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172
Chapter-V
BIOCHEMICAL STUDIES
173
BIOCHEMICAL CHANGES IN THE SELECTED TISSUES OF FISH CHANNA PUNCTATUS EXPOSED TO QUINALPHOS TECHNICAL
GRADE AND 25% EC
The onset of rapid industrialization has resulted in the application of chemicals for human
welfare. This has led to the introduction of various chemicals for controlling different
agricultural and household pests. Under recommended conditions of use and application rates, it
is unlikely that insecticides and their degradation products will attain significant levels in the
environment. But the indiscriminate use has resulted in the presence of insecticides in the living
systems (Shivakumar, 2005).
Although application of pesticides is desirable in the management and control of pests, its
injudicious and indiscriminate use has resulted in freshwater pollution (Kiziewicz & Czeczugu,
2002). A number of investigations have reported that most of the synthetic organic pesticides of
organochlorines, organophosphates and carbamates are extremely toxic to non-target populations
of freshwater fauna, adversely affecting the complex food-web, population dynamics and food-
web energetics (Chandra et al., 2001; Imbaraj & Haider, 1988; Nagender Reddy et al., 1991).
These investigations linked the pesticides to number of biochemical reactions, which could
explain their adverse effects on the morphology and physiology of a number of freshwater
organisms (Ghosh et al., 2001).
The natural physiological functioning of an organism gets disturbed on exposure to
toxicant stress. It induces its effect first at cellular or even at molecular level, but ultimately
causes physiological, pathological and biochemical alterations. It is, therefore necessary to focus
attention on changes in biochemical composition of organisms, which are constantly under
pollutant threat. When the pesticides come in contact with internal organs, irreversible changes
in metabolic activities take place that eventually cause biochemical changes. Pesticide pollutants
act as stress inducing agents which affect the functional state of tissues of the exposed
organisms, all pollutants are not toxic but all pesticides are toxicants. Many pesticides have been
reported to produce a number of biochemical changes in fish both at lethal and more often at
sublethal levels. Changes in ion concentrations, organic constituents, enzyme activity,
endocrinal activity and chemoregulators in fish have been attributed to pesticides. Since aquatic
environment is the ultimate sink for all pollutants, aquatic toxicity testing has became an integral
part of the process of environmental hazard evaluation of the toxic chemicals. Generally, the
174
potential impact of pollutants is more on the aquatic organisms because in the hydrosphere,
pesticides and such other substances are transported to a greater distance and hence many more
non-target organisms are likely to be exposed to them than in the terrestrial environment (Murty,
1986).
The use of biochemical measurements in organisms as indicators of pollution, give
information about the adaptive or deleterious responses in organism exposed to a certain amount
of chemicals. Such analysis provide early warning signals before other toxicological points,
including death are evident (Livingstone, 1998)
Carbohydrates are considered to be the first among the organic nutrients to be depleted
and degraded in response to stress conditions imposed on animals. Carbohydrates are important,
since these provide the energy for the animal required for performing different processes
(Lehninger, 2004; Harper, 2003). Alteration in carbohydrate metabolism is prone to have
deleterious effect on the survival of the animal (Srinivasa murthy, 1983; Radhaiah, 1988; Rama
Murthy, 1988; Veeraiah, 2002 and Madhavi, 2005).
Carbohydrates serve as a reservoir of chemical energy required by the animal. The
carbohydrate metabolism is a complicated process consisting of various steps, such as:
• Aerobic breakdown of glycogen or glucose through glycolysis to form pyruvate which
undergoes oxidation via krebs cycle followed by reduction of co-enzymes.
• An alternative degradative pathway for glucose through hexose monophosphate shunt.
• The synthesis of glycogen from glucose by glycogenesis and also through
glyconeogenesis.
• The conversion of lactate to glycogen through cori cycle.
All these steps constitute the main pathways of carbohydrate metabolism. Alteration in
carbohydrate metabolism is prone to have deleterious effect on the survival of the animal
(Harper, 2003). Impairment of carbohydrate metabolism is one of the outstanding biochemical
lesions caused by the action of toxic compounds (Matias 1983; Srinivasa murthy, 1983;
Radhaiah, 1988; Rama Murthy, 1988). Alterations in biochemical components like protein,
carbohydrate and lipid as response to environmental stress are authenticated by many
investigators; Ramakrishna and Sivakumar (1993) in Oreochromis mossambicus, Malla Reddy
and Bashamohideen (1995) in Cyprinus carpio , Singh, et al. (1996) in Heteropneustes fossilis,
Tilak et al., (2001) in Labeo rohita , Kumar and Saradhamani (2004) in Cirrhinus mrigal,
175
Saraswathi (2004) in Labeo rohita, Arockia Rita and John Mitton (2006) in Oreochromis
mossambicus and Prabhakara Rao and Radhakrishnaiah (2006) in Cyprinus carpio.
Understanding of the protein components of cell becomes necessary in the light of the
radical changes taking place in protein profiles during pesticide intoxication. Both the protein
degradation and synthesis are sensitive over a wide range of conditions and show changes to a
variety of physical and chemical modulators. The physiological and biochemical alterations
observed in an animal under any physiological stress can be correlated with the structural and
functional changes of cellular proteins. Proteins occupy a unique position in the metabolism of
cell because of the proteinaceous nature of all the enzymes which mediate at various metabolic
pathways (Lehninger, 2004; Harper, 2003).
The induced stress and pathological conditions on protein metabolism showed alterations
in sub-cellular proteins, enzyme activity levels which are found to be dependent on the protein
making up to the cytosol fraction. The general nature of protein make up was studied to ascertain
whether such relations do exist under induced stress. Enzyme bioassays however remain a useful
technique in studying or diagnosing sublethal effects of toxic pollutants.
Lactate dehydrogenase (LDH) is one of the most sensitive enzymes to environmental
pollutants. An alteration in its functioning would indicate the occurrence of pathological
conditions in the organisms. LDH is the key enzyme located at the vital point between glycolysis
and TCA cycle. Because of its strategic location and its relation to cori cycle, it is likely that any
fluctuations in the cellular environment alters the activity of this enzyme. Lactate dehydrogenase
enzyme is widely distributed in the tissues, more significantly in the metabolically active tissues
and it catalyses the reversible oxidation-reduction reaction involving lactate, pyruvate, NAD+
and NADH. Lactate dehydrogenase occurs in animal tissues as five different isozymes and
activy changes under pathological conditions (Martin et al., 1983).
Aminotransferases mobilise the aminoacids into carbohydrate and lipid metabolism.
There exists a rapid turnover of free aminoacids from cell to cell, tissue to tissue through the
circulating fluid and utilize for various purposes through interconversions. Transaminases form
an important group of enzymes mediating carbohydrates, protein and lipid metabolism.
Transamination represents the mechanism causing eventual deposition of nitrogenous waste
products like ammonia and urea resulting in the production of carbon compounds, which
contribute towards gluconeogenesis and fatty acid formation. AAT and ALAT are two important
176
enzymes mainly involved in the inter-conversion of important compounds such as pyruvate,
oxaloacetate, α-ketoglutarate and aminoacids thus bringing the protein and carbohydrate
metabolism on one hand and alanine, aspartic acid and glutamic acid on the other (Moore, 1964;
Knox and Greengard, 1965). Aminotransferases also act as precursors of gluconeogenesis and
probably during the period of stress, they meet the energy demands by channeling aminoacids
into carbohydrate metabolism (Watts and Watts, 1974; Martin et al., 1983). The aspartate
aminotransferase catalyses the interconversions of aspartic acid, and α-ketoglutaric acid to
oxaloacetic acid and glutamic acid, while alanine, amino transferase catalyses the
interconversion of alanine and α-ketoglutaric acid to pyruvate and glutamic acid.
The phosphatases, Acid phosphatase (ACP) and Alkaline phosphatase (ALP) are active at
specific pH and are usually termed phosphomonoesterases. Pesticide poisoning increases ACP
activity in the fish (Tejendra et al., 1990). The ACP is a lysosomal enzyme and the raise in its
activity is probably related to the cellular damage. It is difficult, however, to relate the decrease
in ACP activity with tissue damage. Increase in acid phosphatase activities can be interpreted as
a shift of the tissues emphasis on energy breakdown pathway from normal ATPase system to
phosphate system. Pesticides are reported to reduce glycogen levels and increase phosphorylase
activities (Mishra and Srivatsava, 1984). In the event of decreased ATPase system,
phosphorylation may be preceded by activated phosphates to catalyze the liberation of inorganic
phosphates from phosphate esters. Acid activities also serve as diagnostic tool to assess toxicity
stress of chemicals in the living organisms (Harper, 1991). Any change in phosphates activity
will affect the physiological and biochemical pathways of animals (Ramana Rao et al., 1996).
