Genotoxic and Biochemical Biomarker Responses in
Meretrix Casta Exposed to Environmentally Relevant
Concentrations of Cadmium
Avelyno D’costa, Soorambail K. Shyama*, Praveen Kumar M. K. and Swizzle Furtado
*Department of Zoology, Goa University, Goa, India
S.K. Shyama, Department of Zoology, Goa University, Goa-403206, India, Tel.: +91- 0832-6519364 (O);
Received: January 24,2017; Accepted: March 27,2017; Published: April 16,2017
Avelyno D’costa, Shyama Soorambail K, Praveen Kumar M. K. and Swizzle Furtado (2017) Genotoxic and Biochemical Biomarker Responses in Meretrix Casta Exposed to Environmentally Relevant Concentrations of Cadmium. J of Biosens Biomark Diagn
2(1):1-7. DOI: 10.15226/2575-6303/2/1/00111
Cadmium (Cd) enters estuarine water by surface runoffs from
mines, phosphate fertilizers from agricultural fields and other
anthropogenic sources which may pose a threat to the fauna inhabiting
these waters. Bivalve molluscs which are a source of seafood may
accumulate Cd leading to deteriorated health of the organisms and
may also cause various health consequences in man. The present study
was carried out to assess the toxicity of Cd in the estuarine backwater
clam, Meretrix casta. In the experimental setup, the bivalves were
exposed to three environmentally relevant concentrations of CdCl2 (0.75 μg/L, 1.5 μg/L, 3 μg/L) for a period of 15 days. Genotoxicity
tests [Micronucleus Test (MN) and comet assay], oxidative stress
parameters [Catalase Assay (CAT) and Lipid Peroxidation Assay
(MDA)], neurotoxicity test [Acetylcholinesterase Assay (AChE)] and
physical condition (condition index) were employed to evaluate the
effects of Cd in M. casta. A dose-dependent increase of DNA damage
was seen at all the concentrations of Cd. Catalase activity was not
significantly changed at the lowest concentration compared to the
control, but increased significantly at the higher concentrations.
Lipid peroxidation was found to be significantly increased at all the
concentrations of Cd. However, the levels of AChE were found to
decrease in a dose-dependent manner. The condition index ratio was
also found to be lowered with increasing dose. DNA damage was highly
correlated with oxidative stress suggesting the mechanism of action of
Cd on DNA. Oxidative stress was negatively correlated with AChE and
may possibly be a contributing factor to neurotoxicity. M. casta can
therefore be used as a potential sentinel species for monitoring the Cd
in the estuarine environment using these biomarkers.
Cadmium (Cd) occurs naturally in sedimentary rocks
and soils in the environment and is also a constituent of zinc, lead
and copper ores. It is used extensively in various applications
such as anticorrosive agents, stabilizers in PVC products, in
pigments, a neutron-absorber in nuclear power plants and the
manufacture of nickel-cadmium batteries. It is also present in
phosphate fertilizers (1). Cd can therefore be released into the
aquatic environment from sources such as rainwater runoffs from
metal mining sites, mine drainage water, phosphate fertilizers,
sewage treatment plants landfills and hazardous waste sites
(2,3). Cd is known to be implicated in carcinogenesis either
through oxidative stress or inhibition of DNA repair processes
(4). The Environmental Protection Agency has thus classified
Cd as a Group B1 carcinogen and is considered to be a probable
human carcinogen (5). The toxicity of Cd is well documented in
plants and is known to affect various important processes (6).
In animal models such as fish, acute and sub-chronic exposure
to Cd leads to alterations of gill epithelium, liver and kidneys
and also affects enzymes such as acetyl cholinesterase (7,8). In
molluscs, Cd exposure results in reduction of growth rate and
mortality due to impairment of several metabolic functions
(9,10). Several studies have also reported the genotoxicity of Cd
in various animal models (11-15). The effect of Cd on DNA may be
indirect, via the action of reactive oxygen species thus leading to
oxidative DNA damage (16). Further, metal-induced genotoxicity
is predominantly due to the inhibition of the DNA repair process
Molluscs, particularly bivalves have been popularly
used as “sentinels” to detect pollution caused by a wide array
of contaminants in the environment (18). The advantage of
using bivalves is due to their intimate association with the
sediment, filter- or suspension-feeding habit and their ability
to bioaccumulate various contaminants. Bivalves can selectively
concentrate metal ions several hundred times from their
surrounding water by several mechanisms such as the ingestion
of particulate substances from suspended material, ingestion
of food material that have acquired these metals, uptake by
exchange onto mucous sheets of siphons and gills resulting in
their incorporation into important physiological systems and
formation of metal complexes with other organic molecules
within the body (19). Cd, along with other hazardous metals is
known to bioaccumulate in the tissues of bivalve molluscs which
can pose a serious threat to the seafood consumers (20,21). Cd
also affects the early development of bivalves causing growth
abnormalities and reduced survival (22). The International
Agency for Research on Cancer has reported that regular
consumers of bivalve molluscs are estimated to have weekly
dietary cadmium exposures of 4.6 μg/kg of body weight (3).
