploid
sperm. The induction of aneuploidy showed linear dose–
responses between 0 and 24 mg/kg of teniposide. This in vivo
observation is in line with an earlier in vitro report on the CHO
cells that teniposide induces the formation of quadriradial
chromosomes (21). Polyploidy induced by teniposide was also
demonstrated by flow cytometry techniques in murine eryth-
roleukemic cells (48). Furthermore, Morse-Gaudio and Risley
(51) reported that incubation of isolated spermatogenic cells
Xenopus laevis with teniposide led to dose-dependent induction
of DNA breaks in all spermatocytes and spermatid stages to
nuclear elongation stages, as analysed by alkaline single-cell gel
electrophoresis. To determine if subacute treatment with low
doses of teniposide would have an effect because earlier prophase
stages would be included in teniposide treatment. Individual
doses of 0.5, 1 and 2 mg/kg of each compound were injected on
12 consecutive days and sperms were sampled 23 days after the
last treatment with teniposide. It was found that a total of 24
mg/kg teniposide applied to the entire prophase of meiosis
significantly increased disomic and diploid sperm frequencies,
while a total dose of 6 or 12 mg/kg was negative. In contrast,
a single dose of 6 mg/kg teniposide applied to spermatocytes

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DNA to be repaired before cells move to the next cell cycle
stage (50).
Fig. 3. Dose–response curves of the frequencies of abnormal sperm from mice
after treatment with teniposide. *P , 0.05, **P , 0.01 compared with the
concurrent control (Mann–Whitney U-test).
during the first meiotic division (meiosis I; MMI)/the second
meiotic division (meiosis II; MMII) gave a positive result. These
data suggest that earlier prophase stages contribute relatively less
to teniposide-induced aneuploidy in male germ cells.
The sperm-FISH assay for disomy or diploidy is capable of
detecting effects induced during both meiotic divisions and to
compare the sensitivity of both meiotic divisions (52,53). In the
current study, it was found that teniposide caused noticeable
increases above the control of autodiploid sperm (XX88 and
YY88). After treatment with teniposide, autodiploid sperm
resulting from arrest of MMII were more frequent than diploid
sperm resulting from arrest during MMI (XY88). Thus, the
second meiotic division was more sensitive to teniposide
treatment than the first meiotic division. The conclusion that
second meiotic divisions were more sensitive to teniposide
treatment than the first meiotic divisions is also supported by
the observed frequencies of sex chromosome disomic. Sperm
with signals of XX8 or YY8 were more frequent than sperm
with signals of XY8.

Table VI. Results of the multicolour FISH with epididymal sperm of mice treated with teniposide (daily exposure for 12 consecutive days)

Control Teniposide (0.5 mg/kg) Teniposide (1 mg/kg) Teniposide (2 mg/kg)

Number of animals 5 5 5 5
Sperm scored 50078 50072 50171 50012
X/Y-bearing sperm 1.0044 0.9912 0.9952 1.0049
Disomic sperm
X-X-8 7 9 5 11
Y-Y-8 4 8 7 10
X-Y-8 2 3 3 3
X-8-8 8 5 6 12
Y-8-8 5 4 7 8
Total 26 29 28 44
% Disomies  SD 0.052  0.017 0.058  0.008 0.056  0.015 0.088**  0.01
Diploid sperm
X-Y-8-8 0 1 2 2
X-X-8-8 1 1 2 6
Y-Y-8-8 0 1 1 5
Total 1 3 5 13
% Diploidies  SD 0.004  0.005 0.006  0.005 0.01  0.007 0.026**  0.005

*P, 0.05, **P, 0.01 compared with the concurrent control (Mann–Whitney U-test).

