International Journal of Zoology

and Research (IJZR)

ISSN(P): 2278-8816; ISSN(E): 2278-8824
Vol. 5, Issue 6, Dec 2015, 7-16
TJPRC Pvt. Ltd.

INFLUENCE OF QUINALPHOS, AN ORGANOPHOSPHOROUS PESTICIDE,

ON THE ANTIOXIDANT DEFENSE SYSTEM IN HEPATIC SUBCELLULAR
FRACTIONS OF FISH, OREOCHROMIS MOSSAMBICUS (PETERS, 1852)
P. SURYA, P. V. VIDYA & K. C. CHITRA
Department of Zoology, University of Calicut, Thenhipalam, Kerala, India
ABSTRACT
The freshwater fish, Oreochromis mossambicus were exposed to quinalphos at sub lethal concentration, i.e.,
0.5 g/ L for 24, 72 and 96 h. Treatment groups showed no significant (P<0.05) changes in the body weight of animal,
however, a slight decrease in the hepatosomatic index were observed in treated fishes. Mucous deposition was
significantly increased at 72 and 96 h of quinalphos exposure which indicate the first line of defensive mechanism of the
exposed fishes to quinalphos. Activity of antioxidant enzymes as superoxide dismutase, catalase, glutathione reductase
and the level of lipid peroxidation was observed in mitochondrial, nuclear and microsomal fractions of both control and

subcellular fractions of treatment groups with concomitant increase in the level of lipid peroxidation. These findings
indicate that quinalphos induced oxidative stress and altered antioxidant defense system in subcellular fractions of fish
hepatocytes. In addition, one of the marker enzymes in liver, alanine aminotransferase was found to be increased in all
subcellular fractions of every treatment groups which reveal that the increase is to meet the energy demand by the
additional supply of glucose under the toxic condition. Therefore, acute toxicity of quinalphos at sub lethal
concentration imbalance antioxidant defense system in hepatic subcellular fractions of fish, Oreochromis mossambicus.

Original Article

treated groups. The activity of superoxide dismutase, catalase and glutathione reductase decreased significantly in all

KEYWORDS: Quinalphos, Oreochromis Mossambicus, Liver, Subcellular Fractions, Oxidative Stress, Antioxidant
Enzymes, Lipid Peroxidation, Alanine Aminotransferase

Received: Nov 17, 2015; Accepted: Nov 25, 2015; Published: Nov 28, 2015; Paper Id.: IJZRDEC20152

INTRODUCTION
In recent years, human intervention has brought major changes in the aquatic ecosystem. One of the
important such intervention is the application of pesticides in agriculture in order to enhance the food production.
However, pesticides are found to reach the aquatic ecosystems and their deleterious effects are often noticed in
non-target organisms including fish. Fish contribute to the economy of many nations and provide recreational and
psychological value to the naturalists, sports, enthusiast and aquarist, which also serve as a direct link to human
through food chain. Fish are able to uptake and retain different environmental contaminants dissolved in water via
active or passive processes. The interest in understanding the physiological mechanisms associated with fish
responding to environmental contaminants has been growing in recent years. Sub-lethal concentrations of
pesticides in aquatic environments have been shown to cause structural and functional changes in aquatic
organisms and this is more common than the mortality of the animals (Sancho et al., 2003).
Organophosphates has been shown to inhibit oxidative phosphorylation coupled with high-energy

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P. Surya, P. V. Vidya & K. C. Chitra

consumption, which widely affects the metabolism of cell/ tissues by altering cytochrome P450s and generation of reactive
oxygen species in large amount, which eventually leads to oxidative stress. Quinalphos, an organophosphorous pesticide
has been extensively applied in Indian agriculture for pest eradication, as well it is pertinent to study its hazardous effect on
the aquatic system as it is assumed that its residue might affect the health status of the fish. It is well documented that the
fish may serve as good biomonitoring tools, and, in addition to mammals, may provide useful models for further research
in understanding fish antioxidant system. Researches in fish demonstrated that mammalian and piscine systems exhibit
similar toxicological and adaptive responses to oxidative stress (Kelly et al., 1998). There are several advantages in using
piscine model to study oxidative stress, such as the reduction in the number of mammals used in research, the potential for
a reduction in the cost of animal maintenance, and the ability to increase the power of the experiment by increasing the
number of organisms per study. Furthermore, the use of piscine models to study oxidative stress allows for the evaluation
of environmental issues from both a human health and ecological perspective.
Therefore, the main objective of the present study is to evaluate the sublethal effect of quinalphos on the
antioxidant defense system in subcellular organelles such as mitochondrial, nuclear and microsomal fractions, which
provide an effective tool to diagnose the acute toxic effect of the organophosphorous compound in freshwater fish,
Oreochromis mossambicus.

