Mutation Research 520 (2002) 141–150
Chromosome aberration assays for the study of
cyclophosphamide and Bacillus thuringiensis in
Oxya chinensis (Orthoptera: Acrididae)
Zhumei Ren, Enbo Ma∗ , Yaping Guo
College of Life Science and Technology, Shanxi University, Taiyuan 030006, PR China
Received 30 October 2001; received in revised form 28 June 2002; accepted 16 July 2002
Abstract
Chromosome aberrations induced by an anti-neoplastic drug, cyclophosphamide (CP) and a bioinsecticide, Bacillus
thuringiensis (B.t.) were examined using grasshoppers as an animal model, with injection as the route of exposure. Oxya
chinensis (Thunberg), having a small number (2n = 23) of large-sized chromosomes in males, was used for this purpose. The
fifth instar nymphs were treated with various concentrations of CP (2, 5 and 10 mg/ml) and B.t. (0.55, 1.83 and 5.50 IU/ml) by
injection into the abdomen, using physiological saline and distilled water as negative controls, respectively. The chromosomal
preparations were made from the spermatogonia of the specimen testis at different intervals after dosing (24 and 48 h). The
effect of the high dose of CP (10 mg/ml) in O. chinensis was also analyzed at the 42-h time point. The chromosome aberrations
observed were mainly chromatid and chromosome breaks. CP induced a dose- and time-dependents increase in the number of
chromosome aberrations (CAs) per cell and in the percentage of aberrant cells. The strongest effect was seen when grasshoppers were injected with the highest dose and cells were analyzed at the 48-h time point. The results show that CP induced a
significant increase in the frequency of CAs in testicular cells of O. chinensis with the three doses employed, compared to the
negative control. Our results suggest that there exists in the grasshopper an enzyme system analogous to liver-S9 fraction, and
that CP may be used as a positive control in genotoxicity test in this species. In addition, the evaluation of the chromosome
aberrations induced by B.t. in the grasshoppers’ testicular cells showed that B.t. may induce chromosome aberrations, mainly
chromatid and chromosome breaks, in spermatogonia. By statistical analysis, B.t. showed significant dose–effect relationships
and it may be mutagenic in this species. Recent research has focused on the development of biological insecticides to protect
cereal crops against damage by insect species, such as beetles and grasshoppers. The present studies may contribute to our
knowledge of entomological genotoxicity in grasshoppers and provide reference for the research on the mechanism of B.t.
toxicity.
© 2002 Elsevier Science B.V. All rights reserved.
Keywords: Cyclophosphamide; Bacillus thuringiensis (Berliner); Anti-neoplastic; Bioinsecticide; Chromosome aberration; Oxya chinensis
(Thunberg)
1. Introduction
∗ Corresponding author. Tel.: +86-351-7018871;
fax: +86-351-7011981.
E-mail address: [email protected] (E. Ma).
Cyclophosphamide (CP) is an anticancer drug that
is widely used in anti-neoplastic therapy as well as in
the treatment of some non-malignant diseases, such
as rheumatoid arthritis. It is also used as an immuno-
1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 3 - 5 7 1 8 ( 0 2 ) 0 0 1 9 9 - 7
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Z. Ren et al. / Mutation Research 520 (2002) 141–150
suppressive agent prior to organ transplantation [1].
In mammals, the drug can be transformed by liver-S9
fraction to generate active metabolites, which can react with the DNA and ultimately kill cancer cells [2].
However, CP is also a known carcinogen in humans
and produces secondary tumors. There is little absorption either orally or intravenously and 10% of the drug
is excreted unchanged. In somatic cells, CP has been
shown to produce gene mutations, chromosome aberrations, micronuclei and sister chromatid exchanges in
a variety of cultured cells in the presence of metabolic
activation as well as sister chromatid exchanges without metabolic activation. The compound has also produced chromosome damage and micronuclei in rats,
mice and Chinese hamsters [1]. In many genotoxicity
assay systems to evaluate chemical safety, CP is often
used as a positive control [3]. It has been shown to
produce micronuclei and chromosomal aberrations in
rat bone marrow and spleen cells using in vivo and
in vivo/in vitro methodologies [4], and chromosome
aberrations and sister chromatid exchange in CHO-K1
cells [5]. It is both a mutagen and a carcinogen
[6].
