Ecotoxicology and Environmental Safety 119 (2015) 140–147

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Inhibition of cadmium- induced genotoxicity and histopathological

changes in Nile tilapia fish by Egyptian and Tunisian montmorillonite
clay
Karima F. Mahrous a,n, Aziza M. Hassan a,b, Hasnaa A. Radwan a, M.A. Mahmoud c
a
Cell Biology Department, National Research Centre, 33 El-Bohooth Street, Dokki, Giza, Egypt
b
Biotechnology Department, Faculty of Science, Taif University, Taif, Saudi Arabia
c
Department of Pathology Faculty of Veterinary Medicine, Cairo University, El Giza Square, Giza, Egypt

art ic l e i nf o a b s t r a c t

Article history: Cadmium (Cd) is an important inorganic toxicant widely distributed in the environment because of its
Received 1 January 2015 various industrial uses. The aims of the current study were to investigate the efficacy of purified Egyptian
Received in revised form and Tunisian montmorillonite clays (EMC and TMC) to inhibit genotoxicity and histological alterations
28 April 2015
induced by cadmium chloride (CdCl2) utilizing the Nile tilapia fish as an in vivo model. Chromosomal
Accepted 29 April 2015
aberrations (CAs), micronucleus (MN) frequencies and DNA fingerprinting profile were genotoxic end
Available online 19 May 2015
points and histopathological changes that were used in this investigation. Six groups of fish were treated
Keywords: for 2 weeks and included control group, CdCl2-treated group and groups treated with EMC or TMC alone
Fish or in combination with CdCl2.
Cadmium
The present results revealed that, treatment of fish with CdCl2 exhibited significant increased in the
Clays
number of micronucleated erythrocytes (MnRBCs), frequency of CAs and instability of genomic DNA.
RAPD-PCR
Cytogenetics Treatment of EMC and TMC in combination with CdCl2 significantly reduced the frequency of MnRBCs by
Histopathology the percentage of 53.28% and 60.77% and the frequency of CAs by 43.91% and 52.17% respectively. As well
as, normalized DNA fingerprinting profile and significantly improved histopathological picture induced
by Cadmium treatment. It is worth mention that both clays have the ability to tightly bind CdCl2 and
decreased its cytotoxicity and genotoxicity; however, Tunisian clay was more efficient in binding with
the CdCl2 than Egyptian clay.
& 2015 Elsevier Inc. All rights reserved.

1. Introduction levels of the trophic chain (Stoeppler, 1991). It is well known as

The contamination of food and feedstuff with heavy metals
represents many problems for human and animal’s health, thereby
continuously attracting worldwide attention (Satarug et al., 2003;
FAO/WHO, 2003). Heavy metal contamination in the terrestrial as
well as the aquatic environment is a worldwide problem of in-
creasing magnitude. Heavy metals can affect aquatic organisms
through water, sediment or food chain (Zyadah, 1995).
Cadmium (Cd) is an important inorganic toxicant widely dis-
tributed in the environment because of its various industrial uses
and it is a nonessential element to all living organisms (Randi
et al., 1996; Besirovic et al., 2010). Also contamination of ground-
water from smelting and industrial uses as well as the use of
sewage sludge as a food-crop fertilizer. Cadmium was known by
their bioaccumulation and caused harmful effects in different
n
Corresponding author. Fax: þ20 2 33370931, þ20 2 3766187.
E-mail address: kfmahrous@hotmail.com (K.F. Mahrous).
carcinogen (Banerjee and Flores-Rozas, 2005), teratogenic (Hov-
land et al., 2000), caused sterility (Bench et al., 1999), nephrotoxic
(Ahmed and Abdel-Wahhab, 2000; Cai et al., 2001), hepatotoxic
(Horiguchi et al., 2000; Ahmed and Abdel-Wahhab, 2000), geno-
toxic and apoptotic (Kim et al., 2005; Mondal et al., 2005) and also
inducing pancreatic activity changes (Shimada et al., 2000). For
this fact, excessive exposure to Cd results in diseases and occa-
sionally death (Othumpangat et al., 2005).
Cadmium that enters aquatic environments accumulates
within the bodies of aquatic organisms (Habib and Samah 2013).
The growth, osmoregulation and reproduction of fish are affected
by exposure to this metal (Kim et al., 2004). Cd obstructs numer-
ous reproductive processes in fish such as sexual maturation,
spermatogenesis, fertilization success and development of the
embryonic and post embryonic stages (Jezierska and Witeska,
2001; Dietrich et al., 2011). It is also causes DNA single-strand
breaks, DNA–protein crosslinks, chromosome aberrations (CAs),
and apoptosis (Hovland et al., 2000). Previously, Muley et al.
(2000), found significant alterations in the DNA and RNA contents

