Content uploaded by Anwarul Azim Akhand
Author content
All content in this area was uploaded by Anwarul Azim Akhand on Nov 11, 2014
Content may be subject to copyright.
Received: 14 December, 2008. Accepted: 16 March, 2009. Original Research Paper
Terrestrial and Aquatic Environmental Toxicology ©2009 Global Science Books
Arsenic and Mercury Induce Death of Anabas testudineus
(Bloch) Involving Fragmentation of Chromosomal DNA
Mosammat Salma Akter1 • Md. Kawser Ahmed1* • Anwarul Azim Akhand2 •
Nazmul Ahsan2 • Md. Monirul Islam1 • Md. Shahneawz Khan1
1 Department of Fisheries, University of Dhaka, Dhaka-1000, Bangladesh
2 Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka-1000, Bangladesh
Corresponding author: * kawser_du@yahoo.com
ABSTRACT
Heavy metals are considered as devastating environmental pollutants that cause serious pollution of water bodies affecting aquatic
inhabitants, including fishes. The objective of this work was to examine the toxicological effects of two major heavy metal pollutants,
sodium arsenite (NaAsO2) and mercuric chloride (HgCl2), on fresh water climbing perch, Anabas testudineus (Bloch). HgCl2 was found to
be more toxic than NaAsO2 and when fishes were exposed to different concentrations of these two metals, they required less time to
induce fish death as their concentration increased. The highest concentration (1 mM) tested in this study induced fish death as early as 2
hours in HgCl2 and 8 hours in NaAsO2 treatments. Both heavy metals decreased total protein content of the exposed fishes in a
concentration-dependant manner; however, no significant change was observed in fat, moisture and ash content. Liver cell viability was
reduced to about 32 and 48% by HgCl2 and NaAsO2, respectively. The death of the liver cells was accompanied by chromosomal DNA
fragmentation. We later investigated whether the heavy metals could induce any change in protein expression and found that both heavy
metals induced higher expression of a relatively high molecular weight protein detected on the upper portion of the gel. We conclude that
mercury and arsenic showed their toxic effect by causing death of the fishes or fish cells involving fragmentation of chromosomal DNA
and expression of certain high molecular weight proteins.
_____________________________________________________________________________________________________________
Keywords: apoptosis, climbing perch, heavy metals, liver cell, protein expression
INTRODUCTION
Bangladesh, as a developing country, is at a high risk of
environmental pollution. Uncontrolled industrial effluents
and indiscriminate use of inorganic fertilizers and chemicals
in agriculture are mostly responsible for the accumulation
of various pollutants, including heavy metals, in inland
water bodies causing the highest aquatic pollution problem
in Bangladesh (D’Monte 1996). Among different toxicants
of water, heavy metals are gaining importance for their non-
degradable nature. Contamination of water bodies with
heavy metals may have devastating effects on the ecologi-
cal balance of the aquatic environment, and it may limit the
diversity of aquatic organisms (Suzuki et al. 1988). Mer-
cury, lead and arsenic are the top three toxic pollutants of
environmental concern (Gonzalez et al. 2006). Arsenic is a
metalloid element that is widespread in the aquatic envi-
ronment as a result of both geogenic processes and anthro-
pogenic disturbances (Bears et al. 2006; Gonzalez et al.
2006) whereas mercury is primarily introduced into aquatic
systems through atmospheric deposition and anthropogenic
pathways, including effluent containing dyes, fungicides,
mining, industrial wastes, combustion of fossil fuels, muni-
cipal and medical wastes (Clarkson 1990; Porcella 1994;
Watras 1994).
In aquatic environments, several species of microorga-
nisms make arsenic and mercury biologically available to
organisms, including fish (Duker et al. 1995; Gonzalez et al.
2006). In vitro experiments have shown multiple effects at
the molecular level following arsenic and mercury exposure
including differential expression of genes involved in cell
cycle regulation, signal transduction, stress response, apop-
tosis, cytokine production, growth-factor and hormone-
receptor production (Chen et al. 1992; Shen et al. 2002;
Yang et al. 2002; Hossain et al. 2003; Scholz et al. 2005;
Tabellini et al. 2005). Several studies indicate that exposure
of both metals has deleterious effects on aquatic organisms.