In entomological genotoxicity tests, Drosophila
melanogaster is commonly used as an experimental
organism. CP has been shown to produce significant
increases in wing spot frequency [7], in the appearance of visible mutant clones of ommatidia in the
eyes of the emerging adult flies [8], and in the frequency of reversions [9]. However, there are only
few reports that use other insect species as model for
entomological genotoxicity tests.
Grasshoppers (Orthoptera: Acrididae) have long
been known as highly harmful pests to crops and
forage plants. Therefore, the insects have been the
subject of numerous studies throughout the world
[10–15]. Oxya chinensis, belonging to Catantepinae,
is one of the most common and widespread insects
in Asia and is abundantly found in rice paddies, in
sugar cane and other gramineous plants. Because this
organism causes extensive damage in an agriculturally important sector, this species has received much
attention at different levels [16–20].
In recent years, grasshoppers continue to be a major
economic pest of cereal crops in China. Major outbreaks have occurred in different areas every year and
O. chinensis (Thunberg) has caused significant damage to rice and other gramineous crops. The classical
method to control these outbreaks has been the use of
inexpensive and fast-acting chemical insecticides, but
recently, more and more biological insecticides were
used in the pest management in view of the three ‘Rs’:
resistance, residue and resurgence of chemical insecticides. At present, Bacillus thuringiensis (Berliner,
B.t.) is one of the most widely used biological pesticides, especially to control various lepidopterous
(butterfly), dipterous (flies and mosquitoes) and individual coleopterous (beetle) pests [21]. In order to
widen the scope of its application, the toxicity of B.t.
to O. chinensis was tested and its genotoxicity investigated in order to provide further background data
for the development of B.t. as a biological insecticide.
In addition, research is aimed at the isolation of new
strains specific for Orthoptera, grasshoppers, and to
establish a new model for entomological genotoxicity
testing.
The testis of O. chinensis may serve as a suitable
tissue to study the genotoxic effects of pesticides,
because the chromosomes can be prepared with little effort, readily be karyotyped due to their large
size and small number (2n = 23). It is well known
that CP is clastogenic in many organisms and that it
must be activated by hepatic mixed-function oxidase
to produce mutagenic activity. However, no studies
on the effects of CP in the grasshopper have been
published so far. In the work reported here, the clastogenic effect of CP and B.t. in O. chinensis cells was
evaluated using the testis of the fifth instar nymphs
to observe chromosome numbers and morphosis of
mitotic metaphase after treatment with the two agents
via intraperitoneal injection.
2. Materials and methods
2.1. Chemical
Cyclophosphamide (CP, purity 99.9%) is an
anti-neoplastic agent. Its chemical name is 2H-1,3,2oxazaphophorin-2-amine,N,N-bis(2-chlorethyl)-tetrahydro-,2-oxide,monohydrate and see formula on the
next page.
CP is a white, crystalline powder. It was purchased from the Institute of Biochemical Research in
Shanghai, China. It was dissolved in physiological
saline.
Z. Ren et al. / Mutation Research 520 (2002) 141–150
143
2.6. Chromosome preparation
2.2. Pesticide
B. thuringiensis (HD-1) emulsion (2750 IU/ml),
which contains live bacteria, was provided by the
New Technological Corporation of Biological Pesticides in Taiyuan, Shanxi Province, China and diluted
before use.