http://dx.doi.org/10.1016/j.ecoenv.2015.04.054
0147-6513/& 2015 Elsevier Inc. All rights reserved.
 K.F. Mahrous et al. / Ecotoxicology and Environmental Safety 119 (2015) 140–147
in gills, liver and brain of the common carp, Cyprinus carpio ex-
posed to cadmium chloride and lead acetate. Also Bais and Lo-
khande (2014) found reduction of DNA in Ophiocephalus straatus
fish.
Many of common clays and zeolitic minerals may be non-
selective in their action and may pose significant hidden risks due
to interaction with nutrients and heavy metals. Several reports
have indicated that the phyllosilicate montmorillonite clay, which
is currently used as an anti-caking agent for animal feed borne
metals uptake, may remove heavy metals from aqueous solutions
(Subramanian and Gupta 2006; Da Fonseca et al., 2006). In the
aquatic environment, Montmorillonite Clay binds toxins and
heavy metals to a degree that far outperforms charcoal and other
filter products, plus it supports beneficial bacteria also, the Mon-
tmorillonite Clay has a protective effect against some mycotoxins
(sterigmatocystin and aflatoxin) and could be used to protect
cultured tilapia from such toxins (Mahrous et al., 2006; Hassan
et al., 2010). Scientifically speaking, most bentonite clay minerals
have peculiar adsorption arising from their layered structure,
charged layers and active edges. The layered structure provides
inter-layer space to host guest molecules and ions. Charged layers
and “broken edge” sites attract varieties with opposite charges
through Van der Waals force. Such features allow clay minerals to
be used as adsorbents for the removal of heavy metal ions from
water. Calcium Montmorillonite Clay can also be effective ad-
sorbents and absorbents of oily pollutants and animal waste
(Mckinnon, 2013).
Therefore, the application of the purified montmorillonite clay
collected from Tunisian and Egypt environments as adsorbents for
remediation of Cd toxicity is of great national and international
interest. The aim of the present study was to evaluate the pro-
tective effect of Egyptian and Tunisian clay on the chromosomal
aberrations (CAs), micronuclei (MNs), DNA and histo-pathological
changes in tilapia exposed to cadmium.
2. Materials and methods
2.1. Chemicals
2.1.1. Cadmium chloride
Cadmium chloride was purchased in a pure standard solution
in concentration 1000 mg /L from Merck, 64271 Darmstadt
(Germany).
2.1.2. Tunisian montmorillonite clay
The results of the chemical analysis of TM revealed that was
composed of 52.61% SiO2, 10.11% Al2O3, 5.10% Fe2O3, 0.44% TiO2,
1.05% MgO, 5.75% CaO, 2.22% Na2O, 2.30% K2O, 20.19% FL, and 0.19%
SO3 based on the dry weight.
2.1.3. Egyptian montmorillonite clay
It was kindly supplied from the Ceramic Department, National
Research Center, Dokki, Egypt. The results of the chemical analysis
of EM revealed that was composed of CaO 0.99%, SiO2 43.90%,
Al2O3 18.64%, Fe2O3 9.50%, MgO 2.04%, SO3 0.07%, K2O31.08%,
Na2O 2.10% and Cl‾ 4.21%.
2.2. Fish
Sixty apparently healthy, two-month-old Nile tilapia fish (Or-
eochromis niloticus) with an average body weight of 907 10 g were
purchased from El-Nobarya Fish Farm (El-Nobarya, Egypt) and
transported alive in a large plastic water container supplied with
battery aerators as a source of air. During transportation fishes
were treated with lidocaine (5 mg/L) to reduce stress. Fish were
141
maintained on a standard fish diet at the Animal House, Faculty of
Veterinary Medicine, Cairo University (Giza, Egypt). Feeding was
done once daily using a pelleted diet (32% protein ration) at rate of
3% of the fish body weight. The water in aquaria was changed daily
to avoid metabolite accumulations in glass aquaria (static system).
After an acclimation period of one week, the fish were divided into
six experimental groups (10 fish/group) and each group was