For example, dietary arsenic exposure resulted in patholo-
gical injury to both the liver and gallbladder in lake white
fish, Coregonus clupeaformis (Pedlar et al. 2002) and in
rainbow trout (Onchorhynchus mykiss) (Cockell et al. 1991),
while waterborne arsenic decreased hemoglobin and packed
cell volume in walking catfish, Clarius batrachus (Tripathi
et al. 2003). Embryos of medaka (Oryzias latipes) exposed
to arsenic had a reduction in hatching success as well as
reduction in time to hatching (Ishaque et al. 2004). Arsenic
exposure can interfere with the normal expression of gluco-
corticoid receptor-mediated gene in the common killi fish,
Fundulus heteroclitus (Bears et al. 2006) and increase mor-
phological abnormalities in their offspring (Gonzalez et al.
2006). Depending upon different parameters like dose and
exposure route the toxicological effects of mercury includes
organ lesions (kidney, liver, and lung), neurological effects
and haematological alterations (Kotsanis et al. 2000; Spal-
ding et al. 2000; Iliopoulou-Georgudaki et al. 2001; Sweet
et al. 2001). Immunological effects of mercury toxicity have
also been described, both in mammals (Thuvander et al.
1996; Spalding et al. 2000; Institoris et al. 2001) and in fish
(Voccia et al. 1994; MacDougal et al. 1996). Among im-
munological effects in fish, alterations of MLR and blastic
transformation (Low et al. 1996), phagocytosis and respi-
ratory burst (Voccia et al. 1994; MacDougal et al. 1996).
Limited data are available on the impact of agriculture,
domestic and industrial pollution, especially heavy metals,
on the aquatic environment. In Bangladesh the concentra-
tion of heavy metals in aquatic animals, water and sediment
were studied, which covered mostly coastal area, parts of
the GBM river system and some rivers of central parts
(Ahmed et al. 2002, 2003; Haque et al. 2006; Ahmed et al.
2009). In Bangladesh, the fisheries sector is now considered
®
Terrestrial and Aquatic Environmental Toxicology 3 (1), 42-47 ©2009 Global Science Books
as an emerging potential sector and, in particular, has an
invaluable contribution of 4.92% to the GDP (BBS 2004).
However, to date no systematic study yet has been carried
out at the molecular level on the impact of heavy metals on
fish.
Fish are considered ideal organisms for toxicological
studies due to economic considerations. Fish models can be
used to establish biomarkers of aquatic pollutants because
their genomes have many sequence similarities with humans
(Ballatori and Villalobos 2002). We performed experi-
ments to examine the toxic effect of arsenic (NaASO2) and
mercury (HgCl2) in fresh water climbing perch, Anabas
testudineus, whose liver we chose to focus on because it is a
significant site of heavy metal accumulation and bio-trans-
formation (Pedlar and Klaverkamp 2002). We also exa-
mined the toxic effects of NaASO2 and HgCl2 on muscle
proximate composition and on the chromosomal DNA as
well as the cellular protein profile of liver cells.
MATERIALS AND METHODS
HgCl2 and NaAsO2 exposure
Fishes (climbing perch, Anabas testudineus) were either purchased
from the local market or collected from a fish farm, and almost
same sized fish (length 12.05 ± 0.21 cm, weight 19.05 ± 0.35 g)
were used for the experiment. All fishes were acclimatized for a
week in well aerated tap water with no food and the water was
changed once daily. After acclimatization, only the healthy fishes
were selected for the experiment. Small aquaria of 1m3 with 1L
water (same water which used in acclimatization) were used as ex-
perimental unit. Three different doses of HgCl2 and NaAsO2 (BDH,
UK) (0.1, 0.5 and 1.0 mM) each with three replicates were pre-
pared using the experimental water and then inoculated in the
experimental units. After mixing the dose one climbing perch,
Anabas testudineus per unit was exposed to it and the physiologi-
cal responses were observed. The survival period (period between
exposures to death) of fishes were recorded for each concentration
of heavy metal used.
Determination of proximate composition
The muscles were collected from the dorsal, ventral and caudal
portions of the fishes being died due to HgCl2 and NaAsO2 expo-
sures as soon as possible and macerated in a blender to have a
homogenous mass. Moisture and ash content of fish muscle were
determined by the AOAC (1965) method on a wet weight basis.
Crude protein content and fat were determined as total nitrogen by
the Kjeldhal method and ether extractions of the dry material
method, respectively (AOAC 2006).
Preparation of single cell suspension
Fish liver was isolated after being killed by exposure to HgCl2 and
NaASO2. The fishes were dissected by cutting the ventral aorta;
the liver was aseptically removed and pushed through 70 μm
nylon mesh (BD FalconTM, USA) to make single cell suspension
with normal saline (sterile isotonic saline).
