Vol.2, No.4, 292-297 (2010) Natural Science
Copyright © 2010 SciRes. OPEN ACCESS
DNA damage in hemocytes of Schistocerca gregaria
(Orthoptera: Acrididae) exposed to contaminated food
with cadmium and lead
Hesham A. Yousef1*, Amira Afify1, Hany M. Hasan2, Afaf Abdel Meguid1
1Entomology Department, Faculty of Science, Cairo University, Cairo, Egypt;heshamyousef.eg@gmail.com
2Agriculture Research Center, Ministry of Agriculture, Cairo, Egypt
Received 9 December 2009; Revised 4 January 2010; accepted 5 February 2010.
We measured in a comet assay the damage of
DNA in the hemocytes of various stages of the
grasshopper Schistocerca gregaria after ex-
posing them to various doses of Cd and Pb in
the food. The mechanisms of Cd and Pb toxicity
for grasshopper are discussed. The accumula-
tion of heavy metals and stage of the insect may
play important roles in causing the DNA damage.
S. gregaria may be considered a valuable bio-
indicator for evaluation the genotoxicity of en-
vironmental pollutants.
Keywords: Comet Assay; Heavy Metals (Cd, Pb);
DNA Damage; Schistocerca gregaria
Heavy metals are among the most problematic causes of
water, soil and plant pollution. Genetic and biochemical
effects of pollutants on organisms are important in estab-
lishing species as bioindicators for environmental haz-
ards [1,2]. Heavy metals have been found to induce
genotoxic effects in chironomids which are used as a
good bioindicator group for aquatic pollution [3]. Ter-
restrial insects that develop in the soil are also exposed
directly to metal ions present in the soil. Grasshopper
species may provide good systems to evaluate the
mutagenic effects of some environmental contaminants
Cadmium and lead are widespread and dangerous
heavy metals that are released into the environment from
many sources. Their accumulation in the soils can be-
come dangerous to all kinds of organisms, including
plants and human life, causing many genotoxic effects
[7-9]. They are highly toxic and have been recognized as
poison and a probable carcinogen [10]. Clinically, they
can adversely affect human health, especially the blood
and the renal system [11].
Changes in the cell genome caused by genotoxic
agents leading to mutations and possibly tumor forma-
tion are some of the lethal or sub-lethal effects induced
by a complex mixture of pollutants. Among recently
used methods to identify DNA damage is the comet as-
say (SCGE-single cell gel electrophoresis). The comet
assay provides a rapid, sensitive, and inexpensive
method to detect DNA strand breaks in individual eu-
karyotic cells [12]. Despite some difficulties in obtaining
cell/nuclei suspension, this method has been used to
detect and evaluate DNA damage caused by double
strand breaks, single strand breaks, alkali labile sites,
oxidative base damage, and DNA cross-linking with
DNA or protein. It has been successfully applied to cells
of various animal groups [13]. Only a few studies have
been reported on DNA damage in insects, including D.
melanogaster [14], and in the weevil Curculio sikkimen-
sis [15], and in grasshoppers Chorthippus brunneus [16].
The aim of the present work was to determine the
genotoxic effect of cadmium and lead on the locust S.
gregaria and to evaluate its potential as a biomonitor for
detecting a heavy-metal polluted environment.
2.1. Colonization of S. Gregaria
Locusts were reared in wooden cages at 32 ± 2C°, 50-
60% RH and 16 hrs day light in our Entomology De-
partment since about 10 years ago. A daily supply of
fresh grass, clover plant was supplied to the locusts.
Packed moist sterilized sand in suitable glass containers
about 7 cm in diameter and 10 cm deep were prepared
for egg-laying.
2.2. Heavy Metals Treatment and Sample
Preparation for Alkaline Single Cell Gel
(SCG) Assay
Living individuals of S. gregaria of the 4th, 5th instars,
H. A. Yousef et al. / Natural Science 2 (2010) 292-297
Copyright © 2010 SciRes. OPEN ACCESS
and newly emerged (NEA)(4 days old) and mature (15
days old) adult (MA), fed on treated clover (their stems
were previously immersed for 24 hrs in distilled water
containing 25 mg and 50 mg/L of CdCl2 and PbCl2, to
allow the clover to absorb contaminated water) or on
untreated clover, were collected from their respective
cage. Haemolymph samples were withdrawn from the
collected insects by means of micropipettes at incision
made near the 3rd coxae. Five insects were used for each
2.3. Detection of DNA Damage Using
Alkaline SCG Assay
Biochemical techniques for detecting DNA single strand
breaks (frank strand breaks and incomplete excision re-
pair sites), alkali-labile sites, and cross-linking with the
single cell were done according to the alkaline (pH 13)
SCG assay, and developed [17].