In the present study, Meretrix casta (Chemnitz)
commonly called the backwater or estuarine clam, was selected
due to its occurrence in the backwaters or estuaries of both the
east and west coast of India (23). It is consumed as local seafood
in many parts along the coast of India and is available throughout
the year. Despite its consumption, few studies are available on
the toxicity of various contaminants that predominantly occur
in these regions in M. casta. The presence of Cd in the waters
along the coast of Goa may be attributed to the discharge of
effluents from agrochemical industries (24). Oysters (Crassostea
sp.) collected from a polluted estuary in Goa were found to
have high concentrations of Cd in their soft tissues which are
dependent on the speciation of Cd in the water and sediment
(25,26). High concentrations of Cd were also observed in oysters
(C. madrasensis, C. gryphoides and Saccostrea cucullata) collected
from three different polluted sites in Goa and were found to be
consistently high in all the seasons (27).
Materials and Methods
Quality Assurance and Quality Control
The appropriate quality assurance methods of sample
preparation, handling and preservation were carried out in
accordance with US EPA procedures. All chemicals used were of
analytical grade from Himedia (Himedia, India) unless specified
Maintenance of Meretrix casta
Meretrix casta (Estuarine backwater clam) was selected
for the present study as it is consumed by a majority of the
coastal population and also due to its availability in the Goan
estuaries throughout the year. The bivalves (both sexes) were
collected from the intertidal zone with the help of skilled local
fishermen from Palolem, a pristine location in Goa. This site is
a clean, pristine beach with no known industrial activity or
anthropogenic stress (28,29). They were stored in a bucket with
water from the study site and transported alive to the laboratory.
They were then allowed to acclimatize in ordinary seawater from
Palolem for 30 days. The water conditions were maintained as
follows: temperature 25°C, pH 7.5, salinity 25 ppt, dissolved
oxygen 7.5 mg/L. The water was changed once daily to reduce
Bivalves were distributed in groups, each containing
10 individuals and were used for dose-response studies.
Concentrations of CdCl2 were selected based on the 96h LC50
values in M. meretrix (30) and the environmental levels along the
Goan coast (31). Accordingly, three sub-lethal concentrations of
CdCl2 (0.75 μg/L, 1.5 μg/L and 3 μg/L) were selected and were
exposed to the bivalve groups for a period of 15 days. A group of
bivalves was maintained in parallel without any Cd treatment and
served as the negative control.
Analysis of Samples
The bivalves were dissected open, their gill and muscle
tissues were cleaned thoroughly prior to the genotoxicity and
Prior to the comet assay and micronucleus test, the cell
count and cell viability of the peripheral blood were checked to
ensure that there were enough living cells to perform the assay
employing trypan blue dye exclusion test. The samples showing
more than 90% viability and a cell count of a minimum of 106
cells/ml were used for the tests.