Liver cell viability assessment
An equal volume of 0.2% Trypan blue was added to the cell sus-
pension and mixed well. The resultant cell suspension (~10 l)
was added into the counting chamber of a haemocytometer
(Neubauer®, Germany) covered with a cover slip.While observing
under the light microscope, the cells which take up the blue stain
of Trypan blue, were considered as dead cells, whereas those
having yellow nuclei were considered as viable cells. The subse-
quent cell concentration per ml was determined using the fol-
lowing formula:
The percentage viability is calculated as follows:
Analysis of DNA fragmentation by electrophoresis
100 μl of liver cell suspension (from 1×108 cells/ml) was added to
an equal volume of hypotonic lysing buffer [50 mM Tris-HCl
(SIGMA, St. Louis, USA), 10 mM EDTA (Merk, Germany), 0.5%
SDS (LOBA, India)] followed by the addition of 2 μl of proteinase
K (20 mg/ml) (SIGMA) and 6 μl of RNAse (10 mg/ml) (SIGMA)
according to Akhand et al. (1998). 10 μl of the DNA sample
mixed with 1 μl dye was loaded on a 1% agarose gel (SIGMA)
with 0.1 μg/ml ethidium bromide (SIGMA). The sample was run
for about 1 h at 50 mV. Gels were observed under UV light for
viewing DNA bands and photographs were taken (Kodak,
EDAS290).
SDS-PAGE and cellular protein analysis
Liver cell suspension (1 × 108 cells/ml) was placed in an Eppen-
dorf tube and washed twice with equal volume (100 μl) of PBS
through centrifugation (Mikro 22R; Hettich Zentrifugen) at 4°C
for 5 min at 1500 rpm. An equal volume of a two-fold concen-
trated sample buffer [125 mM Tris-HCl, pH 6.8; 20% w/v glycerol
(Robinson Wagner Co., New York, USA); 4% w/v SDS; 0.02%
bromophenol blue (SIGMA); 10% 2-mercapto ethanol (Nacalai
Tesque, Kyoto, Japan)] was added with cell suspension to lyse the
cells and dissolve the cellular proteins. Thereafter the dissolved
proteins were denatured by heating for 3 min in boiling water.
Liver protein samples were run at 30 mA on a 10% polyacryl-
amide gel (SIGMA) using PAGE buffer (196 mM glycine (BDH),
0.1% SDS and 50 mM Tris-HCl pH 8.3). The gel was stained with
Coomassie Brilliant Blue (Merck, Germany) followed by de-
staining with destaining buffer [methanol 25% (Merck), water
65% and glacial acetic acid 10% (Merck)] and finally a photo-
graph of the dried gel was taken by a digital camera (Nikon D60,
Japan).
Experimental design and data presentation
Three replicates for each treatment of both heavy metals were per-
formed in this study. Treatments were distributed following a com-
pletely randomized design (CRD) among the 18 (3 × 3 × 2) expe-
rimental tanks. Data were expressed as the mean ± standard devi-
ation of three measurements for each treatment. Differences
between the treatments (dose levels) were compared by one way
ANOVA with Tukey’s post-hoc for multiple comparisons. Statis-
tical software for windows SPSS version 12 was used to analyze
the data with the levels of significance at p<0.05.
RESULTS AND DISCUSSION
HgCl2 was more toxic than NaAsO2
While treating fish with different concentrations of HgCl2
and NaAsO2 it was found that both heavy metals have sig-
nificant toxic effect on the survivability of the exposed
fishes, F(5,12) = 913.581, p < 0.05. However, HgCl2 was
more toxic than NaAsO2 to induce death of the fishes (Fig.
1). When fishes were exposed to lower concentration of
HgCl2 (0.1 mM), time required for induction of death was
about 9 h. The same concentration of NaAsO2 (0.1 mM)
induced fish death in more than 18 h. As the concentrations
of the heavy metals were increased, the time required for
death induction was decreased. The highest concentration (1
mM), tested in this study, induced death of the fishes as
early as 2 h by HgCl2 and 8 h by NaAsO2.
Muscle total protein, fat, moisture and ash content
of the exposed fishes
Little variation was observed in the level of fat, moisture,
ash content of the exposed fishes, however it was found that
both the heavy metals had significant toxic effect on the
muscle total protein level of Anabas testudineus (F(7,16) =
counted area slip undercover fluid ofDepth
factordilution squareper count average The
mlper Cell u
u
counted cells o
f
no.Total
100 cells unstained of No.
viabilityPercentage u
43
Arsenic and mercury induce death of Anabas testudineus. Akter et al.
20.258, p < 0.05). The protein content was declined with
increasing concentrations of NaAsO2 and HgCl2 (Fig. 2).
