Academic Sciences International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 6, Issue 1, 2014

Research Article

GLUTATHIONE-S-TRANSFERASE AND CATALASE ACTIVITY IN DIFFERENT TISSUES OF

MARINE CATFISH ARIUS ARIUS ON EXPOSURE TO CADMIUM

R. MANI1, B. MEENA2*, K. VALIVITTAN1 AND A. SURESH2

1Department of Biotechnology, St. Peters University, Chennai- 600 054, Tamil Nadu, India, 2Department of Zoology, Presidency College,
Chennai 600005, Tamil Nadu, India. Email: [email protected]
Received: 04 Oct 2013, Revised and Accepted: 01 Nov 2013

ABSTRACT
Objective: Pollution by heavy metals is a serious problem due to its toxicity and its ability to accumulate in the biota. In vivo effects of Cadmium (Cd)
levels of expression on antioxidant enzymes such as Glutathione-S-transferase (GST) and Catalase (CAT) were investigated in liver, kidney, gill and
brain tissues of marine Catfish Arius arius.
Methods: The various concentrations of CdCl2 such as 5.0 ppm, 10.0 ppm and 15.0 ppm exposed to a period of 24, 48, 72 and 96 hrs. GST and CAT
enzyme levels were studied in the tissues of liver, kidney, gill and brain in A. arius. The GST enzyme levels were analyzed using the method of Habig
et al., 1974 and measured using spectrophotometrically at 340 nm. Catalase levels were evaluated by the method of Sinha et al., (1972) and
spectrophotometrically read at 570 nm.
Results: GST is one of the intensely investigated conjugation enzymes and is the second stage of xenobiotic detoxification. CAT is a common
antioxidant enzyme which is produced naturally in almost all living organisms. The catalytic action of CAT, are important to life because it helps the
body to break down hydrogen peroxide (a powerful and harmful oxidizing agent) into oxygen and water, and thus preventing the accumulation of
carbon dioxide bubbles in the blood. The results showed the role of GST and CAT in antioxidant defense system to protect the animals from
oxidative stress and tissue damage in the tissues of exposed fishes.
Conclusion: In conclusion, our results indicate that antioxidant enzyme assays can be used as a bioindicator for acute exposure to Cd in marine
catfish A. arius and other fishes. Hence the GST and CAT activity can be considered as a sensitive biomarker for bioindicator of the antioxidant
defense system in the aquatic organisms, contaminated with heavy metals and this may provide a useful data for future investigations.
Keywords: Glutathione-S-transferase, Catalase, Cadmium, Arius arius, Antioxidants.