placed in a fully prepared aquarium containing de-chlorinated tap
water, the average water temperature was 20 73.7 °C and the pH
was in the range 7.17–8.19.
2.3. Experimental design
Fish within different treatment groups were treated for
2 weeks as follows:
Group 1, untreated control group fed on the standard diet;
Group 2, treated orally with Egyptian Montmorillonite clay (EMC)
alone at level (400 mg kg  1 BW); Group 3, treated orally with
Tunisian Montmorillonite clay (TMC) alone at level
(400 mg kg  1 BW); Group 4, treated orally with CdCl2
(2.5 mg kg  1 BW) in water; Group 5, treated orally with CdCl2
(2.5 mg kg  1 BW) simultaneously with EMC (400 mg kg  1 BW)
and Group 6, treated orally with CdCl2 (2.5 mg kg  1 BW) si-
multaneously with TMC (400 mg kg  1 BW).
2.4. Cytogenetical investigations
2.4.1. Micronucleus test
A drop of blood from the gills was mixed with a drop of fetal
calf serum and smeared directly on slide then air dried, fixed in
absolute methanol for 5 min and stained with 5% Giemsa for
7 min. Two thousand of red blood cells per fish were analyzed for
the frequency of MN erythrocytes. The diameter of the micro-
nucleus (MN) was less than one-third of the main nucleus, sepa-
rated from or marginally overlapped with main nucleus and had
similar staining as the main nucleus. The number of micro-
nucleated erythrocytes (Mn-RBCs) was expressed per 2000 ery-
throcytes (De Flora et al., 1993).
2.4.2. Chromosomal preparation
Chromosomal preparation of kidney tissues was carried out
according to the method described by (Al-Sabti, 1986) with some
modification. In brief the anterior kidney from each fish was ex-
cised and cut into fine particles in 5–7 ml of RBMI medium and
0.2 ml of 0.05 colchicine was added to each tube in vitro. Cultures
were incubated at 37-38 °C for 1 h then the cells were centrifuged
at 1000 rpm for 10 min and resuspended in pre warmed (37 °C)
hypotonic solution (KCl 0.5%) for 30 min at 37 °C. The sample were
centrifuged and fixed; the slides were produced by the conven-
tional method and stained with Giemsa stain. Chromosome ana-
lysis was carried out in 100 metaphase spreads for each fish.
2.5. Molecular genetics analysis
2.5.1. DNA extraction
DNA was extracted from the liver fish according to the method
described by Sambrook et al. (1989). The genomic DNA was iso-
lated using phenol/chloroform extraction and ethanol precipita-
tion method with minor modifications.
2.5.2. Random amplified polymorphism DNA (RAPD- PCR)
RAPD-PCR was carried out with the pooled and the individual
genomic DNA samples. A total of six random decamer primers of
arbitrary sequences with 60-70 GC% content were used as listed in
Table (1). The amplification conditions and PCR mixture were set
according to Williams et al. (1990) and Plotsky et al. (1995),
142 K.F. Mahrous et al. / Ecotoxicology and Environmental Safety 119 (2015) 140–147
Table 1
Sequence of primers used to amplify DNA.
Primer Sequence (5′–3′) CG %
B02 TGATCCCTGG 60
B04 GGACTGGAGT 60
C18 TGAGTGGGTG 60
C20 ACTTCGCCAC 60
D01 ACCGCGAAGG 70
D06 ACCTGAACGG 60
thermal cycling (ABI 9700) was carried out by initial denaturation
at 94 °C for 2–5 min, followed by 34–45 cycles each at 94 °C for
30–60 s, annealing temperature at 28–54 for 30–60 s, poly-
merization temperature at 72 °C for 30–60 s and final extension at
72 °C for 10 min. The samples were cooled at 4 °C.
2.5.3. Agarose gel electrophoresis
The amplified DNA fragments were separated on 2.0% agarose
gel, stained with ethidium bromide, visualized on a UV transillu-
minator and photographed by Gel Documentation system Gel Pro
(version 3.1 for window 3).
2.6. Histopathological examination
Tissue specimens from kidneys, liver and gills were collected
after clinical and gross examination and immediately fixed in
neutral buffered formalin 10% and dehydrated in ascending grades
of ethanol (70%, 80%, 90%, and 100% for 1 h each). The specimens