Heavy metal-untreated fish (control fish) contained about
20% muscle total protein, whereas HgCl2 decreased the
protein level in a concentration dependent manner. It was
observed that a relatively high concentration of HgCl2 (1
mM) decreased the protein level to 16.3%, therefore,
around one fifth of the total muscle protein content was
reduced. However, the effect of arsenic in reducing protein
was less than that of HgCl2. High concentration of arsenic (1
mM) reduced the total muscle protein content to 18.31%
that is one tenth of the muscle total protein content was
reduced. The muscle total fat content of the control sample
was 4.85%. There was a slight reduction in fat content with
increasing the concentration of heavy metals but it was not
statistically significant (p < 0.05) marked change compared
with the control (Fig. 3). The moisture content of the con-
trol sample was near about 71% and in case of both heavy
metal treatments, an increasing trend in muscle total mois-
ture content was observed with increasing the treatment
dose but it was not statistically significant (p < 0.05) (Fig.
4). However, a significant (p < 0.05) variation in muscle
total ash content was found between the treated and un-
treated samples as shown in Fig. 5.
The depletion in protein content was probably caused
by the increased metabolism of proteins to overcome the
stress in HgCl2/NaAsO2-treated fishes (Janna and Bandho-
padhyay 1987; Sivarama and Radha 1998). The increased
metabolism of protein might be due to increased activation
of metabolic enzymes by mercury and arsenic treatment.
Our result is also supported by other reports in which the
investigators showed reduction of muscle total protein in
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
1 mM 0.5 mM 0.1 mM
Dose
Survival period (h)
Arsenic Mercury
c
a
d
b
c
e
Fig. 1 Survival periods (h) of A. testudineus on HgCl2 and NaAsO2
treatments. Fishes were exposed to either HgCl2 or NaAsO2 with indi-
cated concentrations and their survival periods were recorded. Bars repre-
senting the mean ± SD of triplicate assays with different letters are
significantly different (p < 0.05).
0
5
10
15
20
25
1 mM 0.5 mM 0.1 mM
Dose
% of muscle total protein
Cont ro l Arsenic Mercury
a
bc
d
aabc
c
aa
babc
Fig. 2 Effects of HgCl2 and NaAsO2 on muscle total protein content of
Anabas testudineus (Bloch). Fishes were treated with or without HgCl2/
NaAsO2 followed by isolation of muscle total protein by the Kjeldahl
method (2006). Bars representing the mean ± SD of triplicate assays with
different letters are significantly different (p < 0.05).
0
1
2
3
4
5
1 mM 0.5 mM 0.1 mM
Do se
% of mu s c le to tal fat
Control Arsenic Mercury
Fig. 3 Effects of HgCl2 and NaAsO2 on muscle total fat content of A.
testudineus. Fishes were treated with or without HgCl2/NaAsO2 followed
by isolation of total fat by the ether extraction method AOAC (2006). Bars
representing the mean ± SD of triplicate assays with no letters are not
significantly different (p < 0.05).
65
66
67
68
69
70
71
72
73
74
75
1 mM 0. 5 mM 0.1 mM
Dose
% of moisture conten
t
Control Arsenic Mercury
Fig. 4 Effects of HgCl2 and NaAsO2 on muscle total moisture content
of A. testudineus. Fishes were treated with or without HgCl2/NaAsO2
followed by moisture content measurement by AOAC (1965) method on
weight basis. Bars representing the mean ± SD of triplicate assays with no
letters are not significantly different (p < 0.05).
44
Terrestrial and Aquatic Environmental Toxicology 3 (1), 42-47 ©2009 Global Science Books
Cyprinus carpio treated with Cu, Cd and Zn and in Notop-
terus notopterus with mercury (Verma and Tonky 1983;
Shalaby 2000). The reduction in muscle total protein might
also be attributed to the great energy demands and cellular
damage occurred in the tissue of toxicated fish exposed to
heavy metals. With declination in muscle total protein con-
tent, an increasing trend was observed in moisture content
of the treated fishes compared to untreated fishes. This
might be due to increased metabolism of muscle protein as
exposed fishes required energy to fight with the toxic envi-
ronment. However, we observed a little variation in muscle
total fat content after exposure to the heavy metals. This
result could be explained by the fact that activation of
enzymes related to fat metabolism might not be affected by
mercury and arsenic or the fat metabolism process might
need more time to get a visible change.
Liver cell death due to HgCl2 and NaAsO2 toxicity
HgCl2 and NaAsO2 significantly induced liver cell death of
the exposed fishes, F(7,16)= 695.485, p < 0.05. The control
liver cells (collected from fishes that were not exposed to
HgCl2 and NaAsO2) were almost all (98%) alive (Fig. 6).