6

Fig. 4. Dose–response curves of the frequencies of abnormal sperm from mice
23 days after the last treatment with teniposide (daily exposure for 12
consecutive days). **P , 0.01 compared with the concurrent control (Mann–
Whitney U-test).
The data of sperm study indicate that male cancer patients
receiving chemotherapy with teniposide may stand a higher
risk of siring chromosomally abnormal offspring, i.e. with
Down, Klinefelter or Turner syndromes. The chemotherapy
with teniposide may also reduce the fertility of the patients. The
most sensitive period of spermatogenesis to aneuploidy
induction is meiosis. In humans, the time of development
from meiosis in spermatocytes to mature sperm takes 40–57
days (38) with some uncertainty about the retention of sperm in
the epididymis. Baumgartner et al. (54) could show that the
aneuploidy rates in sperm of Hungarian men were elevated
significantly 40–50 days after attempted suicide with diazepam
and returned to control levels when sperm were sampled 100
days after the intoxication. Therefore, the hazard for patients to
foster a chromosomally unbalanced child is only elevated
during 3–4 months after the end of chemotherapy with
aneugenic drugs. Often, cancer patients facing a limited life
expectancy express a great desire to have a child. Genetic
counselling of these patients should take place before the start
of chemotherapy and should take the present results into
consideration.
The present mouse bone marrow MN studies showed that
exposure to 0.5 mg/kg teniposide yielded a significant increase
in MNPCE. However, in the sperm-FISH assay, it was found
that the lowest positive dose, which caused disomic or diploid
sperm, was 6 mg/kg of teniposide. This observation suggests
that MN in bone marrow are induced at lower doses than
disomies or diploidies in sperm; hence, the bone marrow is the
more sensitive tissue. It must of course be noted that the assays
measure different end-points. Chromosome loss and breakage
are measured in the MN test, and non-disjunction is detected in
the sperm-FISH assay. Therefore, the present data confirm the
general paradigm of hazard assessment that the positive
outcome of the bone marrow MN test is an indicator of the
genotoxic potential of a compound in germ cells. However, to
quantify aneuploidy induced in germ cells is important for risk
assessment purposes. Taking into account the fundamental
differences between the meiotic process and the mitotic
process, e.g. requirement for chromosome pairing, formation
of chiasmata to allow recombination, prolonged duration of
meiosis in oocytes (55), it will always be necessary to confirm
the aneugenic potential of a chemical detected in in vitro by
studies in somatic and in germinal cells in vivo. Theoretically,
the differences in cell biology may give rise to qualitative
differences in response of germ cells and somatic cells to
Aneugenic effects of teniposide
aneugens and the possibility of unique germ cell aneugens
should not be neglected.
DNA topoisomerase inhibitors have a clear tendency to
cause stranded DNA breaks, which primarily result in the
formation of centromere-negative MN (32). Similarly, the
demonstration that teniposide is effective topoisomerase
inhibitor suggests that teniposide elicit its clastogenic effects
through this mechanism. Teniposide metabolites could also
activate cells to enhance their intracellular production of
reactive oxygen species, of which the stable and diffusible
forms could damage nuclear DNA (56). If the cellular repair
mechanisms are overloaded, primary DNA damage may lead to
structural or numerical chromosomal aberration and eventually
cause tumours (57). However, the induction of centromere-
positive MN by teniposide indicates that there may be another
mechanism through which teniposide can induce its genotoxic
effect, an observation that also underscores the importance of
using the FISH modification of the MN assay to determine the
origin of the induced MN. The possible mechanism by which
teniposide may exert its aneugenic effect is through inhibition
of topoisomerase II which could result in a mal-segregation of
chromosomes during cell divisions. As mentioned in In-
troduction, topoisomerase II has been demonstrated to be
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necessary for the proper separation of sister chromatids during
cell divisions (2) with both non-disjunction and breakage
occurring in its absence (58). Thus, teniposide may be able to
inhibit two key roles of topoisomerase II, its ability to properly
segregate newly replicated chromosomes as well as its function
to religate transient double-stranded DNA breaks.
In summary, by using FISH assay with centromeric and
telomeric DNA probes for erythrocyte MN, it could be shown
that teniposide is not only clastogenic but also aneugenic in
somatic cells in vivo. The assay also showed that chromosomes
can be enclosed in an MN before and after centromere
separation. By using the BrdU incorporation assay, it could be
shown that the meiotic delay caused by teniposide was
48 h.
With the sperm-FISH analysis, it could be shown that teniposide
induce aneuploidies during meiosis that result in disomic sperm
as well as complete meiotic arrest that results in diploid sperm.
The prevalence of autodiploid (XX88, YY88) sperm and
disomic XX8 or YY8 sperm indicate that the second meiotic
division was more sensitive to teniposide than the first meiotic
division. The results suggest also that earlier prophase stages
contribute relatively less to teniposide-induced aneuploidy. Both
the clastogenic and the aneugenic potential of teniposide can
give rise to the development of secondary tumours and abnormal
reproductive outcomes in cancer patients and medical personnel
exposing to drug regimens that include teniposide. The results of
the current study and our previous data base show that the
aneuploidy assays such as the sperm-FISH assay and MN test
complemented by FISH with centromeric DNA probes have
been sufficiently validated to be recommended for the
assessment of aneugenic effects of chemicals.
Funding
Deanship of Scientific Research at King Saud University
through the research group project No. (RGP-VPP-120).
Acknowledgements
The author is grateful to Mr Müller, M. for assistance with certain techniques.
Conflict of interest statement: None declared.
7
S. M. Attia
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