were then cleared in 2 changes of xylene, embedded in paraffin
and sectioned at 4–5 mm thickness. The sections were stained
using routine Hematoxylin and Eosin stain (Bancroft et al., 1996).
3. Statistical analysis
All values were expressed as mean 7 SE in each group. Sig-
nificant differences between the groups were determined with
SPSS 10.0 software (SPSS Inc., Chicago, IL, USA) by performing one-
way ANOVA with post hoc comparisons followed by Duncan’s
multiple range test. The results of cytogenetic study were statis-
tically analyzed using t-test. A difference was considered sig-
nificant at the p o0.05 level.
4. Results
4.1. Cytogenetical results
4.1.1. Micronucleus result
The size and position of micronuclei in the cytoplasm showed
slight variation and one or more micronuclei per cell was ob-
served. The frequencies of micronuclei recorded in all groups are
summarized in Table 2. The results showed that Egyptian clay or
Tunisian clay induced non significant increase in the mean value of
micronuclei in erythrocytes of fish as compared to the control
group. Cadimum at dose 2.5 mg/kg BW induced a statistically
significant (P o0.05) increased in the frequency of micronuclei. On
the other hand, treatment of EMC and TMC in combination with
Cd significantly (P r0.05) reduced the frequency of micronuclei
induced by cadmium alone and the reduction percent in the mi-
cronuclei incidence was calculated to be 53.28% and 60.77% re-
spectively (Fig. 1).
4.1.2. Chromosomal aberrations results
Table 3 showed the mean 7standard errors of types and total
Table 2
The effect of Egyptian and Tunisian clays on the induction of micro-nucleated red
blood cells in cadmium treated fish.
Treatment No of Mn-RBCs Mn-RBCs mean 7 SE
Control 20 4.3 7 0.25d
Egyptian clay (EMC) 18 3.6 7 0.31d
Tunisian clay (TMC) 14 2.8 7 0.37d
Cadmium 241 48.4 7 1.72a
EMC þ cadmium 126 24.9 7 1.72b
TMC þcadmium 102 21.6 7 1.3c
Means superscript with different letters are significantly different (P o 0.05).
Fig. 1. Histogram showing the inhibition percent of both EMC and TMC clays on the
frequencies of micronucleated erythrocytes and chromosomal aberrations in
Tilapia.
chromosomal aberrations induced in all experimental groups.
Various types of structural chromosomal abnormalities recorded
were chromatid breaks, chromatid deletions and fragments
(chromosomal breaks), centromeric attenuations, Centromeric
fusion and polyploidy whereas chromatid gaps were excluded.
The results showed that the mean value of chromosomal
aberrations induced by both Tunisian and Egyptian clays was
found to be statistically insignificantly changed as compared with
control group. However, the mean value of chromosomal aberra-
tions induced by cadmium was statistically significant (P o0.05)
increased when compared with control group. On the other hand
combined treatment of Egyptian or Tunisian clay resulted sig-
nificantly (P r0.05) reduction in the frequency of chromosomal
aberrations in compared to the Cd-treated group. The reduction
percent in the chromosomal aberrations incidence was calculated
to be 43.91% and 52.17% respectively (Table 3). The effect of Tu-
nisian clay was more pronounced than Egyptian clay in the pre-
vention of induction of micronuclei and chrmomosomal
aberrations.
4.2. RAPD-PCR fingerprinting profiling
Six random 10-mer primers (B02, B04, C18, C20, D01 and D06)
were used to analyze instability in the genome of hepatic treated
tilapia using RAPD-PCR fingerprinting and the presence of changes
in the RAPD profiles obtained from the exposed population de-
pending on the primer used. From the six primers, only three
produced reproducible and scorable amplification fingerprints.
Primers produced similar RAPD fingerprints for controls and
 K.F. Mahrous et al. / Ecotoxicology and Environmental Safety 119 (2015) 140–147 143