2.3. Insects
O. chinensis (Thunberg) were collected from the
farms of the Southern suburbs in Taiyuan, China and
groups of the fifth instar nymphs of male, about 25 mm
in length and 0.5 g in weight were reared in self-made
cages at least for 2 days with fresh wheat seedlings
and foliage, which were replaced twice daily, and fresh
water sprayed to control humidity [22].
2.4. CP treatment
CP was prepared by dissolving 20, 50 and 100 mg
powder in 10 ml physiological saline before treatment.
Groups of four individuals per time point each were
simultaneously injected a single dose of 3 ␮l of 2,
5 and 10 mg/ml compounds by use of a microsyringe into the abdomen between the first and second
sternum. Four negative-control grasshoppers for every time point received 3 ␮l physiological saline using the same method. Four hours before dissection,
each grasshopper was injected 3 ␮l freshly prepared
colchicine (0.05%) [18].
The grasshoppers’ testes were used for chromosomal preparation. The insects were dissected from the
ventral side at 24, 42 and 48 h after dosing. The testes
were collected and connective tissue attached to the
testes removed, then immersed in 0.075 M potassium
chloride (hypotonic solution) for 30 min and fixed in
3:1 methanol–glacial acetic acid. The fixed samples
were washed twice in fixative and stored at 4 ◦ C for
up to 1 month at most. The samples were used within
1 month using the following method of chromosomal
preparation.
A testis was transferred onto a clean, chilled slide
and the terminal filaments of the sperm tubes were
retained, other parts being removed. After being softened for 5 min by acetic acid (45%), the terminal filaments were cut into pieces by tip-head tweezers. With
the larger pieces removed, two drops of fixative were
added and dispersed by puffing, then the slides were
flame-dried. Finally, the slides were stained in buffered
Giemsa (5%) for 15 min [17,18], rinsed and air-dried.
The slides were coded. At least 50 well-spread
mitotic metaphases per individual (at least 200
metaphases for every dose) were observed for the
presence of CAs. The numbers and types of CAs were
scored under the microscope with an oil-immersion
objective at 100× magnification, following the published criteria [6]. Recorded data were evaluated as
the numbers of CAs per cell (excluding cells with
gaps only). Chromatid breaks, chromosome breaks,
chromatid deletions, fragments, minutes and acentric
fragments were recorded as chromosome abnormalities [6,23].
Different dose-gradients of B.t. and CP were used
to assess a dose-response relationship. In order to test
a time-response relationship, CP was tested also at
42 h after injection. Originally, we wanted to obtain
the result at 36 h, but due to the longer preserved
time of fixed testes, not enough metaphases were
observed.
2.5. B.t. treatment
2.7. Statistical analysis
Using the same methods as with CP, the fifth instar
nymphs were treated using different concentrations of
B.t. (0.55, 1.83, 5.50 IU/ml) and distilled water as negative control.
Two-sided trend test was used for determining the
significance of the difference between data. A P-value
of <0.05 was considered to indicate statistical significance [24].
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Z. Ren et al. / Mutation Research 520 (2002) 141–150
3. Results
The diploid chromosome number in males of O.
chinensis has been found to be 23, the sex determination being XO. There are 11 pairs of eu-chromosomes
and a sex chromosome, all of which are acrocentric.
These observations are in agreement with earlier data
[17,18]. The results obtained from the present investigation of structural chromosome aberrations induced
by CP and B.t. in O. chinensis testicular cells are
presented in Table 1. We intended to investigate the
dose– and time–responses relationships in the treatments with CP and B.t., but due to the longer storage
time of fixed testes treated for 42 h it was very difficult
to find enough good-quality disperse division figures
to obtain a statistically reliable result. Consequently,
only the 10 mg/ml dose of CP was analyzed at 42 h.
This may show the time–response relationship of CP
to O. chinensis to a certain extent (Fig. 1).
The dose–response curves (concentration versus
percentage of aberrant cells and numbers of chromosome aberrations per cell, excluding gaps) at different
time points (24 and 48 h) are illustrated in Figs. 2 and
3 for CP, and in Figs. 4 and 5 for B.t. .