The alkaline version of comet assay was used to ana-
lyze the level of DNA damage in the hemocytes of S.
gregaria to estimate the genotoxic effects of Cd2+ and
Pb2+. 20 µL of hemolymph from the pool of 5 insects
were centrifuged at 1000 rpm for 10 min. Isolated hemo-
cytes were immediately suspended in cooled 50 μL
Ringer solution and kept on ice, in darkness. 10 µL of
isolated cells were mixed with 90 µL of 0.75% low
melting point agarose (LMPA), and placed on a micro-
scope slides, pre-coated with 1.5% normal melting point
agarose (NMA). A cover slip was added, and the slides
were immediately placed on ice. After agarose solidified,
cover slips were removed, and the slides were immersed
in a lyses buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris,
0.25 M NaOH, 1% TritonX-100, and 10% dimethylsul-
foxide (DMSO), pH 10.0) for 2h at 4°C. After the lysis,
the slides were placed in a horizontal gel electrophoresis
tank and DNA was allowed to unwind for 20 min in
electrophoresis buffer (300 mM NaOH and 1 mM EDTA,
pH 13). Electrophoresis was carried out at 21 V and
270 mA, at 4°C, for 15 min. Then the slides were neu-
tralized in 0.4 M Tris–HCl (pH 7.4), fixed with methanol
and allowed to dry overnight at room temperature before
staining with ethidium bromide (2 µg/mL). Comets were
analyzed with Axio fluorescence microscope (Carl Zeiss,
Germany) with an excitation filter of 524 nm and a bar-
rier filter of 605 nm. Three replicates were prepared and
each of them consisted of a pool of 5 individuals.
2.4. Evaluation of DNA Damage
DNA damage was visualized with fluorochrome stain of
DNA with the fluorescent microscope and a 40X objec-
tive (depending on the size of the cells being scored). A
Komet analysis system 4.0 developed by Kinetic Imag-
ing, LTD (Liverpool, UK) linked to a CCD camera was
used to measure the length of DNA migration (Tail length)
(TL), and the percentage of migrated DNA (DNA %). To
distinguish between populations of cells differing in size
nuclear diameter was measured. Finally, the program
calculated tail moment. 50-100 randomly selected cells
are analyzed per sample (at least 25 cells per slid and 3
slide per treatment were evaluated).
Statistical analysis for data was done using ANOVA
and T-test analysis, based on a minimum of 4 individual
insects per group. In addition, numbers of cells were
analyzed to exhibit values greater than the 95 or 99%
confidence limits for the distribution of control data.
3.1. Comet Assay of DNA Damage
The typical DNA damage of haemolymph cells of S.
gregaria exposed to low and high concentrations of
cadmium chloride (CdCl2) and lead chloride (PbCl2) in
the food can be seen in Figure 1. The haemolymph cells
of the control showed almost rounded nuclei (Figure
1(a)). In the haemolymph cells of the heavy metals con-
taminated insects, the nuclei with a clear tail like exten-
sion were observed indicating that the haemolymph cells
of the insect were damaged and DNA strand breaks had
occurred (Figure 1).
The typical DNA comet for hemocytes of S. gregaria
showed illustration of rounded nuclei of control and
maximum length of tail formed and migration of DNA in
this tail under the effect of contamination with different
concentrations of CdCl2 and PbCl2 (Figure 1).
The DNA damage of the hemocytes of different stages
of S. gregaria fed on clover exposed to low and high con-
centrations (25 and 50 mg/L) of CdCl2 and PbCl2 was
analyzed quantitatively by comet assay and expressed as
tail length (TL), DNA % and tail moment (TM) (Table 1,
and Figures 2 and 3). It was found that low concentration
(a) (b) (c)
(d) (e)
Figure 1. Typical DNA comet from haemocytes of 4th instar S.
gregaria. (a) Control; (b,c) Low and high concentrations of
CdCl2 respectively; (d,e) Low and high concentrations of
PbCl2, respectively.