The Micronucleus (MN) test was performed following
the protocol outlined by Baršiene et al. (32). A portion of the gill
tissue was placed in a drop of methanol acetic acid mixture (3:1)
on a clean glass slide. This tissue was then gently nipped with
tweezers for a few minutes and the resulting cell suspension was
then smeared and air-dried. The smears were fixed in methanol
for 10 min, stained with 5% Giemsa for 15 mins and allowed to
dry. The frequency of Micronuclei (MNi) was recorded by scoring
2,000 intact cells per bivalve at 1000x magnification using an
Olympus BX53 trinocular research microscope. Micronuclei
(MNi) were identified according to the following criteria: (1)
spherical or ovoid-shaped extra nuclear bodies in the cytoplasm
(2) a diameter of 1/3 - 1/20 of the main nucleus (3) non-refractory
bodies (4) colour texture and optical features resembling those
of the nucleus, and (5) the bodies completely separated from the
Single Cell Gel Electrophoresis (Comet Assay)
The comet assay was carried out as per Lee and Steinert
(33). All steps were carried out in dim light to prevent photooxidation
of DNA. Gill tissue (0.1g) was homogenized gently with
phosphate buffer saline (pH 7.4) and the resulting cell suspension
was passed through a muslin cloth to filter out tissue debris. This
cell suspension was then embedded in Low Melting Agarose
(LMA) on frosted microscopic slides. The cells were then lysed by
placing the slides in a cold lysing solution (2.5 M NaCl, 100 mM
Na2EDTA, 10 mM Tris, 10% DMSO and 1% Triton-X pH 10) at 4°C,
overnight. Following lysis the slides were placed in unwinding
buffer (electrophoresis buffer, pH 10) for 15-20 min to allow the
DNA to unwind. Electrophoresis was then performed for 30 min
at 300 mA, 25 V (Biorad electrophoresis unit). The slides were
placed in neutralization buffer (400 mM Tris base, pH 7.5) for 5
min. The gel containing DNA was stained with ethidium bromide
and examined using a fluorescence microscope (Olympus BX53)
with a green filter at 200x magnification. Randomly selected nonoverlapping
cells were screened and their comets were analyzed
with the help of computer software, CASP (34) and the % tail DNA
was recorded. The % tail DNA is the amount of DNA (in percent)
present in the tail of the “comet” and is used as a measure of DNA
Bivalves were dissected and their whole soft bodies
were collected and homogenized in 50 mM of Tris buffer (pH
7.4) containing 0.3 M sucrose and 1 mM EDTA. This suspension
was then centrifuged at 10,000 xg for 20 min at 4°C and the
supernatant was collected. Catalase activity was carried as per
Aebi (36) based on the decrease in absorbance of the test sample
by the decomposition of H2O2. The reaction mixture consisted of
13.2 mM H2O2 in 50 mM phosphate buffer (pH 7.0) and 0.1 ml of
the homogenate. The reduction in absorbance was measured at
240 nm using a multiwall plate reader (Analytical Technologies
Ltd.) at 25°C over 3 minutes. Total protein concentration was
measured by Bradford’s method (37). The activity of Catalase
(CAT) was expressed as μmol H2O2
The Acetylcholinesterase (AChE) activity in whole soft
bodies of bivalves was determined using the Ellman et al. (38)
with modifications as described by Galloway et al. (39). Briefly,
50 μL of sample homogenate was incubated in microtitre plates
with 150 μl DTNB (270 μM in 50 mM sodium phosphate pH
7.4) at 25°C for 5 min. The enzyme activity was initiated by the
addition of 3 mM acetylthiocholine iodide and the absorbance
was measured at 412 nm. The activity of AChE was expressed as
The Malondialdehyde (MDA) assay which is used to
test lipid peroxidation in the whole soft bodies of bivalves was
carried out using a commercial kit (North West Life Science
Specialities- NWK-MDA01). The assay is based on the reaction
of MDA with Thiobarbituric Acid (TBA) forming a pink coloured
MDA-TBA2 adduct that absorbs strongly at 532 nm. Butylated
Hydroxytoluene (BHT) and EDTA are also added to the reaction
mixture containing the sample homogenate to minimize oxidation
of lipids. The activity of MDA was expressed as nmol MDA1min-
Bivalves were cleaned, dissected and the soft tissues
were carefully separated from the hard shells. Both the soft tissue
and the shells were placed separately in an oven (REMI) at 60°C
overnight to determine their dry weights (40). Condition index
(CI) was then calculated as follows:
Statistical analyses of the data were carried out using
IBM SPSS 23 statistical software package. The data were tested
to meet the assumptions of normality and homogeneity prior to
subsequent analyses by linear models. The data of the MN test
and comet assay are expressed as percentage values and were
therefore arc sine transformed whereas the data of CAT, AChE
and MDA assays were log transformed. A one-way ANOVA was
applied to test the effect of treatment on the % MNi, % Tail DNA,
CAT, AChE, MDA and CI with a post hoc Dunnet’s test to compare
the different groups with the control within the same treatment
group. Pearson’s correlation with scatter plots were also used to
test the relationship between the all the parameters. The data
were considered to be statistically significant at p < 0.05.