In general, the chromosome morphology and structure in the mitotic metaphase of the cell cycle is the
most characteristic and easiest to identify and analyze, especially after treatment with colchicine prior
to the preparation of the chromosome slides. In addition, the types of chromosome aberration may be
easily confirmed according to the common definition
[23]. A sufficient number of mitotic metaphases were
analyzed in the current paper. The aberrations induced
Fig. 1. CP (10 mg/ml) time-response curve for induction of aberrant
cells of Oxya chinensis. Each data point is the mean of four insects.
Fig. 2. CP dose-response curves for induction of aberrant cells in
O. chinensis: (䉬) 24 h and (䊏) 48 h. Refer to Fig. 1 for further
details.
Fig. 3. CP dose-response curves for induction of numbers of
chromosomal aberrations/cells: (䉬) 24 h and (䊏) 48 h. Refer to
Fig. 1 for further details.
by CP were found to be centric, chromatid, and chromosome gaps, chromatid, and chromosome breaks,
chromatid deletions, fragments, acentric fragments
and minutes (Fig. 6). Chromatid and chromosome
Fig. 4. B.t. dose-response curves for induction of aberrant cells in
O. chinensis: (䉬) 24 h and (䊏) 48 h. Refer to Fig. 1 for further
details.
Z. Ren et al. / Mutation Research 520 (2002) 141–150
145
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Z. Ren et al. / Mutation Research 520 (2002) 141–150
Fig. 5. B.t. dose-response curves for induction of chromosomal
aberrations/cells: (䉬) 24 h and (䊏) 48 h. Refer to Fig. 1 for further
details.
breaks were present in higher frequencies than the
other types and complex types of CA, such as rings
and exchanges [26] were not observed. In the control
preparations, the damaged forms were for the main
part simple chromatid and chromosome breaks. The
incidence of acentric fragments and fragments was
very low at the 24-h time point, but only chromatid
and chromosome breaks were observed at the 48-h
time point.
The aberrations induced by B.t. were mainly chromosome and chromatid breaks. In addition, the incidence of fragments was very low and other types of
CAs were not observed. The only forms of damage observed in the control preparations were chromosome
and chromatid breaks, but the incidence was very low.
The numbers of chromosome aberrations and the
percentage of aberrant cells induced by CP increased
with increasing dose and with prolongation of treatment (Figs. 2 and 3). Fig. 2 and Table 1 show that the
increasing trend in the percentage of aberrant cells and
in the number of CAs per cell was similar at different
time points. The numbers of chromosome and chromatid breaks evidently increased, others except acentric fragments all decreased with prolonged treatment
time. CP induced the highest rates of chromosome
aberration at the largest dose (10 mg/ml) and longest
treatment (48 h).
The effect of CP in this species was also observed
in the 10 mg/ml dose group after 42 h (Table 1). The
time–response curve is illustrated in Fig. 1. There was
no statistically significant difference (P > 0.05) when
the result was compared with the 24-h time point but
there was a significant difference (P < 0.05) compared with the results at 48 h. The effect (treatment
time–percentage aberrant cells) was quite evident.
At all dose groups, significantly higher effects were
seen at 48 h than at 24 h. At the same time point, the
higher dose induced higher effect. In all, the percentage of aberrant cells induced by CP in O. chinensis
cells at any treatment dose and duration were significantly different (P < 0.001) as compared with the
physiological saline control. The only exception to the
above results was at 2 mg/ml (P > 0.05) and 5 mg/ml
(0.01 < P < 0.05) at the 24-h time point.