INTRODUCTION additional research before monitoring applications can be

undertaken [14].
The aquatic environment is contaminated with innumerable organic
and non-organic pollutants [1] of municipal waste, industrial, Trace metals at the cellular level are often involved in oxidative
agricultural and mining industrial origin [2]. Such pollutant not only stress, which results in the production of Reactive Oxygen Species
affects the integrity of the ecosystems and also affects the (ROS). ROS include the superoxide radical (O2-), hydrogen peroxide
physiological functions of animals [1] and human, as consumers [3]. (H2O2) and the hydroxyl radical, all of which affect mainly lipids,
The Cd is a ubiquitous heavy metal present in aquatic environments proteins, carbohydrates, and nucleic acids [15]. The importance of
due to natural and anthropogenic sources and is usually present in antioxidant enzymes is generally emphasized in the prevention of
trace amounts. Cd is naturally found in the earth's crust or occurs in oxidative stress by scavenging ROS [16]. Excessive ROS production
combination with other elements such as Zn and Cu. Cd is primarily in response to heavy metal pollution is the natural defense
used for electroplating with other metals and in nickeled batteries mechanism of biomolecules, leading to a cumulative damage [17].
because of its relative resistance to corrosion and high electrical and When the antioxidant enzymes fail or are insufficient, an increase in
thermal conductivity. These inputs may result in increased Cd levels ROS production may result in oxidative damage [18]. Recent studies
of in the aquatic ecosystems, which can be potentially toxic to have suggested that certain GST isoenzymes may be involved in the
organisms such as fish. Fishes have long been used as aquatic regulation of stress-activated cell signaling pathways [19, 20, 21].
contamination indicators for many years. Cd accumulates in
Fish tissues are endowed with antioxidant defense systems
drinking water and air then eventually accumulates in the body,
consisting of CAT, GST enzyme to protect them from oxidative stress
causing a number of diseases such as Hypertension, Osteomalacia,
caused by metals [22]. CAT a primary antioxidant defense
gastric dysfunction, Central nervous system dysfunction, and
component, eliminates hydrogen peroxide (2H2O22H2O+O2) a non-
endocrine disorders [4, 5] in human.
radical reactive oxygen species which can penetrate through all
Cd causes significant metabolic alterations and injuries in biological biological membranes and directly inactivate few enzymes. CAT
systems at different levels [6]. Cd after entering into the organism of activity is considered as a sensitive biomarker of oxidative stress
fishes through the gills binds to albumins and erythrocytes in the before hazardous effects occur in fish [23, 24]. Hence, in the present
blood and is then transferred into tissues and organs where it is study, the effect of Cd on the enzyme GST and CAT activity in a
bound to proteins of low molecular weight producing marine water catfish, A. arius were analyzed.
metallothioneins by the induction of metallothionein mRNA
MATERIAL AND METHODS
synthesis [7]. About 75 % of the total accumulated Cd in the
organism is deposited in the liver and kidney [8, 9], but it can also be The marine water Catfish, A. arius was collected from Chennai along
deposited in the heart, gill and other tissues [10, 11]. The majority of the Coromandel Coast of the Bay of Bengal, Tamil Nadu, Southern
aquatic studies has been directed toward using GSTs as biomarkers India. The fishes were acclimatized in the laboratory in a stone tank
of exposure to environmental chemicals [12]. One of the potential (100L) at room temperature (30 20C) for 7 days. The CdCl2
biomarkers is GST, which is a key phase II detoxification enzymes. (Merck, Mumbai, India) solution was prepared in distilled water for
The phase II metabolism involves the conjugation of xenobiotics acute toxicity studies. The various concentrations of CdCl2 such as 0
with endogenous substrate, thus facilitating their excretion [13]. In (Control), 5.0 ppm, 10.0 ppm and 15.0 ppm exposed to a period of
1994 GSTs have been recommended by the International Council 24, 48, 72 and 96 hrs. Eighty fishes with similar size, length, weight
for the Exploration of the Sea as biomarker which requires about (approx. 40-60 g) were selected and divided into four groups.
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Int J Pharm Pharm Sci, Vol 6, Issue 1, 326-332

Each group has 20 fishes. One group was kept as a control, the other Statistical analysis
three groups were transferred to stone tanks (100 L) containing 5.0
mg L-1, 10.0 mg L-1, and 15.0 mg L-1 of CdCl2, respectively. The test Statistical analyses of data were carried out using Graphpad Prism
solutions were renewed daily to maintain the waterborne Cd Version 5.0. The values are reported as mean SD. One-way analysis
concentration. In our biological experiments, GST and CAT enzyme of variance was utilized to test the differences between the control,
levels were studied in the tissues of liver, kidney, gill and brain. The GST and CAT levels exposed for each sampling. The data of different
content of Cd and other heavy metals were analyzed before the start hours of sampling were compared by ANOVA, Unifactorial analysis
of the study and found to be below detectable levels (BDL) to rule was used to test the differences between the control and treated
out their role or influence in the experiments. groups.