The dose response data of the MN test, comet assay,
catalase assay, acetyl cholinesterase assay, malondialdehyde assay
and condition index are presented in figure 1 (a-f). A significant
dose dependent increase of DNA damage in the form of % MNi
was observed in the gill cells of M. casta at all the concentrations
of Cd (p < 0.05).
Figure 1: Variations of biomarker responses (a-f) in M. casta exposed to different concentrations of CdCl2 (0.75, 1.5 and 3 μg/L; Number of replicates= 3). % MNi: Percentage Micronuclei, CAT: catalase, AChE: acetylcholinesterase, MDA: Malondialdehyde, CI: condition index. * p < 0.05, ** p < 0.01, *** p < 0.001
DNA damage in the form of % tail DNA was also found to be
significantly high at the 0.75 μg/L and 1.5 μg/L of Cd concentrations
and was extremely significant at the 3μg/L concentration (p<
0.001). The CAT activity did not change significantly at the 0.75
μg/L concentration but increased significantly at the 1.5 μg/L (p
< 0.05) and the 3 μg/L (p < 0.05) of Cd. AChE activity was found
to decrease in a dose-dependent manner whereas MDA activity
showed an increasing trend with an increase in the concentration
of Cd and was significant at all the doses (p < 0.05). The CI ratio
also decreased significantly with an increase in the concentration
of Cd (p < 0.05).
The effects of different concentrations of Cd on the
variance of different tests are indicated in the one-way ANOVA
(Table 1).between DNA damage and the CI ratio. The scatterplots (Figure
2) further illustrate the associations between DNA damage and
the other parameters.
Table 1: one-way ANOVA of different concentrations of Cd on
different biomarker responses in M. casta
Independent variable (Treatment)
< 0.001 ***
% Tail DNA
< 0.001 ***
< 0.001 ***
< 0.001 ***
< 0.001 ***
< 0.001 ***
A high positive correlation was observed between
the % MNiand the % tail DNA (R = 0.95) as well as between
both the genotoxicity parameters and the activities of CAT
and MDA. However, a high negative correlation was observed
between DNA damage and AChE levels (R = -0.93, -0.94) as well
as Different concentrations of Cd effects contributed the most to
the % Tail DNA (F = 1551.42, p < 0.001) followed by the % MNi
(F = 926.86, p < 0.001). The activities of CAT, AChE and MDA and
the condition index ratio were also significantly influenced by the
different concentrations of Cd.
The correlation matrix indicating the association
between the different parameters is given in (Table 2).
Table 2: Correlation matrix between the associations of the
biomarker responses in M. casta
% Tail DNA
Figure 2: Scatter plots depicting the associations of DNA damage with other biomarker responses in M. casta exposed to different concentrations of CdCl2 (0.75, 1.5 and 3 μg/L; Number of replicates = 3). % MNi: Percentage Micronuclei, CAT: catalase, AChE: acetyl cholinesterase, MDA: Malondialdehyde,
CI: condition index.
The present study demonstrates the genotoxicity,
neurotoxicity, oxidative stress and deteriorated condition
induced by Cd in M. casta as indicated by the MN test, comet
assay, AChE assay CAT assay, MDA assay and condition index. Cd
was found to induce DNA damage in the gill cells of M. casta at all
the concentrations studied. This is in agreement with the studies
of Slobodskova et al. (41) where in they observed significant
DNA damage induced by Cd in the gill cells of the clam, Corbicula
japonica. Our observations were also on par with that of Sarkar
et al. (31) in which they reported a significant increase of DNA
damage with a concurrent decrease of DNA integrity in the gill
cells of a marine gastropod, Nerita chamaeleon exposed to various
concentrations of CdCl2 . In another study, Cd was found to be
clastogenic in the Pacific oyster (Crassostrea gigas), affecting the
number of chromosomes in somatic cells significantly compared
to control groups (42).
Significant increases in CAT activity were observed at
the 1.5 μg/L and 3 μg/L concentrations. Similar observations
were also reported by Macías-Mayorga et al. (43) in which
Crassostrea angulata exposed to Cd showed an increase of CAT
activity up to 7 days of exposure after which it was found to
decrease significantly. Further, they attributed this oxidative
stress in bivalves to the exposure to Cd. Liu et al. (44) proposed
that Cd may generate free radicals by interfering with cellular
antioxidant systems such as CAT.