In case of B. thuringiensis, the numbers of chromosome aberrations and the percentage of aberrant
cells increased with increasing dose of active ingredient and prolonged treatment time. The overall trend
of the dose–response curves (Figs. 4 and 5) was the
same as for CP. But the trend was different at different treatment periods and a statistically significant difference (P < 0.05) was observed in all dose groups
at 48 h and in the top-dose group at 24 h, when compared with the control (distilled water). In all groups,
significantly higher effects were seen at 48 h than at
24 h. After the 48-h treatment period, the highest dose
group showed the highest effect on the percentage of
aberrant cells and the numbers of aberrations, which
were statistically significantly different (P < 0.01)
compared to the control.
4. Discussion
CP and B.t. were tested to study their effect on the
chromosomes of testicular cells in grasshoppers, using physiological saline and distilled water as negative
controls, respectively.
Although there are differences in the results obtained with physiological saline and distilled water,
no significant difference is indicated between these
two controls by statistical analysis. In the chromosome
aberration assays of inorganic fluoride in cultured rat
bone marrow cells, a high dose of sodium chloride
was used as the negative control. It has been shown to
produce lower efficacy, which according to the authors
may have been caused by technical artifacts [25].
If the length of chromosome damage is shorter than
the chromatid width, the lesion is called a gap. In
the present paper, most of the gaps belong to micro
Z. Ren et al. / Mutation Research 520 (2002) 141–150
147
Fig. 6. The normal chromosome complement and the types of chromosome aberrations in O. chinensis (Thunberg): N, normal chromosomal
complement; see Table 1 for other abbreviations.
damages and they may be repaired during the cell cycle. They may have been caused by the agents’ action,
the preparation of the slides or by artifacts. Based on
this, although the number of gaps is higher than that
of other types of chromosome aberrations in all the
groups, they were not taken into account when illus-
trating the dose– and time–responses curves and analyzing the agents’ genotoxicity.
In previous studies on genetic toxicology, CP was
often used as positive control, mainly in experiments in
vivo or in cell cultures in the presence of liver-S9 fraction. The types of chromosome aberrations induced by
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Z. Ren et al. / Mutation Research 520 (2002) 141–150
CP as a positive control were reported to be chromosome breaks, chromatid breaks, chromatid exchange,
chromosomal exchange and ring chromosomes [5,6].
CP must be activated by hepatic mixed-function oxidases and the metabolites are delivered to different
cells via the bloodstream to show mutagenic activity.
Phosphoramide mustard is thought to be the major
anti-neoplastic metabolite of CP while acrolein, which
is produced in equimolar amounts, is thought to be
responsible for most of the toxic side effects. DNA
adducts have been found after CP treatment in a variety of in vitro systems as well as in rats and mice using
[3 H]-labeled CP. In mammals, the half-life of CP was
3–4 h and about 99% of the total amount was eliminated after 48 h. Generally, CP induced the maximum
number of chromosome aberrations at the 48-h time
point [1]. The results of the present observation were
consistent with this. However, no exchanges and rings
were observed in the testicular chromosomes of O. chinensis. This difference compared to previous studies
may be due to the different species used. Among animal classes, there are dissimilarities in the metabolic
system, in the mechanism of DNA-damage repair, in
the composition of chromosomes, or other unknown
variables. The results showed significant dose–effect
relationships.
In conclusion, the fact that O. chinensis (Thunberg)
treated with various doses of CP exhibited significant
levels of genetic damage as compared to negative control and with a clear dose–response, demonstrates that
CP shows strong clastogenic effects on grasshopper
cells in vivo. From this, we can infer that there exists
an oxidase in the grasshopper capable of bioactivating
CP, analogous to the enzymes of the liver-S9 activating
system. This may be the reason that CP induces chromosome aberrations of grasshopper testicular cells.
Based on the above results, it is concluded that CP
is a clastogen in the spermatogonial chromosomes of
O. chinensis and may be useful as a positive control
in genotoxicity tests in grasshoppers. However, no information is available on activity of CP in in vivo micronucleus and SCE tests. Thus, further work, using
various grasshopper species, different materials and
combinations of cytogenetic end points is worth pursuing.