Many organophosphates are potent neurotoxins, functioning by inhibiting the action of
acetylcholinesterase (AChE) in nerve cells. Neurotransmitters such as acetylcholine (which is
affected by organophosphate pesticides) are profoundly important in the brain’s development,
and many OPs have neurotoxic effects on developing organisms even at low levels of exposure.
The primary effect of OPs on vertebrate and invertebrate organisms is the inhibition of AChE
activity, the enzyme that degrades the neurotransmitter acetylcholine in cholinergic synapses
(Pan and Dutta, 1998). Duration of exposure, type of OP, as well as species of fish has an effect
on the extent of AChE expression. Acetylcholine (ACh) is the only classical neurotransmitter
that after release into the synaptic cleft is inactivated by enzymatic hydrolysis rather than by
reuptake. As a consequence, ACh has a turnover rate in vivo that is much higher than that of any
177
other transmitter, including catecholamines and amino acids (Haubrich and Chippendale, 1977).
AChE activity is a biomarker extremely used in aquatic ecotoxicology studies (Kirby et al.,
2000), and is a fairly sensitive enzyme to low environmental concentrations of
organophosphorus compounds.
The inhibition or activation of physiological activities by pesticides is due to the
interaction between the animal and the chemical nature of the pesticides. The stress induced
biochemical changes are described as secondary responses of the fish. According to Abou-Donia
et al., (1988), the biochemical analysis of DNA, RNA and protein are considered as markers in
the toxicity study.
Considering the role of above biomarkers in the field of eco-toxicology, the present study
has been undertaken to understand the biochemical alterations induced by quinalphos technical
grade and 25% EC on exposure to sublethal and lethal concentrations to fish Channa punctatus
in different tissues exposed. The bioassays include, Glycogen, Total Proteins, Lactate
Dehydrogenase (LDH), Aspartate Aminotransferase (AAT), Alanine Aminotransferase (ALAT),
Acid phosphatase (ACP), Acetylcholinesterase (AChE) and nucleic acids (DNA & RNA)
Protein profile
Fish constitutes one of the major sources of protein for human beings (Bhaqowati and
Rath, 1982). The nutritional value of different tissues of fish depends on their biochemical
composition like protein, amino acids, vitamins, mineral contents, etc.
The primary structure of a protein molecule with its aminoacid sequence, is genetically
determined and it is very likely that the specific folding and cross-linking of polypeptide chain
results largely, if not entirely, from the primary structure (Anifisnsen, 1961). The reasoning is
that ‘the primary structure dictates the secondary, tertiary and quaternary structures
(conformation) in any given environment. The existence of multiple forms of proteins has
interested many biochemists and biologists (Markert and Moller, 1959; Shaw, 1965). In addition
to the multiple forms resulting from the differences in the primary structure of the fundamental
protein unit, there are also multiple forms arising due to other reasons. For example, one type of
multiple molecular form results from the molecules of proteins having the same primary
structure which exists in several physio-chemical forms when the structure gets influenced by the
environment. These are termed as ‘conformational forms` (Lumry and Erying, 1954).
178
The potential value of electrophoresis in this study is based on the hypothesis that stress
conditions may cause significant changes in the proteins of different tissues exposed to the
toxicant. Such changes might reflect an altered antibody synthesis, protein biosynthesis, cellular
leakage or perhaps other events resulting directly or indirectly from the stress.
MATERIALS AND METHODS
The fish Channa punctatus measuring 6 to 8 cm in length and 6.5 to 7.5 gm in weight
irrespective of the sex were used in the experiment. Fish were washed with 0.1% KMnO4
solution to avoid dermal infection. All the precautions laid down by APHA et al., (1998) are
followed, for maintaining the fish. The fish were exposed to organophosphorus pesticide
quinalphos technical and 25% EC to 96 hours LC50 Technical lethal (2.9136 mg L-1), Technical
sublethal (1/10th of 96 hr LC50 i.e., 0.2913 mg L-1), 25% EC Lethal (2.3228 mg L-1) and 25% EC
sublethal (1/10th of 96 hr LC50 i.e., 0.2322 mg L-1) concentrations for 8 days. If mortality occurred
during the experimental period, dead fish were removed immediately to avoid depletion of
dissolved oxygen (DO) level which adversely affects other fish (Schreck and Brouna, 1975). The
vital tissues like muscle, brain, liver, gill and kidney of the fish were taken for the estimation of
Glycogen, Total proteins, Lactate Dehydrogenase (LDH), Aspartate Aminotransferase (AAT),
Alanine AminoTransferase (ALAT), Acid phosphatase (ACP), Acetylcholine esterase (AChE)
and nucleic acids (DNA& RNA).
Estimation of glycogen
The glycogen was estimated by the method of Kemp et al., (1954). 5% homogenates of
gill, brain, muscle and 2% homogenates of liver and kidney tissues were prepared in 80%
methanol and centrifuged at 3000 rpm for 10 minutes. The tissue residue was suspended in 5 ml
of trichloroacetic acid (TCA) and boiled for 15 minutes at 1000C and then cooled in running
water. The solution was made up to 5 ml with TCA to compensate for evaporation and then
centrifuged. From this, 2 ml of supernatant was taken into the test tube and 6 ml of concentrated
H2SO4 was added and the mixture was boiled for 10 minutes. The mixture was cooled and the
optical density was measured at 520 nm in a spectrophotometer (ELICO Model SL171) against a
blank. The standard graph was plotted with D-glucose (Analar supplied by B.D.H. Bombay) by
the aforesaid method. The glucose obtained was converted to glycogen by the multiplication
factor 0.98 (Hawks, 1951) and is expressed as mg of glycogen/gr wet weight of the tissue.
179
Estimation of total protein content
Total protein content was estimated by the modified method of Lowry et al., (1951). 5%
homogenates of gill, muscle and brain and 2% homogenates of liver and kidney were prepared in
5% trichloroacetic acid and centrifuged at 3000 rpm for 10 minutes. The supernatant was
discarded. The suspended protein residue was dissolved in 1 ml of 1N NaOH. From this 0.2 ml
of the extract was taken into the test tube and 5 ml of alkaline copper solution (50 ml of 2%
Na2CO3 and 1ml of 0.5% CuSO4. 5H2O in 1% sodium potassium tartrate) was added. The
contents were mixed well and allowed to stand for 10 minutes. To this 0.5 ml of 50% folin
phenol reagent (diluted with distilled water in 1:1 ratio) was added. After 30 minutes, the optical
density was measured at 540 nm in a spectrophotometer (ELICO Model SL171) against a blank.
The standard graph was plotted by the method of Lowry et al., (1951) with bovine serum
albumin supplied by Sigma chemical Company, U.S.A. The values were expressed as mg/gr wet
weight of the tissue.
Estimation of Lactate Dehydrogenase (LDH)
The Lactate Dehydrogenase activity (LDH) was estimated by the method of Srikanthan
and Krishna Murthy (1955). Two percent homogenates of the tissue were prepared in 0.25 M
ice-cold sucrose solution and centrifuged at 1000 rpm for 15 minutes. The supernatant served as
the enzyme source. The reaction mixture of 2 ml contains 0.5 ml of lithium lactate, 0.5 ml of
phosphate buffer, 0.2 ml of INT [2-p-idophenol-3-(P-nitrophenyl)-5-(phenyl tetrazolium
chloride)], 0.2 ml of NAD and 0.6 ml of supernatant. The reaction mixture was incubated at 370C
for 30 minutes. The reaction was stopped by the addition of 5 ml of acetic acid. Zero time
controls were maintained by adding 5 ml of acetic acid prior to the addition of homogenate. The
formazan formed was extracted overnight in 5 ml of cold toluene. The intensity of colour
developed was read at 495 nm against a reagent blank in a spectrophotometer (ELICO Model
SL171). The activity was expressed as µ moles of formazan formed/mg protein/hr.
Estimation of aminotransferases activity
The activity of AAT and ALAT were determined by the method of Reitman and Frankel
(1957). The selected tissues were homogenized in 5% ice-cold 0.25 M sucrose solution. The
supernatants were used for the analysis of the enzyme activities.