Viable cell number gradually increased with decreasing
concentrations of HgCl2 (for 0.5 mM = 62% viable cells and
for 0.1 mM = 84% viable cells), however the count de-
creased drastically to 32% after exposure of the fishes to 1
mM of HgCl2. Viable cell number gradually increases with
decreasing concentrations of NaAsO2 (for 0.5 mM = 72%
viable and for 0.1 mM = 92% viable cells); however, it de-
creased to 48% after the exposure of the fishes to 1 mM of
NaAsO2.
Characterization of liver cell death by HgCl2 and
NaAsO2
Liver cell DNA was isolated to investigate whether or not
HgCl2/NaAsO2-induced liver cell death was accompanied
by DNA fragmentation. The isolated DNA was resolved on
agarose gel and chromosomal DNA of control fish sample
was detected in relatively on the upper portion of the gel,
whereas, the chromosomal DNA of either mercury or ar-
senic exposed fishes was fragmented as revealed by the
appearance of the DNA bands in lower portion of the gel
(Fig. 7). The effect of 0.5 mM HgCl2 in inducing DNA frag-
mentation was most extensive as demonstrated by the
detection of the DNA band in the lowest position. On the
other hand, 1mM concentration of NaAsO2 induced DNA
fragmentation, although the fragmented band appeared
relatively on a higher position to that of HgCl2 (0.5 mM).
Lower concentrations of both HgCl2 (0.1 mM) and NaAsO2
(0.5 mM) also induced fragmentation of DNA, however, the
fragmented band appeared relatively closer to that of con-
trol. When the fishes were exposed to 1 mM of HgCl2, no
DNA fragmentation was observed (data not shown). This is
probably due to the necrotic death of the cells by the toxic
effect of the higher concentration. Usually cell death due to
apoptosis is accompanied by fragmentation of DNA show-
0.0 0
0.2 0
0.4 0
0.6 0
0.8 0
1.0 0
1.2 0
1.4 0
1.6 0
1 mM 0.5 mM 0.1 mM
Do se
% of muscle total ash
Control Ars e ni c Mercury
c
a
c
b
a
c
c b
c
Fig. 5 Effects of HgCl2 and NaAsO2 on muscle total ash content of A.
testudineus. Fishes were treated with or without HgCl2/NaAsO2 followed
by ash content measurement by AOAC (1965) method on weight basis.
Bars representing the mean ± SD of triplicate assays with different letters
are significantly different (p < 0.05).
0
10
20
30
40
50
60
70
80
90
100
1 mM 0.5 mM 0.1 mM
Dose
%of liver cell viability
C
ontro
l
A
rsen
i
c
M
ercur
y
a
f
a
c
d
a
a
b
e
Fig. 6 HgCl2 and NaAsO2 induced liver cell death. Fishes were exposed
to either HgCl2 or NaAsO2 with indicated concentrations. Bars represen-
ting the mean ± SD of triplicate assays with different letters are signifi-
cantly different (p < 0.05).
NaAsO
2
0.5 mM
HgCl
2
0.5 mM
HgCl
2
0.1 mM
NaAsO
2
1 mM
Control
Fig. 7 Visualization of NaAsO2- and HgCl2-induced chromosomal
DNA fragmentation by agaroge gel electrophoresis. Fishes were ex-
posed to either HgCl2 or NaAsO2 with indicated concentrations. Liver cells
collected after the death of the fishes (for Arsenic 12-18 h and for mercury
4-8 h) and DNA was isolated and resolved on 1% agarose gel. A represen-
tative of three experiments with consistent results is shown.
45
Arsenic and mercury induce death of Anabas testudineus. Akter et al.
ing a ladder (Akhand et al. 1998; Hossain et al. 2000). In
this study we did not observe any such ladder; however,
fragmentation was obviously occurred as the DNA was
detected at a lower position.
It is known that both HgCl2 and NaAsO2 induce apop-
totic cell death in mammalian cells (Akhand et al. 1998;
Hossain et al. 2000, 2003; Tabellini et al. 2005). We ob-
served that both HgCl2 and NaAsO2 induced the death of
liver cells involving DNA fragmentation. This result indi-
cated the death of the cells through apoptosis. It has been
shown that cadmium exposure induced apoptosis in oyster
hemocytes (Sokolova et al. 2004). A recent report by Wang
et al. (2004) also demonstrated arsenic-mediated DNA-
fragmentation and cell cycle arrest in two fish cell lines (JF
and TO-2) that might involve oxidative stress as a causative
factor.
Induction of higher expression of certain protein
Later it was examined whether HgCl2 or NaAsO2-induced
death could have any effect on cellular protein profile. In
control fish sample, we observed a clear protein band on the
upper portion of the gel as indicated by an arrow mark (Fig.