Table 3
The effects of Egyptian and Tunisian clays on the frequency of chromosomal aberrations in kidney of cadmium treated fish.

Treatment Total aberration excluding gap Chromosomal breaks Cent. attenuation Cent. fusion Polyploidy Gap

Break Deletion Fragment

Control 2.5 7 0.29e 0 0 1.75 7 0.25cd 1.757 0.25cd 0 0.5 7 0.29e 1.57 0.30bc
Eg cl 4.27 0.25d 0 0.25 7 0.2c 1.8 7 0.31c 1.87 0.31c 0 1.75 7 0.24d 1.57 0.27bc
Tun cl 3.757 0.48de 0 0.5 7 0.28c 1.5 7 0.29d 1.57 0.29d 0 1.5 7 0.20d 1.27 0.31c
Cadmium 25.57 1.93a 5.07 0.41a 4.75 7 0.48a 5.75 7 0.63a 5.757 0.63a 1.07 0.41a 4.3 7 0.48a 3.757 0.25a
Eg þ Cad 15.47 1.5b 2.57 0.29b 2.9 7 0.31b 3.0 7 0.57b 3.07 0.57b 0.57 0.29b 3.25 7 0.48b 1.57 0.29bc
Tunþ Cad 13.57 0.65c 2.07 0.1b 3.0 7 0.41b 3.0 7 0.40b 3.07 0.40b 0.257 0.2c 2.5 7 0.29c 1.757 0.25b

Means superscript with different letters are significantly different (Po 0.05).
treatment groups rendering it uninformative in revealing altera-
tions in hepatic DNA. A total of 36 loci of different bands were
amplified by the three primers (B02, C20 and D01), with an
average about 12 loci per primer. The 36 loci that were amplified,
14 bands were polymorphic giving (38.8%) polymorphism, the si-
zes of bands ranged from 420–1530 bp. DNA profiles presented in
(Fig. 2A–C) were generated using three primers and a mixture of
three individuals from each replicate. Profiles generated by these
primers revealed differences between control and exposed in-
dividuals, with visible changes in the number and size of amplified
DNA fragments. Moreover, the most obvious result was the ap-
pearance or absence of new bands in DNA samples of the group
treated with cadmium alone which resulted from the genetic al-
teration in DNA in cadmium-treated group whereas none of these
new bands were found in the genomic DNA of the samples col-
lected from hepatic fish of the control group or that received CdCl2
in combination.
interstitial infiltration with oesinophilic granular cells (Fig. 3.1c).
Focal tubular necrosis replaced by leucocytic cells infiltration as
well as oesinophilic granular cells aggregations around the glo-
merular tuft were observed in some examined sections (Fig. 3.1d).
4.3.2. Liver
Microscopically, liver of tilapia from control group and those
treated with Egyptian clay alone revealed no obvious changes.
Meanwhile, examined sections from fish treated with Tunisian
clay showed vacuolar degeneration of hepatocytes. Conversely,
sections from fish treated with cadmium revealed cytoplasmic
vacuolization of hepatocytes, necrosis of hepatopancrease
(Fig. 3.2a) accompanied with infiltration with eosinophilic gran-
ular cells. However, examined sections from groups treated with
combination of cadmium either with Egyptian clay or with Tuni-
sian clay revealed no histopathological alterations except vacuo-
lization of hepatocytes (Fig. 3.2b).