H. A. Yousef et al. / Natural Science 2 (2010) 292-297
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Table 1. Detection of DNA damage by the comet assay, assessed as tail moment (TM) in hemocytes of 4th, 5th, NEA, and MA
of S. gregaria exposed in vivo to CdCl2 and PbCl2, at different doses in the food.
Significant at *P < 0.05; in all cases significance was tested with respect to 0 (control) using t-test, (N = 3). Values are expressed as means ± S.E.
of CdCl2 (25 mg/L) caused a significant increase in the
values of TM in the hemocytes of different stages. While,
the high concentration (50 mg/L) of CdCl2 caused a
lower significance increase in TM in the 4th instar, some-
what insignificant increase or decrease in 5th instar and
led to a significant higher increase of these values in the
adult stage (NEA and MA). Low and high concentra-
tions of PbCl2 caused a significant increase in TM, gen-
erally in all developmental stages with few insignificant
changes. The effect of the PbCl2 concentration was not
clear as in CdCl2 (Table 1).
The damage of hemocyte DNA expressed as TL and
DNA% under the effect of different concentrations of
CdCl2 and PbCl2, analyzed by the comet assay (Figures
2 and 3). It was found that 25 mg/L of CdCl2 caused a
significant increase in the values of TL, DNA% in the
hemocytes of different stages. The high concentration
(50 mg/L) of CdCl2 caused a lower significance increase
in TL in the 4th and 5th instar. Low and high concentra-
tions of PbCl2 caused a significant increase in TL, and
DNA%. The prominent increase in the values of TL and
DNA% in response to contamination with Cd and Pb
was observed in the mature adult stage (MA). The dose
concentration of Cd and Pb had insignificant effect on
the values of TL and DNA % (Figures 2 and 3).
The analysis of variance of the two factors (stage and
heavy metal concentrations) showed that, the stage of
the insect had a clear significant effect on the DNA
damage (TL, DNA % and TM). A less significant effect
of the dose (concentration of heavy metals) was ob-
served (Table 2).
In the present study, the treated clover exposed to CdCl2
and PbCl2, at doses of 10 and 20 mg/L, contained 10 and
20 µg/g plant tissues, respectively to each dose (data not
presented). The exposure of S. gregaria to Cd and Pb in
the food caused an increase in damage (expressed as TL,
TM, and DNA%) of DNA of hemocytes. However, the
obtained data were sometimes ambiguous; for instance,
the TL was not proportional to the Cd dose in the 4th and
5th instars but was true in the NEA and MA (Figure 2).
The available data from the literature are from assays on
cell cultures (mostly human or rat lymphocytes) and
many of them concerning genotoxicity of Cd, As, Pb,
and Hg [13,18,19].
Figure 2. Comet TL data of hemocytes from different instars
of S. gregaria exposed to food with low (25 mg/L) and high
concentration (50 mg/L) of Cd and Pb.
Figure 3. Comet DNA% data of hemocytes from different
instars of S. gregaria exposed to food with low (25 mg/L) and
high concentration (50 mg/L) of Cd and Pb.
Agent / Dose 4th instar 5th instar NEA MA
Control 0.028 ± 4.4 × 10-3 0.095 ± 5.8 × 10-3 0.009 ± 2.3 × 10-3 0.073 ± 3.5 × 10-3
0.092 ± 0.02* 0.16 ± 0.017* 0.04 ± 3.5 × 10-4* 0.29 ± 0.0133*
CdCl2 (mg/L)
50 0.057 ± 3.3 × 10-3* 0.07 ± 6.9 × 10-3 0.08 ± 3.9 × 10-3* 0.45 ± 0.03*
0.081 ± 0.022* 0.124 ± 0.012 0.05 ± 5.8 × 10-3* 0.32 ± 3.5 × 10-3*
PbCl2 (mg/L)
50 0.7 ± 8.8 × 10-3* 0.11 ± 0.017 0.021 ± 8.8 × 10-3* 0.44 ± 0.035*
H. A. Yousef et al. / Natural Science 2 (2010) 292-297
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Table 2. Analysis of variance (Two Way ANOVA) for tail length (TL), DNA %, and tail moment (TM) in S. gregaria with
heavy metal treatment as categorical factors.