180
Estimation of AAT activity
The reaction mixture of 1.5 ml contains: 1 ml of phosphate buffer (pH 7.4), 0.1 ml of L-
aspartate (L-Aspartic acid), 0.1 ml of α-ketoglutaric acid and 0.3 ml of supernatant as enzyme
source. The reaction mixture was incubated at 370 C for 30 minutes. The reaction was stopped by
adding 1 ml of 2, 4-dinitrophenyl hydrazine solution prepared in 0.1 N HCl and was allowed to
stand for 20 minutes at room temperature. The rest of the details were the same as for alanine
aminotransferase. The activity levels were expressed as µ moles of pyruvate formed/mg
protein/hr.
Estimation of ALAT activity
The reaction mixture of 1.5 ml contains 1 ml phosphate buffer (pH 7.4), 0.1 ml of L-
alanine, 0.1 ml of α-ketoglutarate and 0.3 ml of supernatant as enzyme source. The contents
were incubated at 370 C for 30 minutes. The reaction was stopped by the addition of 1 ml of 2, 4-
dinitrophenyl hydrazine solutions. After 20 minutes, 10 ml of 0.4 N sodium hydroxide was
added and the colour developed was read at 545 nm in a spectrophotometer (ELICO Model
SL171) against a reagent blank. The enzyme activity was expressed as µ moles of pyruvate
formed/mg protein/hr.
Estimation of Acid Phosphatase (ACP)
The activity of acid phosphatase was estimated by the method of Bodansky (1932). 2%
homogenates of the tissues were prepared in 0.25 M ice sucrose solution and centrifuged at 1000
rpm for 15 minutes. The supernatant served as the enzyme source. The reaction mixture of 1.5
ml contains 1 ml of phosphate buffer (pH 5.3), 0.1 ml α-napthyl phosphate, Fast Red TR 0.1
ml and tartrate 0.2 ml. The contents were incubated at 370C for 30 minutes. In acidic pH of
buffer system acid phosphatase hydrolyses α-napthyl phosphate to α-napathal and phosphate.
The α-napthal is then coupled with diazotized fast red TR to form a diazo dye which has strong
absorbance at 405 nm. The addition of L-tartrate inhibits the reaction. Zero time controls were
maintained by adding 5 ml of L-tartrate prior to the addition of homogenate. The intensity of
colour developed was read at 405 nm against a reagent blank in a spectrophotometer (ELICO
Model SL171). The activity was expressed as mg pi/g protein/h.
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Estimation of Acetyl cholinesterase activity (AChE)
AChE enzyme assays were performed spectrophotometrically by the method of Ellman et
al., (1961). The principle of the method is the measurement of the rate of the production of
thiocholine as acetylcholine is hydrolysed. This is accomplished by the continuous reaction of
the thiol with 5:5 dithiobis-nitrobenzoate ion to produce the yellow anion of 5-thio-2-nitro
benzoic acid.
The rate of production of colour is measured at 412 nm in a spectrophotometer. The
reaction with the thiol is sufficiently rapid so as not to be rate limiting in the measurement of the
enzyme and in the concentrations used do not inhibit the enzyme hydrolysis. The rate of enzyme
hydrolysis can be recorded by using a recorder (Ellman et al., 1961).
Enzyme preparation
The fish were sacrificed and the tissues like muscle, brain, liver, gill and kidney were
quickly excised into cold solution. The excess blood is washed with 0.15 M KCl (cold) solution.
The tissues were homogenized (10% w/v) in 0.1 M pH 8 tris HCl buffer using potter-Elvehjam
homogenizer fitted with Teflon pestle. The homogenates were centrifuged at 5000 rpm for 10
minutes. The resultant supernatant was again centrifuged at 5000 rpm for 10 minutes. The
resultant supernatants were stored in ice and were used as enzyme source for the estimation of
AChE activity. All the enzyme preparations were carried out at 0-40C. Protein content for
enzyme preparations were estimated by the method of Lowry et al., (1951) using Bovine serum
albumin as standard.
AChE assay: The reactions performed at 370C were initiated by adding small aliquots of varying
concentrations of the substrate (acetyl-choline iodide) to yield a final volume of 3ml. The
absorbances of 412 nm were recorded continuously for 5 min. corresponding blanks lacking
AChE were subtracted to yield the enzymatic activity rate. The typical runs for all experiments
used were 2.7 ml buffer, 0.1 M phosphate buffer (pH 8), 50 µl (0.16mM) DTNB, 100 µl (1
mg/ml) protein and 100 µl substrate.
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Calculation:
3 1 V=▼A/min x ---------- x ------------ = µ moles/min/mg protein Protein 14.3
▼ A/min is changes in optical density
3 is ml of solution in cuvette
14.3 is molar extinction coefficient of DTNB
Estimation of Nucleic acids
The nucleic acids, deoxyribo nucleic acid (DNA) and ribo nucleic acid (RNA) were
estimated by the method of Searchy and Maclinnis 1970 (a&b). 5% homogenates of gill, brain,
muscle, liver and kidney were prepared in 5 ml of 0.5 N perchloric acid and heated at 900C for
20 minutes. After cooling the tissue homogenates were centrifuged at 3000 rpm for 10 minutes.
The supernatant was separated into two volumes and used for DNA and RNA analysis.
DNA: The first half or one half of the homogenate was mixed with diphenylamine reagent and
kept aside for 20 hr. Then the colour developed was read at 595 nm. The standard graph was
plotted with standard DNA (calf thymus) supplied by the Sigma Chemical Company with the
aforesaid method.
RNA: The other part of the homogenate was mixed with dischi-orcinol and heated at 900C for 15
minutes. After cooling at room temperature, the colour developed was read at 655 nm. The
standard graph was plotted with standard RNA (Bakers yeast) supplied by Sigma chemical
company.
Students’t-test was employed to calculate the significance of the differences between
control and experimental means. P values of 0.05 or less were considered statistically significant
(Fisher, 1950).
SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis)
Electrophoresis was carried out by Laemmli (1970) method.
Sample preparation
1% homogenates of gill, liver, kidney, brain and muscle were prepared in 10%
Trichloroacetic acid and centrifuged at 8000 rpm for 10 min in cooling centrifuge. The pellet was
washed twice with ice cold acetone, again centrifuged at 8000 rpm for 10 minutes. The pellet
was dissolved in sample buffer (0.5M Tris-HCl, pH 6.8-2ml, 40% glycerol -1.6 ml, 10% SDS-
183
3.2 ml, α-mercaptoethanol- 0.8 ml, 0.1% (W/V) bromophenol blue-0.4 ml) and boiled in water
bath at 950C for 10 minutes.
Preparation of Gel slab:
The glass plate’s sandwich was assembled using two clean glass plates and 1mm teflon
spacers. The glass plates were sealed with 1% agar solution. Resolving gel solution 12.5 % (1.5
M Tris- HCl, PH 8.8 -2 ml, 30 % Acrylamide-3.2 ml, 10 % SDS-0.5 ml double distilled water-1.8
ml, TEMED-0.015 ml, Ammonium per sulfate-0.5 ml) was prepared and poured in between the
clamped glass plates. To avoid entrapment of any air bubbles, the gel solution was overlaid with
distilled water. The plates were left undisturbed for 30 min for polymerization of the gel. After
gel polymerization, overlaid water was removed and rinsed with stacking gel buffer. Now the 5%
stacking gel solution (0.5 M Tris-HCl, pH 6.8-2 ml, 30% Acrylamide-0.8 ml, 10% SDS-0.5 ml,
double distilled water -1.2 ml, TEMED -0.015 ml, 1.5% Ammonium per sulfate 0.5 ml) was
prepared and poured over the polymerized resolving gel, comb was inserted carefully. The gel
slab was left undisturbed for 15 minutes, after polymerization comb was removed carefully and
0.1 µl from the prepared samples were loaded into the wells and gel was run at 60V.
