DNA damage in hemocytes of Schistocerca gregaria (Orthoptera: Acrididae) exposed to contaminated food with cadmium and lead

doi:10.4236/ns.2010.24037

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Vol.2, No.4, 292-297 (2010) Natural Science

http://dx.doi.org/10.4236/ns.2010.24037

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;[email protected]

2Agriculture Research Center, Ministry of Agriculture, Cairo, Egypt

Received 9 December 2009; Revised 4 January 2010; accepted 5 February 2010.

ABSTRACT

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

1. INTRODUCTION

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

[4-6].

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. MATERIALS AND METHODS

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

293

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

sample.

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. RESULTS

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

294

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).

3. DISCUSSIONS

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*

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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.

TL DNA % TM

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

pollutants.

4. ACKNOWLEDGEMENTS

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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