4.3. Histopathological results 4.3.3. Gills

4.3.1. Kidney
Examination of kidneys of both control fish and those treated
with Egyptian clay alone revealed no histopathological changes,
whereas, sections from tilapia treated with Tunisian clay alone
showed vacuolar degeneration of renal tubular epithelium. Mi-
croscopically, kidney of tilapia treated with cadmium revealed
vacuolar degeneration of renal tubular epithelium (Fig. 31a), focal
necrosis of renal tubules associated with oesinophilic granular
cells infiltration (Fig. 3.1b). Examined sections from tilapia treated
with combination of cadmium and Egyptian clay revealed im-
provement in histopathological picture as it showed vacuolar de-
generation of renal tubular epithelium without necrosis or evi-
dence of inflammatory cells infiltration. On the other hand, kid-
neys from group treated with combination of cadmium and Tu-
nisian clay revealed severe changes described as vacuolations of
epithelial lining renal tubules, intertubular edema associated with
Sections from control, untreated tilapia revealed no histo-
pathological alterations. Meanwhile, those treated with Egyptian
clay alone showed congestion of secondary gill lamellae. On the
other hand, examined sections from tilapia treated with Tunisian
clay alone revealed hyperplasia and fusion of gill lamellae as well
as infiltration of the brachial tissue with eosinophilic granular
cells. Examined sections from fish treated with cadmium revealed
marked congestion of secondary gill lamellae (Fig. 3.2c), hyper-
plasia of mucous secreting cells, hyperplasia and fusion of gill la-
mellae. Massive infiltration with eosinophilic granular cells was
also noticed in some examined sections (Fig. 3.2d). Meanwhile,
sections from group treated with combination of cadmium and
Egyptian clay showed hyperplasia and congestion of secondary gill
lamellae as well as hemorrhages in the branchial tissue (Fig. 3.3a).
Moreover, gills from fish treated with combination of cadmium
and Tunisian clay revealed marked hyperplasia of mucous se-
creting cells as well as diffuse hyperplasia and fusion of gill

Fig. 2. RAPD fingerprinting profiles of DNA from different groups of fish using primers B02,C20 and D01 in A,B and C. Lane (M): DNA ladder. Lane (1): control. Lane (2):
treatment with Eg cl. Lane (3): treatment with Tun cl. Lane (4): treatment with cadmium chloride. Lane (5): treatment with Eg þCd. Lane (6): treatment with TunþCd.
144 K.F. Mahrous et al. / Ecotoxicology and Environmental Safety 119 (2015) 140–147

Fig. 3. 1-Photomicrographs of kidney of O. niloticus fish: (a) treated with cadmium showing vacuolar degeneration of renal tubular epithelium (H&E 200  ). (b) Treated with
cadmium showing vacuolar degeneration of renal tubular epithelium, focal necrosis of renal tubules associated with oesinophilic granular cells infiltration (H&E 200  ).
(c) Treated with combination of cadmium and Tunisian clay showing vacuolations of epithelial linning renal tubules, intertubular edema associated with interstitial in-
filtration with oesinophilic granular cells (H&E 200  ). (d) Treated with combination of cadmium and Tunisian clay showing oesinophilic granular cells aggregations around
the glomerular tuft (H&E 400  ). 2-Photomicrographs of several organs of O. niloticus fish: (a) Liver of tilapia treated with cadmium showing necrosis of hepato-pancrease
(H&E 200  ). (b) Liver of tilapia treated with combination of cadmium and Egyptian clay showing vacuolization of hepatocytes (H&E 200  ). (c) Gills of tilapia treated with
cadmium showing marked congestion of secondary gill lamellae (H&E 200  ). (d) Gills of tilapia treated with cadmium showing massive infiltration with eosinophilic
granular cells (H&E 400  ). 3-Photomicrographs of gills of O. niloticus fish: (a) Treated with combination of cadmium and Egyptian clay showing hemorrhages in the
branchial tissue (H&E 200  ). (b) Treated with combination of cadmium and Tunisian clay showing marked hyperplasia of mucous secreting cells as well as diffuse
hyperplasia and fusion of gill lamellae (H&E 100  ). (c) Treated with combination of cadmium and Tunisian clay showing necrosis, edema and massive infiltration with
granulated and degranulated eosinophilic granular cells (H&E 200  ).