GST Enzyme Assay RESULTS

The GST levels in response to Cd treatments were analyzed in the GST Enzyme Activity
tissues using the method of Habig et al., 1974 [25]. Enzymatic assay In the present study, GST activity in response to Cd treatments was
was performed on A. arius liver, kidney, gill and brain. The tissues analyzed in the liver, kidney, gill and brain of A. arius for the period
(50mg) were homogenized in 50 mM TrisHCl buffer, pH 7.4, and of 96 hrs. These data were graphically represented in (fig.1). The
containing 0.2 M sucrose and centrifuged at 16,000g for 45 min at highest levels of GST enzyme activity 7.31 0.454 (M/min/mg
4C. The pellet was discarded and the supernatant was used as the protein) was observed in the liver during 72 hrs of Cd exposure with
enzyme source. The reaction mixture in a volume of 3 mL contained 15.0 mg L-1, during 72 hours of Cd exposure, in the kidney GST
2.4 mL of 0.3 M potassium phosphate buffer (pH 6.9), 0.1 mL of 30 levels were 5.661 0.321 (M/min/mg protein) when treated with
mM CDNB and 0.1 mL of 30 mM GSH, as enzyme source. The 15.0 mg L-1 of CdCl2. During 48 hours of Cd exposure, in the gills
reaction was initiated by glutathione. The absorbances were read at GST levels were 3.485 0.164 (M/min/mg protein) when treated
340 nm against the reagent blank. The results were expressed as with 15.0 mg L-1 of CdCl2. During 96 hours of Cd exposure, the brain
M/min/mg protein. The GST levels were measured using GST levels were 2.830 0.11 (M/min/mg protein) when treated
spectrophotometrically. with 15.0 mg L-1 of CdCl2. The GST levels of control tissues in the
Catalase Enzyme Assay liver were 0.298 0.062 (M min/mg protein), kidney 0.219 0.055
(M/min/mg protein), gills 0.179 0.016 (M/min/mg protein) and
Catalase levels in response to Cd treatments were evaluated by the brain 0.163 0.011 (M min/mg protein). The liver and kidney, GST
method of Sinha et al., (1972) [26]. The tissues (50mg) were enzyme levels gradually increased rapidly to reach a peak during
homogenized in 50 mM phosphate buffer, pH 7.0, and centrifuged at 72 hours and then declined gradually during 96 hours. In gill, GST
16,000g for 45 min. The supernatant was used as the enzyme enzyme levels decreased after 48 hours. The brain showed the lower
source. The reaction mixture contained 2 mL of phosphate buffer GST levels than all other tissues. In the brain, GST enzyme levels
(pH 7.0) 0.45 mL H2O2, and 0.025 mL of enzyme source. The gradually increased at 96 hours. The data were subjected to
absorbance was read at 570 nm and the enzyme activity was statistical analysis of one way ANOVA and the values were found to
expressed as micromoles of H2O2 consumed/min/ mg protein. be statistically significant at P < 0.05.

(a). Liver

(b). Kidney

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Int J Pharm Pharm Sci, Vol 6, Issue 1, 326-332

(c).Gill

(d). Brain
Fig. 1: Activity of GST (M/min/ mg protein) in A. arius on exposure to various concentrations of CdCl2 (Control, 5.0 ppm, 10.0 ppm and
15.0 ppm) for a period of 24, 48, 72 and 96 hrs. The results were represented as Mean SD, n=5. Statistical comparisons were made
against Control fish on each sampling day. (*Statistically significant at P < 0.05)