8). We could not detect any other clear protein band on the
gel. It was also observed that both HgCl2 and NaAsO2 inten-
sified the protein band detected on untreated control sample.
This result may suggest higher expression of this particular
protein by HgCl2 and NaAsO2. It was also observed that the
protein band was more intensified by the HgCl2 treatment
compare to NaAsO2 treatment. The intensification of the
band was increased in a concentration dependent manner. It
might be due to the fact that both HgCl2 and NaAsO2 in-
duce expression of some particular protein by the fish to
overcome the adverse or toxic effect created by the heavy
metals. A recent report revealed that arsenic can induce the
synthesis of specific stress proteins in fish (Roy and Bhat-
tacharya 2006). It was reported that heavy metals induce the
synthesis of metal binding protein metallothionein in Tila-
pia (Cheung et al. 2004). This finding should not be taken
as contradictory with our finding of heavy metal-mediated
reduction of total muscle protein content. This reduction
was due to high energy demand to overcome the stress
created by the toxic effect of the heavy metal. On the other
hand, the expression of a particular protein might be due to
detoxify the heavy metal in the liver cell.
The findings from the present investigation can be sum-
marized as both of the metals are reported to decrease the
total muscle protein content, liver cell were forced to death
by HgCl2 and NaAsO2 and these deaths of the liver cells
were accompanied with DNA fragmentation indicating cell
death through a mechanism, termed programmed cell death
or apoptosis. It was also observed that both metals might in-
duce the expression of some particular proteins.
Liver cell death might involve a cascade of signal
transduction that might be common to different cell types.
Therefore, this study could be extended to find out the
whole signal cascades for HgCl2 and NaAsO2 mediated fish
cell death. If we know the exact molecular mechanism of
heavy metal mediated fish cell death we can later get an
idea of designing drugs for their remedy. Moreover, the
interpreted pathway could be used as a model for other
toxic elements and a guide to study signaling pathways.
ACKNOWLEDGEMENT
The authors are grateful to Victory Foundation, Bangladesh as the
present work was partly supported by a grant of that foundation.
REFERENCES
Ahmed MK, Bhowmik AC, Rahman S, Haque MR, Hasan MM (2009)
Heavy metal concentrations in water, sediments and their bio-accumulations
in fishes and oyster in Shitalakhya River. Asian Journal of Water Environ-
ment and Pollution in press
Ahmed MK, Mehedi MY, Haque MR, Ghosh RK (2003) Concentration of
heavy metals in two upstream rivers sediment of the Sundarban Mangrove
Forest. Asian Journal of Microbiology, Biotechnology and Environmental
Sciences 5(1), 41-47
Ahmed MK, Mehedi MY, Huque MR, Ahmed F (2002) Heavy metal concen-
tration in water and sediment of Sundarbans Mangrove Forest, Bangladesh.
Asian Journal of Microbiology, Biotechnology and Environmental Sciences 4
(2), 171-179
Akhand AA, Kato M, Suzuki H, Nakashima I (1998) Level of HgCl2-medi-
ated phosphorylation of intracellular proteins determines death of thymic T-
lymphocytes with or without DNA fragmentation. Journal of Biochemistry
and Cell Biology 71, 243-253
AOAC (1965) Official method of analysis. Association of official agricultural
chemists. (10th Edn.) Washington, DC.
AOAC (2006) Official Method. Washington, DC, pp 984.13 (A-D), pp 920.39
(A)
Ballatori N, Villalobos AR (2002) Defining the molecular and cellular basis of
toxicity using comparative models. Toxicology and Applied Pharmacology
183, 207-220
BBS (2004) Bangladesh Bureau of Statistics. Statistical Year Book of Bangla-
desh. Statistical Division, Ministry of Planning. Govt. of the People’s Repub-
lic of Bangladesh, 69 pp
Bears H, Richards JG, Schulte PM (2006) Arsenic exposure alters hepatic ar-
senic species composition and stress-mediated gene expression in the com-
mon killifish (Fundulus heteroclitus). Aquatic Toxicology 77, 257-266
Chen Q, Yu K, Stevens JL (1992) Regulation of the cellular stress response by
reactive electrophiles: The role of covalent binding and cellular thiols in
transcriptional activation of the 70-kilodalton heat shock protein gene by
nephrotoxic cysteine conjugates. The Journal of Biological Chemistry 267,
24322-24327
Cheung APL , Lam THJ, Chan KM (2004) Regulation of Tilapia Metallothio-
nein gene expression by heavy metal ions. Marine Environmental Research
58, 389-394
Clarkson TW (1990) Human health risks from methylmercury in fish. Environ-
mental Toxicology and Chemistry 9, 821-823
Cockell KA, Hilton JW, Bettger WJ (1991) Chronic toxicity of dietary di-
sodium arsenate hydrate to juvenile rainbow trout (Onchorhynchus mykiss).