lamellae (Fig. 3.3b). Necrosis, edema and massive infiltration with
granulated and degranulated oesinophilic granular cells were no-
ticed in gill rackers of examined sections (Fig. 3.3c).
5. Discussion
Cadmium has been demonstrated to stimulate free radical
production, resulting in oxidative deterioration of lipids, proteins
and DNA, and initiating various pathological conditions in humans
and animals (Shaikh et al., 1999; El-Demerdash et al., 2004). Fishes
are very sensitive to a change in their environment and can play
significant role in assessing potential risk associated with con-
tamination in aquatic environment (Lakra and Nagpure, 2009).
The results of our study showed increasing in the induction of
micronuclei in peripheral erythrocytes of O. niloticus when ex-
posed to cadmium. In line with our findings, several authors have
previously been able to demonstrate that the micronuclei test in
fish erythrocytes and other tissue cells yielded positive results
after the administration of cadmium. Cadmium induced significant
micronuclei formation in different tissues of Oreochromis niloticus
(Ozkan et al. 2011), Oreochromis mossambicus (Chandra and
Khuda-Bukhsh, 2004), Salmo trutta, and Phoxinus phoxinus
(Sanchez-Galan et al., 1999) and rainbow trout (Castano et al.
1998). Similarly, Cavaş et al. (2005) reported an increase in the
induction of MN in peripheral blood erythrocytes, gill epithelial
cells, and liver cells of Cyprinus carpio, Carassius gibelio, and Cor-
ydoras paleatus with exposure to doses of Cd. They suggested that
 K.F. Mahrous et al. / Ecotoxicology and Environmental Safety 119 (2015) 140–147
the mechanism of cadmium genotoxicity is mainly conditioned by
single strand breaks in DNA through the direct cadmium–DNA
interactions as well as by the action of incision nucleases and/or
DNA-glycosylase during DNA repair (Privezentsev et al., 1996;
Lutzen et al., 2004). Also several pathological examinations sug-
gest that cadmium can cause aneuploidy, and it poisons the mi-
totic spindle (Berces et al., 1993). The mitotic spindle has a great
importance in the formation of micronuclei.
Our results also showed that the exposure of fish to Cd induced
elevated levels of chromosomal aberrations including; chromoso-
mal breaks, gaps, centromeric attenuations, Centromeric fusion
and polyploidy. These results are in agreement with previous
studies as Chandra and Khuda-Bukhsh (2004) have revealed that
CdCl2 induced aberrations of various kinds in Oreochromis mos-
sambic, such as break, terminal association, centric fusion, pre-
cocious centromeric separation and C-mitosis. Cd had already
been reported to have adverse effects on chromosomes and Cd
burden in the body has been reported to be directly correlated
with chromosomal aberrations (IARC, 1993). Low dose of cadmium
(1 mg/kg/day) for 30 days resulted in chromosomal aberrations as
manifested by aneuploidy, breaks, gaps, centromeric fusion re-
sulting in formation of submetacentric and metacentric chromo-
somes. However, it was also observed that CdCl2 administration at
a large dose given staggered e.g. 25 mg/kg/day for 20 days and
200 mg/kg/day for 5 days resulted in severe chromosomal damage
if compared to the same dose given singly (Singh et al., 2007).
Singh and Sankhla (2010) have observed that in animals ad-
ministered with CdCl2 there was a significant increase in the
number of chromosomal aberrations and a decline in mitotic in-
dex. Free-radicals can originate from exposure to various en-
vironmental toxicants, cadmium being one of them, resulting in
disturbed homeostasis and induction of biological stress as man-
ifested by a sharp decline in mitotic index and an elevation of
chromosomal aberrations.
The present results, RAPD profile showed genomic DNA al-
teration in cadmium- treated group which represented by the
appearance or absence of new bands in DNA samples which re-
sulted from the genetic instability in DNA in cadmium-treated
group. In this concern, Rocco et al. (2014) stat that the dis-
appearance or the loss of PCR amplification products can reveal a
change in the DNA sequence due to mutations, showing new an-
nealing events and/or large deletions, bringing two pre-existing
sites nearer or separating them farther. Furthermore, the amplifi-
cation pattern of the DNA of the individuals exposed showed the
acquisition or the loss of bands and/or the change of intensity of
the same, caused by a variation in the number of recognition sites
of the sequence of the primer and thus of mutations. The varia-
tions of frequency of the bands could be the result of structural
changes induced by the genotoxic events such as cadmium.
Previous studies (Zhiyi, and Haowen 2004; Atienzar and Jha,
2006) showed that the changes in electrophoretic patterns reflect
the alterations of the DNA due to a single change in the bases
(point mutations) or a more complex chromosomal reorganiza-
tion. In the same manner, Cambier et al. (2010) confirmed that
cadmium treatment producing differential band patterns between