MATERIALS AND METHODS

Standardization Procedures
Fresh water fish, Oreochromis mossambicus weighing 3.5 0.5 g and length 4.5 1 cm were collected from a
fish farm, Kaloos Aquarium, Kottakal, Malappuram District, Kerala. Fishes were acclimatized to the laboratory conditions
in dechlorinated and well-aerated cement tanks of 40 L capacity for four weeks prior to experiments with constant supply
of water and good lighting system. The physico-chemical features of the tap water were estimated as per APHA (1998) by
maintaining water temperature in the test ranged from 28 2C, oxygen saturation of water between 70 and 100 % and pH
between 6.5 to 7.0.
Median Lethal Concentration
The LC50 values of quinalphos for 96 h were determined by probit analysis, with a confident limit of 5 % level
(Finney, 1971). The concentration of any toxicant which kills 50 percentage of the test animals during a specific period is
referred to as median lethal concentration (LC50) or median tolerance limit. For determining the median lethal
concentration, 10 animals were exposed to different concentrations (1, 2, 3, 4, 5, 6 and 7 g/ L) of quinalphos for 96 h
along with separate control group, without toxicant. LC50-96 h that kills 50 % of exposed animals was computed on the
basis of probit analysis, which was 5 g/ L. One-tenth of quinalphos concentration (0.5 g/ L) was chosen as sublethal
concentration and it was used in the present study.
Treatments
Quinalphos - O, O-Diethyl O-2-quinoxalinyl phosphorothioate of 97% purity was used in the experiment.
Animals were grouped into four, three tanks with toxicant and a control tank, each group with ten fishes were exposed to
single sublethal concentration (0.5 g/ L) for three durations (24, 72 and 96 h) and were maintained separately.

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Influence of Quinalphos, an Organophosphorous Pesticide, on the Antioxidant Defense System

in Hepatic Subcellular Fractions of Fish, Oreochromis Mossambicus (Peters, 1852)

Group I: Control group (without toxicant)

Group II: Quinalphos at 0.5 g/ L for 24 h
Group III: Quinalphos at 0.5 g/ L for 72 h
Group IV: Quinalphos at 0.5 g/ L for 96 h
Killing of Animals and Tissue Processing
The fish was caught very gently using a small dip net, one at a time with least disturbance. At the end of each
exposure time, fishes were decapitated and liver tissue was dissected. Different sub-cellular fractions were obtained by the
differential centrifugation method as described by Palade and Siekevitz (1956). A 1% (w/v) homogenate of liver tissue was
prepared in ice-cold 0.25 M sucrose solution with the help of a motor-driven glass Teflon homogenizer on crushed ice for a
minute. The homogenate was centrifuged at 1000g for 10 minutes at 4C to obtain the nuclear pellet. Mitochondrial pellet
was obtained by centrifuging the post-nuclear supernatant at 10,000g for 10 minutes at 4C. As microsomal membranes
can sediment prematurely during traditional pre-clearances (6,000 10,000g), it is evidently not necessary to use
ultracentrifugation (100,000g) to collect microsomes. In fact, it is possible to sediment quantitatively all major
microsomal-type membranes at 21,000g in a normal bench-top microcentrifuge. The obtained nuclear, mitochondrial and
microsomal fractions were used for the following biochemical estimations.
Biochemical Analysis
Protein was estimated by the method of Lowry et al (1951) with bovine serum albumin as the standard. Activity
of antioxidant enzymes as superoxide dismutase (Marklund and Marklund, 1974), catalase (Claiborne, 1985), glutathione
reductase (Carlberg and Mannervik, 1985) was estimated. Level of lipid peroxidation (Ohkawa et al., 1979) and the
activity of alanine aminotransferase were measured as described by Segal and Matsuzawa (1970).
Statistical Analyses
All biochemical estimations were carried out in duplicate and are presented as mean SD for ten animals per
group where the differences were set significant at p<0.05 against the control group which were denoted with asterisk (*)
symbol in the figures. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by
Duncans Multiple Range test using statistical package SPSS 19.0.