Staining Method:
The proteins separated by electrophoresis through SDS-PAGE were fixed by placing the gel in
fixation solution (60 ml of 50% Acetone, 1.5 ml of 50 % TCA and 25 µ l of 37% HCHO) for 5
minutes with gentle shaking. The fixation solution was decanted, and gel was rinsed thrice with
double distilled water for 5 seconds. The gel was allowed to stand in double distilled water for 10
minutes at room temperature with gentle shaking. The fixation solution was decanted, and gel
was rinsed thrice with double distilled water for 5 seconds. The gel was placed in pretreatment
solution-1 (60 ml of 60% acetone) for 5 minutes with gentle shaking. The pretreatment solution-
1 was decanted and gel was placed in pretreatment solution-II (100 µ l 10% Na2 S2O3. 5H2O in
60 ml double distilled water) for few minutes with gentle shaking. The fixation solution was
decanted, and gel was rinsed thrice with double distilled water for 5 seconds. The pretreated gel
was placed in impregnated solution (0.8 ml 20% AgNO3, 0.6 ml 37% HCHO 60 ml double
distilled water) for 8-10 minutes at room temperature with gentle shaking. The fixation solution
was decanted, and gel was rinsed thrice with double distilled water for 5 seconds. The gel was
placed in developing solution (1.2 g Na2CO3, 25 µl 7 % HCHO, 25 µl Na2S2O3. 5H2O stock in
60 ml double distilled water) at room temperature. The gel was washed carefully until desired
184
contrast of protein bands appeared. The developing solution was decanted and the reaction was
quenched by washing the gel in 1% acetic acid for few minutes. Then the gel was washed several
times in double distilled water and the electrophoretogram gel was preserved in water.
Determination of molecular weight of the protein subunits separated on SDS PAGE:
To determine the molecular weight of the individual subunits of the protein, the relative
mobility of the individual subunit was calculated by using the following formula.
Distance travelled by individual subunit Relative mobility Rm value = --------------------------------------------------------- Distance travelled by the marker dye
A standard curve is prepared by plotting migration distances (‘X’-axis) of known protein
standards against their molecular weights (‘Y’-axis) on semilog graph paper. From the migration
distance of an unknown protein, the molecular weight of the protein is being calculated from the
standard curve.
185
RESULTS AND DISCUSSION
Glycogen
The calculated values for glycogen along with percent change over control and standard
deviation are given in Table V.1 and are graphically represented in Fig V.1. In the test tissues of
control fish, Channa punctatus glycogen content is in the order of:
Liver > Muscle > Brain > Gill > Kidney
The main storage of polysaccharide of animal cells is glycogen. It is especially abundant
in the liver and can attain up to 10% of the wet weight. In the skeletal muscle, glycogen only
attains 1-2% (Lehninger, 2004). In fish, the skeletal muscle glycogen is also an important store
but the concentrations found are generally an order of magnitude less than those in the liver
(Heath, 1995). Among the test tissues higher glycogen content was observed in liver. Highest
glycogen content of liver is acceptable due to its involvement in glycogen synthesis and
utilization. Glycogen is the major storage form of carbohydrate in animals which occurs mainly
in liver and muscle. Liver glycogen is largely concerned with storage and export of hexose units
for maintenance of blood glucose. The function of muscle glycogen is to act as a readily
available source of hexose units for glycolysis within the muscle itself (Harper, 2003). Though
brain tissue is metabolically active, lower glycogen content was observed, since it lacks the
inherent potential to store glycogen and is dependent on blood glucose for all its metabolic
activities (Lehninger, 2004).
Under exposure to sublethal and lethal concentrations of quinalphos technical grade, the
percent depletion of glycogen content in the test tissues of the fish Channa punctatus is in the
order of:
Technical sublethal: Kidney > Gill > Liver > Muscle > Brain
Technical lethal : Kidney > Gill > Liver > Muscle > Brain
Under exposure to sublethal and lethal concentrations of quinalphos 25% EC, the percent
depletion of glycogen content in the test tissues of the fish Channa punctatus is in the order of:
25%EC sublethal: Gill > Kidney > Liver > Muscle > Brain
25% EC lethal : Gill > Muscle > Kidney > Liver > Brain
18
6
Tab
le V
.1
Cha
nges
in th
e G
lyco
gen
cont
ent (
mg/
gr w
et w
eigh
t of t
he ti
ssue
) and
% c
hang
e ov
er c
ontr
ol in
dif
fere
nt ti
ssue
s of
fish
Cha
nna pu
nctatus
expo
sed
to s
uble
thal
and
leth
al c
once
ntra
tion
s of
qui
nalp
hos
tech
nica
l gra
de a
nd 2
5% E
C
T
issu
es
T
echn
ical
25
% E
C
Con
trol
Su
blet
hal
% C
hang
e L
etha
l %
Cha
nge
Su
blet
hal
% C
hang
e L
etha
l %
Cha
nge
Gill
29.6
5 ±0
.25
12.6
4 ±0
.54
-57.
34
11
.69
±0.6
4 -6
0.57
13.3
±0
.86
-55.
14
10
.93
±0.1
7 -6
3.13
Liv
er
85
.27
±1.5
9 42
.22
±0.2
6 -5
0.48
38.6
9 ±1
.21
-54.
62
45
.39
±1.9
4 -4
6.76
41
±1.2
1 -5
1.91
Kid
ney
21
.94
±0.9
4 8.
83
±1.3
4 -5
9.73
7.95
±0
.14
-63.
76
10
.69
±0.2
4 -5
1.27
9.85
±0
.38
-55.
10
Bra
in
30
.45
±0.6
4 21
.98
±0.1
4 -2
7.81
20.9
2 ±1
.94
-31.
27
20
.84
±0.4
8 -3
1.55
19.5
6 ±1
.95
-35.
76
Mus
cle
34
.16
±1.2
8 20
.27
±0.5
9 -4
0.64
18.6
9 ±0
.81
-45.
27
19
.12
±0.1
5 -4
4.02
13.3
4 ±0
.28
-60.
94
Val
ues
are
the
mea
n of
five
obs
erva
tions
St
anda
rd D
evia
tion
is in
dica
ted
as (±
) V
alue
s ar
e si
gnif
ican
t at p
< 0
.05
18
7
Fig
V.1
Cha
nge
in th
e G
lyco
gen
cont
ent (
mg/
gr w
et w
eigh
t of t
he ti
ssue
) in
diffe
rent
tiss
ues
of f
ish
expo
sed
to s
uble
thal
and
leth
al c
once
ntra
tions
of q
uina
lpho
s te
chni
cal g
rade
and
25%
EC
0102030405060708090
Con
trol
Subl
etha
lle
thal
subl
etha
lle
thal
Tech
nica
l25
%EC
Gill
liver
kidn
eybr
ain
mus
cle
188
In quinalphos technical grade sublethal exposure, maximum percentage of depletion was
(-59.73%) in kidney and minimum percentage was (-27.81%) in brain. In technical grade lethal
exposure, maximum percentage of depletion was (-63.76%) in kidney and minimum percentage
was (-31.27%) in brain. In quinalphos 25% EC sublethal exposure, maximum percentage of
depletion was (-55.14 %) in gill and minimum percentage was (-31.55%) in brain. In 25% EC
lethal exposure maximum percentage of depletion was (-63.13%) in gill and minimum
percentage was (-35.76%) in brain.
Depletion of glycogen may be due to utilization of stored carbohydrates in liver for
energy production as a result of pesticide-induced hypoxia. A reduction of brain glycogen of fish
exposed to the subacute doses points to deranged intermediary metabolism primary to ATP
production (Soengas and Aldegunde, 2002). This together with a hypoglycemic tendency
deprives the nervous system a supply of metabolic fuel resulting in general debility. The
depletion of glycogen in the tissues is indication of typical stress response in fish challenged with
pesticides. A fall in glycogen levels indicates its rapid utilization to meet the enhanced energy
demands in pesticide treated animals through glycolysis or hexose monophosphate pathway
(Cappon and Nicholas, 1975). Pesticides are known to act on endocrine system (Edwards, 1973).
Hence, it contributes to the decreased glycogen synthesis. Decreased glycogen synthesis is also
attributed to the inhibition of the enzyme glycogen synthetase which mediates glycogen
synthesis.
The liver has vital physiological role to perform under any stress condition. Firstly, the
toxicant in the system should be metabolized, decreased, and eliminated form the organism.
Secondly, there is a necessity for increased energy production for the physical activities
manifested under stress effect. The processes of glycogenolysis and glyconeogenesis are utilized.
In the latter process, mono acids form the precursors in fish liver (Premakumari, 1988).
Therefore the role of liver is important in this process. The liver tissue possesses enzymatic
machinery to carry out the energy production and detoxification. The data obtained in this
investigation clearly shows that liver plays a major role in the physiological reorganization under
the pesticide impact.
Glycogen depletion in liver and muscle after toxic stress has been reported in several
studies with aquatic animals (Bhavan and Geraldine, 1997; Aguiar et al., 2004). The significant
decrease in liver, the vital organ and the site of the metabolism induces the toxicant effect overall
189
affecting the life processes, especially growth and reproduction. In other organs, it will lead to
the disturbance in organ coordination and ultimately and definitely can not lead a normal life.