Similarly, another consequence of oxidative stress was
found to occur in M. casta as observed by the increase of a lipid
peroxidation product, malondialdehyde (MDA). Dovzhenko et
al. (45) also reported a similar increase of MDA in the bivalve
Modiolus modiolus exposed to Cd. The increase in MDA and other
lipid peroxidation products lead to a decrease in the total oxygen
radical scavenging activity. As a result, there is an accumulation
of Reactive Oxygen Species (ROS) leading to oxidative stress in
the organism. These ROS in turn affect DNA causing modification
of DNA bases and DNA strand breaks (46,47). Alternatively,
Malondialdehyde (MDA) which is also highly mutagenic may
form adducts with DNA and induce DNA damage (48). Our
results are also supported by the observations of Xia et al. (30)
in which an increase in the activities of both CAT and MDA in
M. meretrix exposed to different concentrations of Cd, which in
turn induced the apoptosis of hepatopancreatic cells. Dailianis
et al. (49) suggested that Cd may induce the formation of ROS
and DNA damage by stimulating the production of Protein
Kinase C (PKC) via adrenergic receptors. Therefore, based on the
strong correlation between DNA damage and oxidative stress
parameters, the DNA damage observed in M. casta in the present
study may be attributed to oxidative stress as a result of Cd
We also observed a significant positive correlation
between the frequencies of % MNi and % tail DNA (R = 0.95)
which are represented in the form of scatter plots (Figure 2).
The comet assay is able to detect repairable DNA damage such
as DNA strand breakages, whereas the MN test detects more
persistent DNA damage that are more difficult to repair (50,51).
These micronuclei are formed when a whole chromosome or a
fragment of a chromosome does not get incorporated into either
of the two daughter cells during cell division due to aneugenic
agents that affect the spindle apparatus or clastogenic agents
that damage and break the chromosome (52). Thus these two
tests reflect different forms of environmental stress. The positive
correlation in our study may be due to the conversion of the short
term reversible damage to long term irreversible damage as a
result of persistent Cd exposure.
Cd was also found to be neurotoxic in the bivalves as seen
by the decreased concentration of AChE. The primary function of
AChE is to catalyze the rapid hydrolysis of the neurotransmitter
Acetylcholine (ACh) in the synaptic cleft thus terminating
synaptic transmission. Cd may thus disrupt the function of
AChE leading to an accumulation of ACh and overstimulation
of cholinergic receptors. Our results are in agreement with
that of Machreki-Ajmi and Hamza-Chaffai(53) in which cockles
(Cerastoderma glaucum) transplanted from an unpolluted site
to a site contaminated with Cd exhibited a significant inhibition
of AChE activity. Our studies are also comparable with those
of Dellali et al. (54) in which clams (Ruditapes decussatus) and
mussels (Mytilus galloprovincialis) collected from sites polluted
with heavy metals exhibited decreased acetylcholinesterase
activity compared to those collected from unpolluted sites.
Although the exact mechanism by which Cd causes inhibition
of AChE in bivalves is not known, one possible mechanism may
be due to ROS-mediated oxidative stress which is also seen to be
negatively correlated in the present study (44).
A significant negative correlation was observed between
the MN test and CI (R = -0.86) as well as between the comet assay
and CI (R = 0.9). A similar negative correlation between condition
index and tissue levels of environmental contaminants of
Littorina littorea, Mytilus edulis and Cerastoderma edule in a river
system (Milford Haven Waterway) of Wales, UK was reported by
Langston et al. (55). This decrease of condition of the organism
may be attributed to altered DNA function and thereby resulting
in an altered protein function which is ultimately required for
normal physiological processes. The physiological state of the
bivalve can also lead to changes in its feeding activity thereby
altering its life cycle as a consequence (56). Another reason for
the decrease in the CI ratio could possibly be the survival adaptive
response of M. casta wherein they reduce the filtration rate or
closure of the shell on exposure to contaminants (57).
The present study revealed that Cd (0.75 μg/L, 1.5 μg/L
and 3 μg/L) induced DNA damage in M. casta which was caused
as a result of oxidative stress. Increasing concentrations of Cd
also inhibited the activity of AChE and lowered the condition
index ratio. The comet assay and the micronucleus test along
with the biomarkers of oxidative stress such as CAT and MDA,
AChE assay and condition index can be reliably used to assess
the genotoxicity of Cd in M. casta in the environment. Hence, the
regular monitoring of estuaries for contaminants such as Cd is
of utmost importance as the persistence of these contaminants
could lead to significant decline in the natural populations of
bivalves and may also pose a threat to the humans consuming
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