RESULTS
Exposure to quinalphos at the sub lethal concentration of 0.5 g/ L showed no significant changes in the body
weight after the end of 24 h, 72 h and 96 h of treatment when compared to those of the corresponding control group
(Figure 1).

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P. Surya, P. V. Vidya & K. C. Chitra

Figure 1: Effect of Quinalphos on the Body Weights of

Freshwater Fish, Oreochromis Mossambicus
The hepatosomatic index of the quinalphos-treated fishes showed a slight decrease in all treatment groups, but the
result was not significant when compared to that of the control group (Figure 2).

Figure 2: Effect of Quinalphos on the Hepatosomatic Index of

the Freshwater Fish, Oreochromis Mossambicus
At the end of 24 h of quinalphos treatment there was no significant deposition of mucous; however, at the end of
72 h and 96 h of treatment fishes showed a remarkable deposition of mucous over the body with the percentage increased
to 50% when compared with those of control fish (Figure 3).

Figure 3: Effect of Quinalphos on Mucous Secretion in

Freshwater Fish, Oreochromis Mossambicus

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Influence of Quinalphos, an Organophosphorous Pesticide, on the Antioxidant Defense System

in Hepatic Subcellular Fractions of Fish, Oreochromis Mossambicus (Peters, 1852)

The activity of superoxide dismutase remain unchanged after 24 h of quinalphos exposure in mitochondrial and
microsomal fractions, but a significant (p<0.05) reduction was observed in nuclear fractions. However, there was a
significant decrease in the activity of the enzyme in all duration in the nuclear fractions as well as in 72 h and 96 h of
mitochondrial and microsomal fractions than that of the corresponding control group (Figure 4).

Figure 4: Effect of Quinalphos on the Activity of Superoxide

Dismutase in Hepatic Subcellular Fractions in the
Freshwater Fish, Oreochromis Mossambicus
The activity of catalase decreased significantly (P<0.05) in all duration in mitochondrial and nuclear fractions and
only at 96 h in the microsomal fractions (Figure 5).

Figure 5: Effect of Quinalphos on the Activity of Catalase in Hepatic Subcellular

Fractions in the Freshwater Fish, Oreochromis Mossambicus
On the other hand, the activity of glutathione reductase was found to be significantly (p<0.05) decreased in all
durations in mitochondrial and microsomal fractions, but only at 96 h treatment group in nuclear fractions (Figure 6).

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P. Surya, P. V. Vidya & K. C. Chitra

Effect of quinalphos on the activity of glutathione reductase in hepatic

subcellular fractions in the freshwater fish, Oreochromis mossambicus

nmol NADPH oxidized/ min/ mg protein

8
7
6

5
4
*

3
2
1

*
*

0
Mitochondrial fractions

Nuclear fractions
Control

24 h

72 h

Microsomal fractions
96 h

Figure 6: Effect of Quinalphos on the Activity of Glutathione Reductase in Hepatic

Subcellular Fractions in the Freshwater Fish, Oreochromis Mossambicus
The level of lipid peroxidation was increased significantly (p<0.05) at the end of 72 h and 96 h of mitochondrial
and nuclear fractions. However, no such changes were observed in all treatment durations in the microsomal fractions
(Figure 7).

Figure 7: Effect of Quinalphos on the Level of Lipid Peroxidation in the

Hepatic Subcellular Fractions in the Freshwater Fish, Oreochromis Mossambicus
There was a significant (p<0.05) increase in the activity of alanine aminotransferase in the hepatic mitochondrial,
nuclear and microsomal fractions at the end of 24 h, 72 h and 96 h of quinalphos-treated fishes as compared with the
control group (Figure 8).