The earlier reports in this line of observations are Suneetha et al., 2009; Swarna kumar et al.,
2008; Tilak et al.,2005, 2003a; Tilak and Marina Samuel., 2001 ; Anita Susan et al., 1999.
The earlier observation on the effect of pesticides on carbohydrate metabolism in various
species indicates an attenuation of the energy reserve under pesticide stress (Holden, 1973;
Radhaiah, 1988; Rama Murthy, 1988 ). It appears that exposure to quinalphos leads to
enhancement of energy requirement. Since the glycogen is considered to be the first among the
organic nutrients, it initially gets affected and decrease under any physiological stress conditions
imposed on the animal. A drop in tissue glycogen content may also be either due to decreased
synthesis as a consequence of toxic stress or breakdown (Dezwaan and Zandee, 1972).
Swarna kumari et al., (2008) reported decrease in glycogen content in gill, liver, muscle,
brain and kidney of Ctenopharyngodon idella exposed to organophosphorus pesticide dichlorvos
after exposure to lethal and sublethal concentrations for 8 days. According to Venkataramana et
al., (2006) Glossogobius giuris when exposed to sublethal concentrations of (0.05, 0.25 and 0.5
mg L-1) malathion for short duration of 24 to 96 hr, the cardiac muscles showed maximum
depletion of glycogen after treatment with 0.5 mg L-1 concentration. The depletion of glycogen
content in heart might be due to a possible glycogenolysis resulting in anaerobic glycolysis to
cope up with the adverse condition, as reported by Chaudhari (2000).
It was reported earlier that the glycogen content was reduced followed by hyperglycemia
in sumithion exposed Tilapia mossambica (Koundinya and Ramamurthy, 1979a), in malathion
exposed Tilapia mossambica (Rao et al., 1986), and in endosulfan exposed Calarias batrachus
(Venkateshwarlu et al., 1987). The present depletion in glycogen content in the tissues and
increase in blood glucose levels vivid from chapter-IV may be due to the rapid turnover of
glycogen synthesized or due to the decreased rate of glycogenesis.
Long-term exposure to sublethal concentrations of quinolphos decreased the glucose level
in the fish, Channa punctatus (Sastry and Siddiqui, 1984). Monocrotophos exposure to
Channa punctatus reduced the glycogen levels (Miny Samuel and Sastry, 1989) and
phosphamidon on Gambusia affinis (Govindan et al., 1994). The decreased glycogen level is
also attributed to the conversion of carbohydrates into aminoacids (Gaiton et al., 1965).
Koundinya and Ramamurthy, (1979a) reported that stepped up glycogenolysis leads to a
190
decrease in glycogen content. Similar changes were observed in Sarotherodon mossambicus
exposed to endosulfan (Vasanthi and Ramaswamy, 1987) and in Channa striatus to metasystox
exposure (Natarajan, 1981a). Shastry and Dasgupta (1991) reported decrease in the total
carbohydrate content of liver and muscle tissue in Channa punctatus (Bloch) exposed to
sublethal concentration of Nuvacron an organophosphate. The observed decrease in glycogen
content and decrease in O2 uptake of fish vivid from chapter-II suggests the existence of anoxic
and hypoxic condition at tissue level forcing the animal to augment its energy source. This is
also correlated with fall in glycogen content in all the tissues indicating rapid utilization by the
respective tissues as a consequence of pesticide toxic stress.
Sublethal concentrations of cypermethrin induced depletion of glycogen in Tilapa
mossambica (Reddy and Yellamma, 1991) in Labeo rohita (Veeraiah and Durga Prasad, 1998;
Veeraiah, 2002) and in Cyprinus carpio (Ravisankar et al., 1992). Endosulfan 96 hr exposure
decreased the glycogen level in the fish, Clarias batrachus (Asfia Parveen and Vasantha, 1994).
Decrease of glycogen content in liver and muscle tissue in Atlantic salmon was observed under
sublethal exposure of fenvalerate (Haya, 1989); hexachlorocyclohexane exposure on Channa
punctatus (Ganathy et al., 1994); Suneetha et al., (2009) reported depletion of glycogen in
Labeo rohita exposed to endosulfan and fenvalerate in sublethal and lethal exposures for 24, 48
and 96 hours.
In the present study, it was observed that quinalphos exposure to the fish Channa
punctatus caused depletion in the total glycogen level in all the vital tissues estimated, which
may be attributed to toxic stress, resulting in the disruption of enzymes associated with
carbohydrate metabolism. Reduction in oxidation of glucose in the TCA cycle may leads to
anaerobic oxidation of carbohydrates. The carbohydrate metabolism plays an important role in
energy yielding process and its inhibition by OP insecticide stress might lead to severe energy
crisis at the cellular level.
Total Proteins
The calculated values for total proteins and percent changes over control along with
standard deviation are given in Table V. 2 and are graphically represented in Fig V.2. In the
control fish, Channa punctatus the total protein content is in the order of:
Muscle > Liver > Brain > Gill > Kidney
191
The variation in distribution suggests differences in metabolic caliber of various tissues.
The present trend in the tissues is justifiable in the wake of mechanical tissue of muscle intended
for mobility and does not participate in metabolism. The liver is also much in proteins because of
metabolic potential being oriented towards it and is the seat for the synthesis of various proteins
besides being the regulating center of metabolism.
Under exposure to sublethal and lethal concentrations of quinalphos technical grade the
percent depletion of total protein content in the test tissues of the fish Channa punctatus is in the
order of:
Technical sublethal: Liver > Muscle > Gill > Kidney > Brain
Technical lethal : Liver > Muscle > Gill > Brain > Kidney
Under exposure to sublethal and lethal concentrations of quinalphos 25% EC the percent
depletion of total protein content in the test tissues of the fish Channa punctatus is in the order
of:
25%EC sublethal: Liver > Muscle > Gill > Kidney > Brain
25% EC lethal : Liver > Muscle > Gill > Kidney > Brain
In quinalphos technical grade sublethal exposure maximum percentage of depletion was
(-33.17%) in liver and minimum percentage was (-12.03%) in brain. But in technical lethal
exposure maximum percentage of depletion was (-35.31%) in liver and minimum percentage
was (-18.26%) in kidney. In quinalphos 25% EC sublethal exposure maximum percentage of
depletion was (-38.68 %) in liver and minimum percentage was (-13.64%) in brain. In 25% EC
lethal exposure maximum percentage of depletion was (-40.42%) in liver and minimum
percentage was (-14.38%) in brain.
19
2
Tab
le V
.2
Cha
nges
in th
e T
otal
pro
tein
con
tent
(mg/
gr w
et w
eigh
t of t
he ti
ssue
) and
% c
hang
e ov
er c
ontr
ol in
dif
fere
nt ti
ssue
s of
fish
Cha
nna pu
nctatus
exp
osed
to s
uble
thal
and
leth
al c
once
ntra
tion
s of
qui
nalp
hos
tech
nica
l gra
de a
nd 2
5% E
C
T
issu
es
T
echn
ical
25
% E
C
Con
trol
Su
blet
hal
% C
hang
e L
etha
l %
Cha
nge
Su
blet
hal
% C
hang
e L
etha
l %
Cha
nge
Gill
60.2
4 ±0
.45
44.8
5 ±0
.17
-25.
54
43
.35
±0.2
4 -2
8.03
43.8
6 ±1
.87
-27.
19
42
.56
±0.1
9 -2
9.34
Liv
er
75
.92
±1.6
1 50
.73
±1.1
9 -3
3.17
49.1
1 ±1
.64
-35.
31
46
.55
±0.9
4 -3
8.68
45.2
3 ±1
.32
-40.
42
Kid
ney
59
.69
±0.8
4 51
.62
±1.8
6 -1
3.51
48.7
9 ±1
.51
-18.
26
50
.21
±1.5
2 -1
5.87
48.5
6 ±1
.56
-18.
64
Bra
in
69
.65
±0.2
6 61
.27
±1.2
5 -1
2.03
55.9
4 ±1
.43
-19.
68
60
.14
±0.3
1 -1
3.64
59.6
3 ±1
.81
-14.
38
Mus
cle
93
.72
±1.3
3 67
.16
±1.3
4 -2
8.33
62.8
2 ±0
.86
-32.
97
64
.84
±0.2
2 -3
0.81
62.5
2 ±0
.84
-33.