B. thuringiensis is gram-positive, spore-forming
bacterium living in the soil. It is known for its ability
to produce crystalline, proteinaceous, delta-endotoxin
during sporulation. Since the first report of Bacillus sotto [27], numerous related papers about B.
thuringiensis have appeared. In the field of biocontrol of insect pests by this bacterium, after the initial
discovery that several B. thuringiensis isolates were
effective against lepidopteran insects, the isolations
of B.t., specific to dipteran larvae [28], and specific
to some groups of coleopteran insects [29], were important advances. Now, many B. thuringiensis preparations have been shown to kill various lepidopteran,
dipteran and coleopteran insects. Even nematodes
were shown to be a target of this bacterium [30].
However, no data on the toxicity of this bacterium to
O. chinensis were found until now.
In this paper, the genetic toxicity of B. thuringiensis to the fifth instar nymphs of O. chinensis was examined. First, the general toxicity of B. thuringiensis was determined after administration via injection.
One tenth of the LD50 was then chosen as the highest dose (HD) to be tested in the genotoxicity assay,
with 1/3 HD and 1/10 HD as the middle and lowest
dose, respectively, according to Brusick’s protocol on
genotoxicity tests [6]. The actual doses used in the in
vivo study were 0.55, 1.83 and 5.50 IU/ml.
The results presented here show that the B.
thuringiensis emulsion causes chromosome aberrations in the spermatogonia of the grasshopper. This
effect may be a consequence of its functional course
and mode of action. B.t. parasporal crystal bodies are
solubilized in the alkaline environment of the insect
gut to produce an active delta-endotoxin. This toxin
binds to the gut epithelial cells, creating pores in the
cell membranes and leading to exchange of ions. As
a result, the gut is rapidly immobilized, the epithelial
cells lyse, the larvae stop feeding, and the pH of the
gut is lowered by equilibration with the blood pH.
This lower pH enables the bacterial spores to germinate, and the bacterium can then invade the host,
causing a lethal septicaemia [21]. On the other hand,
delta-endotoxin may also restrain the action of RNA
polymerase during DNA transcription. The function
is evident during insect ecdysis and metamorphosis.
This may be a primary reason that B. thuringiensis
induces chromosome aberrations in grasshoppers. As
spermatogonia represent the first stage of spermatogenesis [31], chromosome aberrations in spermatogonia
of O. chinensis may influence sperm formation, hence
affect reproduction of this species. It is well known
Z. Ren et al. / Mutation Research 520 (2002) 141–150
that mutagenesis by pesticides is complicated with
many biological factors. Further studies on the mechanism of the induction of chromosome aberrations in
grasshoppers are still needed.
In recent years, many different strains of B.
thuringiensis have been isolated to control lepidopteran, dipteran and coleopteran insects. The
present finding that a B. thuringiensis emulsion injected directly into the grasshoppers’ abdomen gave
rise to dose-related effects, demonstrates that B.
thuringiensis is active towards Orthopteran grasshoppers. It is obvious that future developments will be
focused on the isolation of new B. thuringiensis
strains specific for the Orthopteran grasshopper. This
will contribute to integrated pest management.
The injection method is one of the recommended
modes of treatment of large-size insects in toxicity
studies in the laboratory [32]. In the preliminarily tests
presented here, the toxicity and genotoxicity of B.t.
to O. chinensis were tested after injection, and the results show a significant toxic effect. In practical applications, the effect of B.t. to O. chinensis should still
need to be tested after spraying or other methods, first
on a small scale, then on a large scale. To a certain
extent, the present results may provide guidance for
such field studies.
Acknowledgements
The financial assistance was provided by the
National-Science Commission in China and Professor
Ziqiang Meng is very thankfully acknowledged for
providing technical instructions, Dr. Yihao Duan for
revising the paper. This work was supported by the
National Nature Science Foundation of China, Grant
39570107.
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