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Influence of Quinalphos, an Organophosphorous Pesticide, on the Antioxidant Defense System

in Hepatic Subcellular Fractions of Fish, Oreochromis Mossambicus (Peters, 1852)

13

Figure 8: Effect of Quinalphos on the Activity of Alanine Aminotransferase in

Hepatic Subcellular fractions in the Freshwater Fish, Oreochromis Mossambicus

DISCUSSIONS
The aquatic environment is exposed to substantial amount of environmental pollutants that have the potential
impact to cause oxidative stress in aquatic organisms through free radical generation. Ecotoxicological studies on aquatic
organisms have focused primarily on redox cycling compounds and their effects on the major organs of biotransformation
using the adult or larval stages of fishes. In the present study quinalphos was selected as the toxicant and exposed at sub
lethal concentration in order to determine its effect on the fish antioxidant system in hepatic subcellular fractions. Some of
our laboratory studies in the past few years provided evidence of reactive oxygen species (ROS) production in various vital
organs as liver, gill, muscle and brain of fish exposed to toxic pollutants (Chitra et al., 2012; 2013; Asifa et al., 2014;
Thulasi and Chitra, 2015). Also, the hepatic marker enzymes as well lipid peroxidation was also estimated to assess the
toxic effect of quinalphos on hepatic subcellular fractions as mitochondrial, nuclear and microsomal fractions in the
freshwater teleost fish, Oreochromis mossambicus.
Several investigations have reported that the changes in the antioxidant defense system can be used as biomarkers
of oxidative stress due to exposure to various pollutants in aquatic organisms. The antioxidant defense system of an
organism can be subdivided into enzymatic antioxidants, such as superoxide dismutase, catalase, glutathione reductase and
glutathione peroxidase; and nonenzymatic antioxidants, such as glutathione, vitamin E, ascorbate, beta carotene, and urate
(de Zwart et al., 1999). The alterations in the enzymatic activities could directly reflect the metabolic disturbances and cell
damage in specific organs. The body weights of fishes were monitored throughout the experiment and it was observed that
there was no significant change in the weight of the animal. Measures of animal growth are routinely evaluated in
toxicology studies and are keys to interpret the compound-related effects. The present results suggest that quinalphos
exposure did not showed toxicant-related effect on the body weight of the treated fishes. However, mucous deposition was
significantly increased at 72 h and 96 h of quinalphos treatment than that of control group. Mucous cells are considered
efficient in seizing the toxic agents and thus help to prevent the entrance of these agents into the gills (Perry and Laurent,
1993). Hypersecretion of mucous may be the consequence of a chronic defensive mechanism of the fish against the
exposure to the environmental toxicant quinalphos.
Tissue somatic indices are commonly reported in fisheries studies because it is easily determined and some
indices, such as the hepatosomatic index can be an excellent predictor of adverse health status of fish. In the present study,
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P. Surya, P. V. Vidya & K. C. Chitra