29
Val
ues
are
the
mea
n of
five
obs
erva
tions
St
anda
rd D
evia
tion
is in
dica
ted
as (±
) V
alue
s ar
e si
gnif
ican
t at p
< 0
.05
19
3
Fig
V.2
Chan
ge in
the
Tota
l pro
tein
con
tent
(mg/
gr w
et w
eigh
t of t
he ti
ssue
) in
diffe
rent
tiss
ues
of
fish
exp
osed
to s
uble
thal
and
leth
al c
once
ntra
tions
of q
uina
lpho
s te
chni
cal g
rade
and
25%
EC
0102030405060708090100
Cont
rol
Subl
etha
lle
thal
subl
etha
lle
thal
Tech
nica
l25
%EC
Gill
liver
kidn
eybr
ain
mus
cle
194
Proteins are indeed of primary and paramount importance in the living world not only
because of their peculiars but also because of the fact that they appear to confer their biological
specificity among various type of cells (Bhushan et al., 2002).
According to Venkataramana et al., (2006) Glossogobius giuris when exposed to
sublethal concentrations of (0.05, 0.25 and 0.5 mg L-1) malathion for short duration of 24 to 96
hr. The cardiac muscles showed significant decrease in levels of proteins after treatment with 0.5
mg L-1concentration. The decreased trend of the protein content as observed in the present study
in most of the fish tissues may be due to metabolic utilization of the ketoacids to
gluconeogenesis pathway for the synthesis of glucose; or due to the directing of free amino acids
for the synthesis of necessary proteins, or, for the maintenance of osmotic and ionic regulation
(Schmidt Nielson, 1975).
Changes in the total protein in the liver and kidney of freshwater catfish Clarias
batrachus have been studied by Shukla et al., (2005) after exposing the catfish to 0.10 and 0.16
mgL-1 Nuvan for 10 and 20 days. The decrease in total protein was found to be highly significant
after 10 days exposure at a concentration of 0.10 mg L-1 when the fishes were exposed to 0.16
mg L-1 of Nuvan the total proteins in the liver were decreased significantly. The decrease was
more pronounced after 20 days of exposure than after 10 days. The total protein contents of the
kidney also showed highly significantly reduction after the exposure of 0.10 mg L-1 for 10 days.
The decrease in the totals protein content after 20 days was non-significant.
Vishal Tiwari (2004) exposed Cirrhina mrigala sublethal concentration of 2 mg L-1 of
malathion for 7, 14 and 21 days and observed decrease in total, structural and soluble proteins
and an increase in free amino acids and protease activity levels in contrast to protein decrement
noticed in 7 and 14 days of exposure. But on 21 days of exposure all the values came to
normalcy. The restoration of different protein fraction to normalcy indicates that after 14 days of
exposure there seems to exist an oscillatory phase in protein turnover towards a more synthetic
phase leading to the establishment of recovery and adaptation phenomena. Aruna Khare et al.,
(2000) observed that the sublethal concentrations of malathion showed a significant increase in
total protein content in kidney of exposed fish, Clarias batrachus during the first week and there
after a gradual decrease in protein content was observed in the later periods of exposure.
Aruna Khare et al., (2000) observed that the sublethal concentrations of malathion
showed a significant increase in the protein content in kidney of exposed fish during the fish
195
week and then after a gradual decrease in protein content was observed in the later periods of
exposure All these investigations support the present study of decreasing trend of proteins due to
metabolic utilization of ketoacids to gluconeogenesis pathway for synthesis of glucose. The
decrease in total protein content could be due to their degradation into amino acids which in turn,
might enter the tricarboxylic acid cycle through aminotransferase pathway, to cope with the high
energy demands due to stress. The decline in total protein was more in the liver than in the other
tissues. Being a major metabolic centre, it is only natural to expect that liver showed greater
variation in total protein compared to other tissues.
Toxicity response generally depends on the toxicant concentration and the duration of
exposure in the tissue (Pickering and Henderson, 1964). The time dependent and tissue specific
response in the present study could be attributed to the concentration of quinalphos in the tissue
and also due to its distribution and elimination. The Kinetics of protein depletion in tissue may
constitute a physiological stress to compensate osmoregulatory problems encountered due to
leakage of ions and other molecules during toxicant stress (Rafat Yasmeen, 1986). The changes
and decrease in protein level might be due to inhibition or induction or induction of metabolizing
enzymes by administration of toxicants (Narayana Swamy, 1995).
Joshi and Desa (1988) observed a decrease in protein content in the liver of Tilapia
mossambica exposed to monocrotophos. Protein being involved in the architecture and also in
the physiology of the cell seems to occupy a key role in the cell metabolism, the decline in
protein level indicates an acceleration of protein catabolism during quinalphos intoxication. The
fall in protein content during stress may be due to increased proteolytic activity and deceased
anabolic activity of protein. It is possible that the protein from the tissues of the fish were utilized
under stressful conditions and released into the circulatory system to meet the increased
metabolic demand of the stressed fish. Moreover, the decreased protein content might also be
due to tissue destruction necrosis of disturbance of cellular fraction and consequent impairment
in protein synthetic machinery (Bradbury et al., 1987).
Jha and Verma (2002) studied the impact of the pesticidal mixture (Endosulfan;
Malathion and Agrafun 1:1:1) on total protein content in the stomach, intestine and ovary of the
fish Clarias batrachus actue (96 hr) subchronic (7 & 14 days) and chronic (21 days) exposures
and found that reduced protein profiles in the exposed fish were dose duration dependent. Jeba
Kumar et al., (1990) reported decrease in protein content of Lipidocephalichthys thermalis
196
exposed to sublethal concentration of fenvalerate. Pandi Bhaskaran (1991) reported depletion in
the protein content in muscle and liver of Tilapia mossambica, Mystus vittatus and Channa
straitus exposed to fenvalerate. Exposure of fish to sublethal concentrations of malathion
decreased the protein content in the gill (2.2 to 3.9%) over the control during the experiment
period of 30 days (Aruna et al., 2000). According to Tilak et al., (2002) the glycogen and
protein values are significantly decreased due to fenvalerate exposure in the fish
Ctenopharyngodon idella.
Tilak et al., (2003a) reported a decrease in protein content in Channa punctata exposed to
sublethal concentration of fenvalerate. The similar decreasing trend in total proteins was also
reported in the liver, brain and gill tissues of Catla catla under sublethal and lethal
concentrations of fenvalerate by Anita Susan et al., (1999). A significant decrease was reported
in the protein content in almost all tissues in Ctenopharyngodon idella by Tilak and Yacob,
(2002). Tilak et al., (2001d) reported that when the freshwater fish, Labeo rohita was exposed to
sublethal concentrations of pesticide mixture of monocrotophos and fenvalerate, the protein
content was decreased.
Casida et al., (1983) reported that there is an increased evidence of pesticide protein
interaction which is relevant to the mode of action of insecticide. The depletion in total protein
content may be due to augmented proteolysis and possible utilization of their product for
metabolic purposes as reported by Ravinder et al., (1988). The depletion in total proteins
observed during present investigation might also be due to the inhibition of nucleic acid
synthesis, which in turn suppressed protein synthesis. Rate of protein synthesis depends on RNA
content and RNA/DNA ratio of the tissue. Rath and Misra (1980) reported decease in DNA,
RNA and protein content in Tilapia mossambica exposed to sublethal concentration of
dichlorvos. Devi (1981) reported that the reduction in total protein may be related to the action of
chemical on nucleic acids. These findings support the results of present study.
A steady depletion of protein on exposure to chronic endosulfan has been reported by
Subbiah et al., (1985). Jayantha Rao et al., (1987) observed a decline in protein content of renal
tissue on exposure of freshwater teleost Tilapia mossambica to heptachlor. Ghosh and Chatterjee
(1988) noted a decrease in the protein content in Anabas testudineus exposed to fenvalerate. The
levels of protein decreased significantly in liver, kidney and muscle of Catla catla treated with
endosulfan (Rao, 1989).
197
The decreased protein levels may be due to their degradation. The degradation products
may in turn be fed into a tricarboxylic acid through the aminotransferase system to cope up with
the high energy demands augmented during malathion stress (Malla Reddy, 1987;
Bashamohideen, 1988). Decline in muscle protein profile in early period of suggests stress in
metabolic process and impairment of protein synthesis machinery in fish; the catabolic process
was initiated by increased proteolysis that led to rapid decline in protein concentration to meet
the energy demand in extremely stressful environment Baruah et al., (2004). Hypoprotenemia
was observed in the selected tissues of fish exposed to organophophorus pesticides by many
investigators, thus supporting the findings of the present study (Ramalingam, 1985 and Deva
Prakash Raju, 2000).