control and metal-exposed genomic DNAs in zebra fish which in
consistent with our results.
From pathological point of view our results indicated that,
obvious pathological lesions were noticed in case of exposure to
cadmium. In liver vacuolar degeneration was a common finding.
Necrosis and inflammatory cells were not uncommon. In this
concern, Kaoud and El-Dahshan (2010) observed vacuolar degen-
eration in kidneys and necrosis in liver as a result of cadmium
toxicity in O.niloticus fish. The degenerative changes in the cells
may be induced exposure of the cells to either toxic or hypoxic
cases which effect on cellular function, Al- Nasser (2000) provide
145
an acceptable explanation for the cytoplasmic vacuolation of the
cells as a result of swelling and inhibition of mitochondrial
function.
The changes in the gills in our study were clear in case of fish
group exposed to cadmium and were characterized by hyperplasia
of gill filaments, congestion and eosinophilic granular cells in-
filtration; the results were go parallel with the findings of Be-
sirovic et al. (2010). The changes in the gills were also noticed in
the treated group; however it is not indicative to inefficiency of
the clays to inhibit the toxicity but it may attributed to failure of
the tissue to regenerate the chronic changes that noticed in
branchial tissue.
In this work we find that there are no significant effects on the
number of MnRBCs in the treatment with Egyptian clay or Tuni-
sian clay alone compared to the control group. As well as no sig-
nificant differences in chromosomal aberrations were observed in
the group treated with Tunisian clay alone compared with control
group, also between Egyptian clay and Tunisian clay. This result
confirmed with several reports indicated that treatment with
montmorillonite or other clay minerals did not show any tox-
icological or physiological effects in rats (Abdel-Wahhab et al.,
2002; Wiles et al., 2004; El-Kady et al., 2009), chicken (Chung
et al., 1990), in mice (Abbès et al., 2006a,b, 2007a), human (Phillips
et al., 2008) and in Tilapia fish, (Abdel-Wahhab et al., 2005; Hassan
et al., 2010). These results confirmed the safety of EM.
Clay minerals have been shown so far to protect against toxicity
of various natural or synthetic chemical products, including my-
cotoxins and heavy metal ions (Abdel-Wahhab et al., 2005; Gupta
and Gardner, 2005; Abbès et al., 2006a). In the present study fish
received the combined treatment of cadmiumplus Egyptian or
Tunisian clay showed a significant improvement in the frequencies
of micronuclei, chromosomal aberrations, DNA alterations and the
histopathological picture of liver and kidney although it failed to
normalize them. The mechanism by which EM induced its pro-
tective effect may be achieved from in vitro study revealed that
EM had a high affinity for Cd2 þ ions in aqueous solutions (El-Kady
et al., 2009). This mechanism appears to involve sequestration of
Cd2 þ ions in the gastrointestinal tract and chemisorption (i.e.,
tight binding) to EM, which resulted in a reduction in metal
bioavailability in the gastrointestinal tract by adsorption of Cd2 þ
ions metal by EM after simultaneous addition. In this regards,
Abdel- Wahhab et al. (2002) reported that montmorillonite did not
affect minerals metabolism in animals that received aflatoxin.
Cd2 þ ions are bound on the clay minerals by cation exchange (Da
Fonseca et al., 2006; Hassan et al., 2010).The mechanism of pro-
tection by clay minerals has been primarily attributed to the ad-
sorption and cation exchange capacity to reduce the bioavailability
of the toxins or the heavy metal ions in the gastrointestinal tract
when added to contaminated diet (Babich and Stotzky, 1977; Ab-
del-Wahhab et al., 2005; Abbès et al., 2006b).
Moreover, the results of this study indicate that, the effect of
Tunisian clay was more pronounced than Egyptian clay in the
prevention of induction of micronuclei and chromosomal aberra-
tions. The absorbability of different clay minerals differs according
to their specific surface area (Sposito et al., 1999). So that Tunisian
clay has a high affinity to adsorb cadmium. The physicochemical
properties of this montmorillonite indicate a high cation-exchange
capacity and high specific surface area (Srinivasan and Fogler,
1990a,b). These results agree with those reported by Phillips et al.
(1995) that montmorillonite effectively sequesters different con-
taminants. Abbès et al. (2007a) indicated that TEM has a good
ability to absorb heavy metals and protect mice against im-
munotoxicological effect of cation.
146 K.F. Mahrous et al. / Ecotoxicology and Environmental Safety 119 (2015) 140–147
Acknowledgments
The authors are indebted to the College of Veterinary Medicine,
Cairo University (Department of Pathology), which generously
accepted to perform laboratory work and scientific sectors tissue.
We express our appreciation to the three anonymous reviewers for
improving our paper.
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