the hepatosomatic index of the quinalphos-treated fishes showed a slight decrease in all treatment groups, but the result
was not significant when compared to that of the control group.
Regulated production of free radicals in higher organisms and maintenance of redox homeostasis are essential
for the physiological health of organisms. But, during these metabolic processes, a small proportion (23%) of free radicals
may escape from the protective shield of antioxidant mechanisms, causing oxidative damage to cellular components.
Biological systems have developed during their evolution an adequate enzymatic and non-enzymatic antioxidant
mechanism to protect their cellular components from oxidative damage. The imbalance between the generation and the
neutralization of ROS by antioxidant mechanisms within an organism is called oxidative stress (Davies, 1995). Free
radicals/ ROS generated in tissues and in sub-cellular compartments are effectively scavenged by the antioxidant defence
system, which constitutes antioxidant enzymes such as, superoxide dismutase, catalase, glutathione reductase and
glutathione peroxidase. Superoxide dismutase (SOD) catalysis the dismutation of superoxide to hydrogen peroxide (H2O2)
and oxygen (O2). The conversion of H2O2 to 2H2O is by the enzyme glutathione peroxidase and the conversion of H2O2 to
O2 and H2O is by the enzyme catalase. Since the reaction catalyzed by glutathione peroxidase requires glutathione (GSH)
as substrate and depends in part on the ratio of glutathione disulfide (GSSG): GSH, the concentrations of these reactants
and their ratio, which is a reflection of the redox state of the cell, are important to ROS detoxification. Glutathione
peroxidase/ reductase directly act as antioxidant enzymes to inhibit lipid peroxidation (Sikka, 2001). In the present study a
decrease in the activity of superoxide dismutase have been shown to increase the level of superoxide anion, which is
known to inactivate catalase activity (Kono and Fridovich, 1982). Similarly, catalase or glutathione peroxidase has been
shown to eliminate hydrogen peroxide from the cell leading to the inactivation of superoxide dismutase and generation of
lipid peroxides (Bray et al., 1974). The observed results suggest that the toxic effect of quinalphos in the generation of free
radicals are more pronounced in mitochondrial and nuclear fractions than that of microsomal fractions.
Molecular biomarkers are used to test oxidative damage in biomolecules and various aspects of oxidative stress by
free radicals in experimental animals. The most frequently used methods to monitor lipid peroxidation are based on
measuring of the end products. One of the methods is by measuring production of malondialdehyde and it is of particular
interest because it is commonly used assay than measured with thiobarbituric acid-reactive substances (TBARS).
Malondialdehyde is one of the end products of lipid peroxidation and it is very attractive to monitor its concentration,
which is a major oxidation product of peroxidized polyunsaturated fatty acids. Therefore, increased malondialdehyde
content is an important indicator of lipid peroxidation (Freeman and Crapo, 1981). There are some relatively new
approaches proposed recently to measure the end products of lipid peroxidation and they are HPLC and
immunotechniques, which are more specific than malondialdehyde and TBARS measurement (Claeson et al., 2001). But,
in the present study the simplified method as the production of malondialdehyde as a marker of lipid peroxidation was
performed and it was observed that the level of lipid peroxidation was increased significantly at the end of 72 h and 96 h in
mitochondrial and nuclear fractions. However, no such changes were observed in all treatment durations in the microsomal
fractions. The end products of lipid peroxidation are considered as a dynamic parameter because they can be further either
catabolized, or interact with other cellular components as proteins. Increased lipid peroxidation may indicate an increased
oxygen free radical generation in the hepatic sub cellular fractions (Thiele et al., 1995).
Aminotransaminases play an important role in the utilization of amino acids for the oxidation and/or for
gluconeogenesis (Samsonova et al., 2005). Alanine aminotransferases, is an enzyme frequently used in the diagnosis of

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Influence of Quinalphos, an Organophosphorous Pesticide, on the Antioxidant Defense System

in Hepatic Subcellular Fractions of Fish, Oreochromis Mossambicus (Peters, 1852)

damage caused by pollutants in various tissues such as liver, muscle, and gills. This enzyme is known to play a key role in
mobilizing L-amino acids for gluconeogenesis and function as links between carbohydrate and protein metabolism under
altered physiological, pathological, and induced environmental conditions (Victor, 1985). The elevation of the activity of
this enzyme provides the oxaloacetate required for the gluconeogenesis pathway to meet the additional supply of glucose
for the production of energy under reduced phase of oxidative metabolism. Elevation in the levels of alanine
aminotransferase in liver of the fish can be considered as a response to the stress induced by quinalphos to generate
ketoacid-like ketoglutarate and oxaloacetate for contributing to gluconeogenesis and/or energy production necessary to
meet the excess energy demand under the toxic manifestation. The increases in activities of aminotransferases as observed
in the present study were agreement with earlier reports (Arshad et al., 2007). The present results revealed, significant
increases in liver enzyme in mitochondrial, nuclear and microsomal fractions of quinalphos-treated fish throughout the
experimental periods may appear to reflect the stress effect of quinalphos.

CONCLUSIONS
As the source of the above mentioned discussion it can be summarized that exposure to quinalphos induced
oxidative stress in hepatic subcellular fractions of fish by inducing ROS generation thereby inhibited the activities of
antioxidant enzymes in the freshwater fish, Oreochromis mossambicus.

ACKNOWLEDGEMENTS
The authors acknowledge UGC-SAP/ BSR for utilizing the equipments during this study.
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