The decrease in protein content of quinalphos intoxicated fish in the present study also
indicates the physiological adaptability of the fish to compensate for pesticide stress. To
overcome the stress the animals require high energy. This energy demand might have led to the
stimulation of protein catabolism. The present analysis also coincides with the findings of Sastry
and Siddiqui (1984) who reported that the protein content was decreased in liver, muscle, kidney,
intestine, brain and gill when C. punctatus treated with quinalphos. Similar reports of Durairaj
and Selvarajan (1992), Anusha Amali et al., (1996); Yeragi et al., (2000) and Tilak et al., (2005)
support the present data. The changes and decrease in protein level might also be due to
inhibition of metabolizing enzymes by administration of toxicants. Several other investigations
also revealed a decrease in protein profiles with organophosphate compounds. All these
investigations support the present study of decreasing trend of proteins in the tissues of the fish
Channa punctatus exposed to quinalphos.
Lactate Dehydrogenase activity (LDH)
The calculated values of LDH activity and the percent change over control along with
standard deviation are given in the Table V.3 and graphically represented in Fig V.3.The LDH
levels of gill, liver, kidney, brain, muscle of control fish were almost stable. The control values
of LDH in different tissues of the fish, Channa punctatus is in the order of:
Liver > Brain > Kidney > Muscle > Gill
Under sublethal and lethal exposure of quinalphos technical grade the activity levels of
LDH were found to increase in all the tissues of the fish Channa punctatus. The percent changes
in the activity levels of LDH in the test tissues are in the order of:
198
Technical sublethal: Muscle > Gill > Liver > Brain > Kidney
Technical lethal : Gill > Muscle > Liver > Brain > Kidney
Under sublethal and lethal exposure of quinalphos 25% EC the activity levels of LDH were
found to increase in all the tissues of the fish, Channa punctatus. The percent changes in the
activity levels of LDH in the test tissues is in the order of:
25%EC sublethal: Gill > Muscle > Liver > Kidney > Brain
25%EC lethal : Gill > Muscle > Liver > Brain > Kidney
The LDH activity levels increased significantly in all exposures during the 8 days exposure
period.
In quinalphos technical sublethal exposure maximum percentage of elevation in LDH
activity was (50.34%) in muscle and minimum elevation was (13.58%) in kidney. But in
technical lethal exposure maximum percentage of elevation was (61.21%) in gill and minimum
percentage of elevation was (18.75%) in kidney. In quinalphos 25% EC sublethal exposure
maximum percentage of elevation in LDH activity was (60.51%) in gill and minimum elevation
was (16.39%) in brain. But in 25% EC lethal exposure maximum percentage of elevation was
(64.02%) in gill and minimum percentage of elevation was (21.53%) in kidney.
Lactate dehydrogenase is an enzyme involved in carbohydrate metabolism and has been
used as indicative criteria of exposure to chemical stress (Wu and Lam, 1997; Sparling et al.,
1998; Ribeiro et al., 1999; Diamantino et al., 2001). In the present study it is observed that the
activity of LDH was highly elevated following quinalphos exposure indicating increased
anaerobic respiration to meet the energy demands where aerobic oxidation is lowered. Lactate
dehydrogenase (LDH) converts lactate to pyruvate and has very important role in carbohydrate
metabolism. LDH activity depends on its five isoenzymes and the activity changes under
pathological conditions (Martin et al., 1983). The elevated LDH activity is a marker for tissue
damage in fish (Ramesh et al., 1993), hypoxic conditions (Das et al., 2004a), and muscular harm
(Balint et al., 1997) and serves as a good diagnostic tool in toxicology. The present increase in
LDH values in the tissues are in support to the tissue demand vivid from chapter-VI.
LDH is the terminal enzyme of anaerobic glycolysis located in the cellular cytoplasm.
The enzyme has been identified as potential biomarkers of metabolic perturbation after exposure
of fish to petroleum hydrocarbons (Gagnon and Holdway 1999). Cohen et al., (2005) observed
anaerobic (LDH) activity increased in the gills, liver, and white muscle of the fish Macquaria
199
novemaculeata after exposure to petroleum hydrocarbon. Stimulation in anaerobic activity also
occurred in the liver and white muscle of fish after exposure to contaminated food. The increase
of LDH activity during conditions favouring anaerobic respiration to meet the energy demands
lowers the aerobic respiration (Martin et al., 1983). The earlier reports on Tilapia mossambica
(Anastasi and Bennister, 1980; Radhaiah and Jayantha Rao, 1988) support the present study.
Lactate dehydrogenase forms the centre of delicately balanced equilibrium between
catabolism and anabolism of carbohydrates (Everse and Kaplan, 1973). LDH, an enzyme located
at a strategic point between glycolysis and citric acid cycle, catalyses the reversible oxidation of
lactate to pyruvate, serving in the terminal step of glycolysis. Lactate dehydrogenase (LDH) is
found in the cellular cytoplasm and is active during glycolysis, converting pyruvate from glucose
to lactic acid (Knox et al., 1994). LDH enzyme has been reported to increase with changes in
growth rates (Pelletier
et al., 1994) and metabolism (Lind, 1992).
Ravinder (1989) have reported elevation LDH activity during decis toxicity in fish
Clarias batrachus similarly David (1995) during fenvalerate toxicity in Labeo rohita,
Muralimohan (2000) during deltamethrin toxicity in Labeo rohita, Hymavathi (2001) during the
toxicity of chloropyrifos and endosulfan in Channa punctatus reported elevation LDH activity
in different tissues. Shaffi (2001) reported that HCH raised LDH in Labeo rohita when exposed
for 16 hrs. It indicates the enhanced metabolic rate to overcome the toxic effect of HCH and
toxicant induced anxiety. Vasudhara Devi and Narayan (2001) studied LDH activity in the ovary
of Channa punctatus spawnning and post-spawnning periods. The maximum increase in LDH
activity was in the direction of lactate formation appears to be a notable feature, when exposed to
mercuric chloride and phenol. Adam Cohen et al., (2001) observed the LDH activity is high in
white and red muscles followed by the gills and the liver due to total petroleum hydrocarbons
released into the water column. De Coen et al., (2001) observed that LDH activity increases
when exposed to Daphnia magna in sublethal concentrations of mercury. While lindane
exposure on the contrary, inhibited the cellular lactate formation and increased the krebs cycle
activity.
The LDH activity increased in gill, brain, muscle and liver tissues of Channa punctatus
exposed to sublethal concentrations of metasystox (Natarajan, 1984). The sublethal toxicity of
organophosphate (phosphamidon) on Clarias batractus showed an elevated LDH activity in gill,
200
brain, liver and muscle tissues (Ghosh, 1987). Fluke (1972), explained that the raise of LDH
activity increases the permeability of cells as well as necrosis. Tilak et al., (2005 & 2003a)
reported elevation of LDH activity in different tissues of brain, liver, muscle, gill and kidney of
the fishes Catla catla, Labeo rohita and Cirrhinus mrigala exposed to chlorpyrifos and in
different tissues of Catla catla and Channa punctatus exposed to fenvalerate.
Similar observations on LDH activity were made under pesticide stress by Veeraiah
(2002); Sastry (1999); Ganathy et al., (1994); Asfia Parveen and Vasantha (1994);
Nagabhushanam et al., (1994).
In the present study also, it was observed that the activity of LDH in the fish Channa
punctatus under exposure to sublethal concentration of quinalphos was elevated indicating that
the anaerobic respirations arrived and aerobic respiration inhibited so as to meet the increased
metabolic stress and to over come the toxic stress.
20
1
Tab
le V
.3
Cha
nge
in th
e sp
ecif
ic a
ctiv
ity
leve
ls o
f Lac
tic
dehy
drog
enas
e (L
DH
) (µ µµµ
mol
es o
f for
maz
an/m
g pr
otei
n/hr
) and
% c
hang
e ov
er th
e co
ntro
l in
diff
eren
t tis
sues
of f
ish Cha
nna pun
ctatus
exp
osed
to s
uble
thal
and
leth
al c
once
ntra
tion
s of
qui
nalp
hos
tech
nica
l gra
de
and
25%
EC
T
issu
es
T
echn
ical
25
% E
C
Con
trol
Su
blet
hal
% C
hang
e L
etha
l %
Cha
nge
Su
blet
hal
% C
hang
e L
etha
l %
Cha
nge
Gill
0.27
±0
.03
0.36
±0
.13
+32.