Archives of Environmental Contamination and Toxicology 21, 518-527
D’Monte D (1996) Filthy flows the Ganga. People Planet 5 (3), 20-22
Duker AA, Carranza EJM, Hale M (2005) Arsenic geochemistry and health.
Environment International 31 (5), 631-641
Gonzalez HO, Roling JA, Baldwin WS, Bain LJ (2006) Physiological chan-
ges and differential gene expression in mummichogs (Fundulus heteroclitus)
exposed to arsenic. Aquatic Toxicology 77, 43-52
Haque MR, Ahmed MK, Mannaf MA, Islam MM (2006) Seasonal variation
of heavy metals concentrations in Gudusia chapra inhabiting the Sundarban
mangrove forest. Journal NOAMI 23 (1), 1-21
Hossain K, Akhand AA, Kato M, Du J, Takeda K, Wu J, Takeuchi K, Liu
W, Suzuki H, Nakashima I (2000) Arsenic induces apoptosis of murine T
lymphocytes through membrane raft-linked signaling for activation of c-Jun
amino-terminal kinase. Journal of Immunology 165, 4290-4297
Hossain K, Akhand AA, Kawamoto Y, Du J, Takeda K, Wu J, Youshihara
M, Tsuboi H, Kato M, Suzuki H, Nakashima I (2003) Caspase activation is
accelerated by the inhibition of arsenic induced, membrane raft-dependant
Akt activation. Free Radical Biology and Medicine 34, 598-606
Iliopoulou-Georgudaki J, Kotsanis N (2001) Toxic effects of cadmium and
mercury in rainbow trout (Oncorhynchus mykiss): a short-term bioassay. Bul-
letin of Environmental Contamination and Toxicology 66, 77-85
Institoris L, Siroki O, Undeger U, Basaran N, Desi I (2001) Immunotoxico-
HgCl2 1 mM
HgCl2 0.5 mM
HgCl2 1 mM
NaAsO2 1 mM
NaAsO2 0.5 mM
C
ontrol
Fig. 8 Observation of toxic effect of HgCl2 and NaAsO2 on cellular
protein profile by polyacrylamide gel electrophoresis. Fishes were ex-
posed to either HgCl2 or NaAsO2 with indicated concentrations. Liver cells
collected after the death of the fishes (for arsenic 12-18 h and for mercury
4-8 h) and cellular protein was isolated as described in the methods and
materials. Cellular protein was resolved on 10% polyacrylamide gel.
46
Terrestrial and Aquatic Environmental Toxicology 3 (1), 42-47 ©2009 Global Science Books
logical investigation of subacute combined exposure by permethrin and the
heavy metals arsenic (III) a nd mercury (II) in rats. International Immuno-
pharmacology 1, 925-933
Ishaque AB, Tchounwou PB, Wilson BA (2004) Developmental arrest in Japa-
nese medaka (Oryzias latipes) embryos exposed to sub lethal concentrations
atrazine and arsenic trioxide. Journal of Environmental Biology 25, 1-6
Jana SR, Bandhopadhyay N (1987) Effect of heavy metals on some bioche-
mical parameters in fresh water fish (Channa punctatus). Environment and
Ecology 53, 488-493
Kotsanis N, Iliopoulou-Georgudakis J, Kapata-Zoumbos K (2000) Changes
in selected haematological parameters at early stages of the rainbow trout,
Oncorhynchus mykiss, subjected to metal toxicants: arsenic, cadmium and
mercury. Journal of Applied Ichthiology 16, 276-278
Low KW, Sin YM (1996) In vivo and in vitro effects of mercuric chloride and
sodium selenite on some non-specific immune responses of blue gourami,
Trichogaster trichopterus (Pallas). Fish and Shellfish Immunology 6, 351-362
MacDougal KC, Johnson MD, Burnett KG (1996) Exposure to mercury
alters early activation events in fish leukocytes. Environmental Health Pers-
pectives 104, 1102-1106
Pedlar RM, Klaverkamp JF (2002) Accumulation and distribution of dietary
arsenic in Lake Whitefish (Coregonus clupeaformes). Aquatic Toxicology 57,
153-166
Pedlar RM, Ptashynski MD, Wautier KG, Evans RE, Baron CL, Klaver-
kamp JF (2002) The accumulation, distribution, and toxi cological effects of
dietary arsenic exposure in lake whitefish (Coregonus clupeaformis) and lake
trout (Salvelinus namaycush). Comparative Biochemistry and Physiology –
Part C: Toxicology and Pharmacology 131, 73-91