Catalase Enzyme Activity subjected to statistical analysis of one way ANOVA and the values
were found to be statistically significant at P < 0.05.
Catalase is an important enzyme in antioxidant defense system
protecting animals from oxidative stress. The effect of Control, 5 DISCUSSIONS
ppm, 10 ppm and 15ppm of CdCl 2 in the four tissues a liver, kidney,
gill and brain of A. arius were graphically represented in (fig.2). GST Enzyme
The highest CAT enzyme activity of 553.8 16.15 (mol H2O2 Glutathione-S-transferases are a family of multifunctional enzymes
consumed/min/mg protein) was observed in the liver during 24 that are involved in the detoxification of both xenobiotics as well as
hrs of Cd exposure with 5.0 mg L -1, during 24 hours of exposure to endogenous reactive compounds of cellular metabolism. GST was
Cd, the kidney CAT activities were 396.4 14.36 (mol H2O2 shown to catalyze essential steps in the biosynthesis of
consumed/min/mg protein) when treated with 5.0 mg L-1 of CdCl2.
prostaglandins and leukotrienes [27]. GST plays a critical role in
During 24 hours of exposure to Cd, in gills, CAT activities were 227
mitigating oxidative stress in all life forms and GST activity also has
10.49 (mol H2O2 consumed/min/mg protein) when treated
been widely used as a biomarker to detect stress. As an antioxidant
with 5.0 mg L-1 of CdCl2. During 24 hours of exposure to Cd, the
brain CAT activities were 167.2 7.69 (mol H2O2 enzyme, a GST activity either has a significant increase or decrease
consumed/min/mg protein) when treated with 5.0 mg L-1 of CdCl2. with different patterns according to the exposed elements or
The CAT activities of control tissues in the liver were showed exposure conditions. GST activity varied in different tissues and
values of 635.5 12.17 (mol H2O2 consumed/min/mg protein), in organs of aquatic animals [28]. The GST activities in the fish tissues
the kidney were showed values of 455.2 10.49 (mol H2O2 of A. arius were in the following order liver > kidney > gill > brain.
consumed/min/mg protein), in the gills were showed values of These results are in accordance with the works of [29] in whitefish
252.3 10.15 (mol H2O2 consumed/min/mg protein) and the tissues. The concentrations of Cd levels in liver and kidney were
brain were showed values of 182.6 14.98 (mol H2O2 much higher than gills and brain in the experimented fishes. This
consumed/min/mg protein). CAT activities decreased on exposure might be due to the fact that the liver and kidney are the major
to increased Cd concentration when compared to the control CAT targets for Cd distribution. Here they Cd are detoxified by binding
activity. The specific activity CAT in the brain was found to be with MT. The gills are the major entry site of heavy metals and act as
lower, when compared to all other tissues. The datas were a transient store for accumulation of metals [30].

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(a). Liver

(b). Kidney

(c). Gill

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Int J Pharm Pharm Sci, Vol 6, Issue 1, 326-332

(d). Brain
Fig. 2: Activity of CAT (mol H2O2 consumed/min/ mg protein) in A. arius on exposure to various concentrations of CdCl2 (Control, 5.0
ppm, 10.0 ppm and 15.0 ppm) for a period of 24, 48, 72 and 96 hrs. The results were represented as Mean SD, n=5. Statistical
comparisons were made against Control fish on each sampling day. (*Statistically significant at P < 0.05)