Source of
Variation Df F P F P F P
Stage (1) 3 181.3 0.00001 133.6 0.00001 358.3 0.00001
Heavy metal
Concentration (2) 4 44.1 0.00001 78.1 0.00001 73.9 0.00001
Interaction 1 × 2 12 12.4 0.00001 29.7 0.00001 36.1 0.00001
The no increased or even decreased DNA migration
using comet assay may reflect the cells with DNA
cross-linking lesions [19,20]. The higher DNA damage
in the mature adults with respect to the long period of
exposure to heavy metals (Figures 2 and 3) reflects the
absence of repair mechanism in this insect at the used
concentrations of CdCl2 and PbCl2.
3.1. Cadmium Genotoxicity
Exposure of S. gregaria to Cd in their food leads to ac-
cumulation of the metal in the insect body of 10, 20, 10,
and zero µg/g insect body in the 4th, 5th, NEA, and MA
(data not presented data). The accumulation factors of
heavy metals in grasshoppers were found in the order
Cd > Hg > Pb indicating a greater affinity for Cd accu-
mulation. With the growth of muscle tissues and fat
bodies during post-embryonic development (nymph to
adult), the concentration of Cd was found to be steadily
increasing [21]. It has been suggested that the molecular
mechanism for the genotoxicity of cadmium may in-
volve either indirect or direct interaction of with DNA
[22], such as DNA strand breaks [10], DNA protein-cross
linking [23], Oxidative DNA damage [24], enhanced pro-
liferation, depressed apoptosis and inhibition of DNA
repair [24-26].
Many metals, including cadmium, in biological sys-
tems form complexes with nucleophilic ligands of target
molecules [27]. The affinity of cadmium is higher for
biomolecules containing more than one binding site such
as metallothionein [7]. Another factor of cadmium toxic-
ity is that it replaces zinc in enzymes, thereby inhibiting
their activity [28]. Some insects as D. melanogaster have
tolerance to heavy metals [27], and their natural popula-
tions differed in amplification of the metallothionein
gene [29]. By binding to plasma membrane receptors,
cadmium stimulates release of calcium from intracellular
storage sites [7]. Moreover elevated cadmium levels may
inhibit Ca-ATPase working in the plasma and endoplas-
mic reticulum membranes, leading to disturbance of cal-
cium homeostasis [7,25]. Also, it nhibits DNA repair
enzymes, such as DNA polymerases by binding to nu-
cleic acids and chromatin [25].
3.2. Lead Genotoxicity
The exposure of grasshoppers to Pb in the food caused
an increase of DNA damage in haemolymph cells. The
increase of TL values was proportional to the Pb dose in
the food in 4th nymphal instars, newly emerged, and ma-
ture adults but not proportional in 5th instars (Figure 2).
This may be due to high retention of metals in the 5th
instar as compared to the other stages. Dietary factors
greatly influence lead retention. Several mechanisms
could intervene in these effects. Low dietary calcium and
lead-binding proteins at the sites of absorption [30] in-
fluence lead retention, because lead interferes with the
regulation of calcium metabolism [31]. An interaction of
lead and calcium can occur on the sites of toxic action
by binding to phosphate groups, or by interfering with
uptake in organelles etc.
There are several mechanisms how lead might inter-
fere with repair process. Their ions may interfere with
calcium regulated processes involved in the regulation of
DNA replication and repair [32], induced genome dam-
age includes DNA single- strand and double-strand breaks,
DNA-DNA crosslinks, induction of reactive oxygen in-
termediates [33], and consequently acts as co-clastogens
or co-mutagens [34].
The present work clearly shows that, the significant
increase of genotoxicity in relation to the development
of nymph to adult stage may be due to accumulation of
heavy metals in the tissues and blood. This suggests that
the DNA damage increased with Pb in blood. Likewise,
a significant correlation was found between Pb accumu-
lated in the blood and genotoxic effects in Pb exposed
workers [35].
In conclusion, the genotoxicity of cadmium and lead
in S. gregaria was very high in the mature adult stage;
irrespective of the heavy metal dose and accumulation in
the cells. So this may reflects the role played by S. gre-
garia as a valuable bioindicator for environmental genotoxic
Authors are grateful to Professor Dr. James. L. Nation (Department of
Entomology and Nematology, University of Florida, USA) and Profes-
sor Dr. John Trumble (Entomology Department, California University,
USA), for valuable reviewing the manuscript.
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