Porcella DB (1994) Mercury in the environment: Biogeochemistry. In: Watras
CJ, Huckabee JW (Eds) Mercury Pollution: Integration and Synthesis, Lewis
Publishers, Boca Raton, Florida, pp 3–19
Roy S, Bhattacharya S (2006) Arsenic-ind uced histopathology and synthesis
of stress proteins in liver and kidney of Channa punctatus. Ecotoxicology
and Environmental Safety 65, 218-229
Scholz C, Richter A, Lehman M, Schulze-Osthoff K, Dorken B, Daniel PT
(2005) Arsenic trioxide induces regulated death receptor independent cell
death through a Bcl-2-control pathway. Oncogene 24, 7031-7042
Shalaby AME (2000) Sublethal of heavy metals copper, cadmium and zinc
alone or in combinations on enzymes activities of common carp (Cyprinus
carpio L.). Egyptian Journal of Aquatic Biology and Fish 4, 229-246
Shen ZY, Shen J, Chen MH, Wu XY, Wu MH, Zeng Y (2002) The inhibition
of growth and angiogenesis in heterotransplanted esophageal carcinoma via
intratumoral injection of arsenic trioxide. Oncology Reports 10, 1869-1874
Sivarama KB, Radha KK (1998) Impact of sublethal concentration of mercury
on nitrogen metabolism of fresh water fish, Cyprinus carpio (Linnaeus).
Journal of Environmental Biology 19 (2), 111-117
Sokolova IM, Evans S, Hughes FM (2004) Cadmium induced apoptosis in
oyster hemocytes involves disturbance of cellular energy balance but no
mitochondrial permeability transition. The Journal of Experimental Biology
207, 3369-3380
Spalding MG, Frederick PC, McGill HC, Bouton SN, Richey LJ, Schuma-
cher IM (2000) Histologic, neurologic, and immunologic effects of methyl-
mercury in captive great egrets. Journal of Wildlife Diseases 36, 423-35
Suzuki KT, Sunaga H, Aoki Y, Hatakeyama S, Sumi Y, Suzuki T (1988)
Binding of cadmium and copper in the mayfly Baetis thermicus larvae that
inhabit in a river polluted with heavy metals. Comparative Biochemistry and
Physiology 91C, 487-492
Sweet LI, Zelikoff JT (2001) Toxicology and immunotoxicology of mercury: a
comparative review in fish and humans. Journal of Toxicology and Environ-
mental Health Part B: Critical Reviews 4, 161-205
Tabellini G, Tazzari PL, Bortul R, Evanquelisiti C, Billi AM, Grafone T,
Baccarani M, Martelli AM (2005) Phosphoinositide 3kinase/Akt inhibition
increases arsenic trioxide-induced apoptosis of acute promyelocytic and T-
cell leukaemias. Journal of Haematology 130, 716-725
Thuvander A, Sundberg J, Oskarsson A (1996) Immunomodulating effects
after perinatal exposure to methylmercury in mice. Toxicology 114, 163-175
Tripathi S, Sahu DB, Kumar A (2003) Effect of acute exposure of sodium
arsenite on some haematologi cal parameters of Clarius batrachus (Common
Indian Catfish) in vivo. Indian Journal of Environmental Health 45, 183-188
Verma SR, Tonk IP (1983) Effect of sublethal concentration of mercury on the
composition of liver, muscle and ovary of Notopterus notopterus. Wa t er, A i r
and Soil Pollution 20, 287-292
Voccia I, Krzystyniak K, Dunier M, Flipo D, Fournier M (1994) In vitro
mercury-related cytotoxicity and functional impairment of the immune cells
of rainbow trout (Oncorhynchus mykiss). Aquatic Toxicology 29, 37-48
Wang YC, Chaung RH, Tung LC (2004) Comparison of the cytotoxicity in-
duced by different exposure to sodium arsenite in two fish cell lines. Aquatic
Toxicology 69 (1), 67-79
Wat ra s CJ (1994) Sources and fates of mercury and methylmercury in Wiscon-
sin lakes. In: Watras CJ, Huckabee JW (Eds) Mercury Pollution: Integration
and Synthesis, Lewis Publishers, Boca Raton, Florida, pp 153-177
Yang C, Frenkel K (2002) Arsenic-mediated cellular signal transduction, trans-
cription factor activation, and aberrant gene expression: implications in car-
cinogenesis. Journal of Environmental Pathology, Toxicology and Oncology
21, 331-342
47