There are various modes of Cd uptake in aquatic organism, where it CAT activity in the liver. The reduction may be associated with the
is most readily absorbed by organisms directly from the water in its direct binding of metal to SH groups in the enzyme molecule. Liver
free ionic form Cd (II). Metal ions are usually absorbed through CAT activity was found to be inhibited following both in vivo and in
passive diffusion or carrier mediated transport over the gills while vitro exposure to dissolved Cd 2+ at a concentration greater than 1
metals associated with organic materials are ingested and absorbed mg/L in the killifish, Fundulus heteroclitus and the authors suggested
by endocytosis through the intestine. It has been suggested that Cd a direct effect of Cd 2+ of high molecular weight compounds like CAT
ions enter the chloride cells in the gills through calcium channels [36]. In previous studies, the liver was found to be stronger in the
[31]. Cd after entering into the organism of fishes through the gills, face of oxidative stress than the other tissues and a uniform organ
Cd binds to albumins and erythrocytes in the blood and then with the highest antioxidant enzyme activities (CAT). This could be
transferred into the tissues and organs where it is bound to proteins related to the fact that the liver is the site of multiple oxidative
of low molecular weight producing metallothioneins by the reactions and the maximal free radical generation [37].
induction of metallothionein mRNA synthesis [7]. The present study
has also shown higher concentrations of GST activity in the liver The highest inhibition was observed in the kidney, this can be
than the gills. About 75 % of the total accumulated Cd in an associated with the effective antioxidant system in this tissue where
organism is deposited in the liver and kidney [8, 9], but it can also be there is higher metal bioaccumulation and is directly related to the
deposited in the heart, gills and other tissues [10, 11]. metal binding protein synthesis and non-enzymatic antioxidant
mechanisms that has been suggested by Dautremepuits et al., [38].
The role of the liver in antioxidant enzyme response as a result of its Moreover, this can be attributed to the possible induction of stress
higher sensitivity to metals when compared to the kidney has been proteins and non-enzymatic antioxidant formation [22]. A gill is first
elucidated by various investigations as the liver has to overcome the affected organ when fish are exposed to metals because its direct
oxidative stress than the other tissues because of the high contact with water medium. In the present study there was a
antioxidant enzyme activities [32]. Liver of vertebrates exhibits a significant change in CAT activity in the gill and this could be
high metabolism and oxygen consumption and it is the main organ associated with the higher activity of GPX, which acts as a defense
of xenobiotic detoxification. It is a particularly rich source of GST against the formation of H2O2 or effective antioxidant responses due
[33]. Gills uptake the heavy metals from the site and directly interact to a higher renovation in the gill epithelium. The lowest activity of
with the toxic medium [34]. But however their GST enzyme levels CAT was measured in the gill tissue; this was explained by the
are low, compared to liver and kidney. The GST activity increases increased generation of H2O2, which led to a decreased CAT activity
steadily during 24 hrs and 48 hrs of exposure and then decline [39].
slowly during 72 hrs and 96 hrs of exposure during the present
study. These variations might due to the time taken to the metal to The brain is susceptible to oxidative damage by free radicals as it
be transported to other detoxifying organs. contains high amounts of unsaturated lipids and utilizes about 20%
of total oxygen demand of the body [40]. The specific activity of
Catalase Enzyme brain CAT was found to be lower, which may be related to the direct
binding of metal ions to SH groups in the enzyme molecule,
CAT being a primary antioxidant defense component, eliminates increased hydrogen peroxide and superoxide radical due to
hydrogen peroxide (2H2O22H2O+O2) a non-radical reactive oxygen oxidative stress. It was indicated that rapid inactivation of CAT at
species which can penetrate through all biological membranes and high hydrogen peroxide concentration was due to the conversion of
directly inactivate few enzymes. CAT is considered as a sensitive active enzyme compound to inactive compounds [41]. In general,
biomarker of oxidative stress before major deleterious effects occur inhibition of CAT enzyme activity in all tissues of A. arius may be
in fish [23, 24]. CAT is an important enzyme in antioxidant defense
resulted due to the direct effect of heavy metal Cd.
system protecting animals from oxidative stress. The highest CAT
activity was marked in liver tissue when compared to other tissues, CONCLUSION
which are in agreement with various authors [35, 24]. These data
are in accordance with those reported in other fish species were CAT In conclusion, our results indicate that antioxidant enzyme assays
activity is seen in a decreasing order, as follows: liver > kidney > can be used as a bio indicator for acute exposure to Cd in the marine
heart> brain > muscle [35]. In the present study, Cd decreased the catfish A. arius and other fishes. This metal stimulated rapidly the

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Int J Pharm Pharm Sci, Vol 6, Issue 1, 326-332

antioxidant system as evidenced by an increase in GST activities in 16. Vieira LR, Gravato C, Soares AMVM, Morgado F, Guilhermino L.
the detoxifying organs such as liver and kidney. GST is one of the Acute effects of copper and mercury on the estuarine fish
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Conflict of Interest Statement 2452-2458.
There is no conflict of interests. 19. Cho SG, Lee YH, Park HS, Ryoo K, Kang KW, Park J, Eom SJ et al.
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ACKNOWLEDGEMENT signals by suppressing apoptosis signal-regulating kinase1. Jou
Biol Chem 2001; 276: 1274912755.
The Authors are thankful to the Department of Biotechnology, St
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Peters University, Avadi, Chennai-54.
P1-1 (GSTP1-1) inhibits c-Jun N-terminal kinase (JNK1)
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