54
0.
43
±0.0
2 +6
1.21
0.43
±0
.22
+60.
51
0.
44
±0.0
5 +6
4.02
Liv
er
0.
51
±0.4
8 0.
62
±0.1
6 +2
2.61
0.66
±0
.27
+29.
64
0.
63
±0.0
7 +2
5.22
0.68
±0
.13
+33.
98
Kid
ney
0.
35
±0.0
8 0.
39
±0.1
8 +1
3.58
0.41
±0
.32
+18.
75
0.
41
±0.2
8 +1
8.78
0.42
±0
.10
+21.
53
Bra
in
0.
46
±0.7
2 0.
52
±0.0
3 +1
4.62
0.55
±0
.08
+19.
54
0.
53
±0.0
7 +1
6.39
0.56
±0
.07
+22.
87
Mus
cle
0.
30
±0.0
5 0.
45
±0.0
31
+50.
34
0.
46
±0.1
8 +5
5.29
0.39
±0
.08
+32.
21
0.
48
±0.0
9 +6
0.74
V
alue
s ar
e th
e m
ean
of fi
ve o
bser
vatio
ns
Stan
dard
Dev
iatio
n is
indi
cate
d as
(±)
Val
ues
are
sign
ific
ant a
t p <
0.0
5
20
2
Fig
V.3
Cha
nge
in th
e sp
ecifi
c ac
tivity
leve
ls o
f Lac
tic d
ehyd
roge
nase
( µ
mol
es o
f for
maz
an/m
g pr
otei
n/hr
) in
diffe
rent
tiss
ues
of fi
sh e
xpos
ed to
sub
leth
al a
nd le
thal
con
cent
ratio
ns o
f qui
nalp
hos
tech
nica
l gra
de a
nd 2
5% E
C
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Co
ntr
ol
Sub
leth
alle
thal
sub
leth
alle
thal
Tech
nica
l25
%E
C
Gill
liver
kidn
eybr
ain
mus
cle
203
Aminotransferases
Aspartate Amino Transferase (AAT) and Alanine Amino Transferase (ALAT) activity
The calculated values of transferases and percent change over control along with standard
deviations were given in Table V.4 & V.5 and are graphically represented in Fig V.4 and V.5.
The changes in the levels of aspartate aminotransferases (AAT) and alanine aminotransferases
(ALAT) were studied in different tissues like brain, liver, muscle, gill and kidney in the test fish
Channa punctatus under lethal and sublethal concentrations of quinalphos technical grade and
25% EC after 8 days of exposure. The values are expressed as micro moles of pyruvate formed
/mg protein /h.
Aspartate Amino Transferase (AAT)
The calculated values and percent change over control along with standard deviation and the
changes for AAT activity are given in Table V.4 and Fig V.4. The AAT activity in the control
fish is in the order of:
Kidney > Gill > Liver > Muscle > Brain
On exposure to sublethal and lethal concentrations of quinalphos technical grade, the
lyotropic gradation series in terms of percent increase in AAT activity is in the order of:
Technical sublethal: Muscle > Kidney > Gill > Liver > Brain
Technical lethal : Muscle > Kidney > Gill > Brain > Liver
Under exposure to sublethal and lethal concentrations of quinalphos 25% EC the lyotropic
gradation series in terms of percent increase in AAT activity is in the order of:
25%EC sublethal: Muscle > Kidney > Gill > Liver > Brain
25%EC lethal : Muscle > Kidney > Gill > Brain > Liver
The AAT specific activity levels increased significantly during the 8 days exposure period.
In quinalphos technical sublethal exposure maximum percentage of elevation in AAT
activity was (72.93%) in muscle and minimum elevation was (31.84%) in brain. But in technical
lethal exposure maximum percentage of elevation was (89.51%) in muscle and minimum
percentage of elevation was (46.31%) in liver. In quinalphos 25% EC sublethal exposure
maximum percentage of elevation in AAT activity was (86.11%) in muscle and minimum
elevation was (39.82%) in brain. But in 25% EC lethal exposure maximum percentage of
elevation was (97.58%) in muscle and minimum percentage of elevation was (48.51%) in liver.
Alanine Amino Transferase (ALAT)
204
The calculated values and percent change over control along with standard deviation and
the changes for ALAT activity are given in Table V.5 and Fig V.5. The ALAT activity in
different tissues of control fish was in the order of:
Gill > Liver > Muscle > Brain > Kidney
On exposure to sublethal and lethal concentrations of quinalphos technical grade, the
lyotropic gradation series in terms of percent increase in ALAT activity is in the order of:
Technical sublethal: Kidney > Gill > Muscle > Brain > Liver
Technical lethal : Kidney > Gill > Muscle > Brain > Liver
Under exposure to sublethal and lethal concentrations of quinalphos 25% EC the
lyotropic gradation series in terms of percent increase in ALAT activity is in the order of:
25%EC sublethal: Kidney > Gill >Muscle > Brain > Liver
25%EC lethal : Kidney > Gill >Muscle > Liver > Brain
The ALAT specific activity levels increased significantly during the 8 days exposure period.
In quinalphos technical grade sublethal exposure maximum percentage of elevation in ALAT
activity was (75.95%) in kidney and minimum elevation was (30.68%) in liver. In technical
lethal exposure maximum percentage of elevation was (89.61%) in kidney and minimum
percentage of elevation was (33.42%) in liver. In quinalphos 25% EC sublethal exposure
maximum percentage of elevation in ALAT activity was (86.21%) in kidney and minimum
elevation was (30.67%) in liver. But in 25% EC lethal exposure maximum percentage of
elevation was (92.34%) in kidney and minimum percentage of elevation was (32.98%) in brain.
Since the pesticide stress was known to induce significant change in protein metabolism,
it is likely that the aminotransferases were also considerably affected. Increased activities of
AAT and ALAT in different tissues of fish suggest either increased operation of transamination
or increased synthesis of amino acids from other sources like glucose or fatty acids during
quinalphos intoxication. The ALAT and AAT are liver specific enzymes and they are more
sensitive measure of hepatotoxicity and histophathalogic changes and can be assessed within a
shorter time (Balint et al., 1997). The increase in ALAT and AAT indicate the tissue damages in
liver, kidney and gill (Rajyasree and Neeraja, 1989; Oluah, 1999).
Aminotransferases are important as they convert amino acids into keto acids and
incorporate them in to TCA Cycle. Both ALAT and AAT level increased in tissues of fish
205
suggesting the conversion of aminoacids released by the proteolysis into keto acids for energy
production. The increase in ALAT and AAT activities in our study supports earlier findings and
serves as indicator of tissue damage (Oluah, 1998; Oluah, 1999; Zikic et al., 2001
Satyaparameshwar et al., 2006).
AAT and ALAT are located in both mitochondrial and cytosol fractions of the cell. A
close relation appears to exist between the mitochondrial integrity and transaminase levels
(Bonitenko, 1974) and any modification in the organization of mitochondria is bound to alter the
enzyme systems associated with it. The alteration in the activities of AAT and ALAT as
observed in the present study may also be due to the mitrocondrial distruption and damage as a
result of quinalphos induced stress.
Tilak et al., (2005) reported elevation in the levels of AAT and ALAT in different tissues
of brain, liver, muscle, gill and kidney of the fishes Catla catla, Labeo rohita and Cirrhinus
mrigala exposed to chlorpyrifos. Anita susan et al., (1999) and Tilak et al., (2003a) also
reported increase in activities of AAT and ALAT in different tissues of fish Catla catla and
Channa punctatus exposed to fenvalerate.
Ghousia Begum (1993) reported that the free amino acid content of the liver tissue
decreased after dimethoate treatment in C.batrachus and the increased amino acids might have
been converted to ketoacid by transaminases which in turn fed into TCA cycle. So there was an
increase in the activity of transaminases. Similar increase in aspartate and alanine
aminotransferease activity was observed in exposed fish (Bakthavatasalam and Srinivasa Reddy,
1982). The elevation in transaminases suggests the existence of heavy drain on metabolites
during dimethoate stress, since stress is known to induce elevation of aminotransferease
(Kulkarni and Mehrotra, 1973).
20
6
Tab
le V
.4
Cha
nge
in th
e sp
ecif
ic a
ctiv
ity
leve
ls o
f Asp
arta
te a
min
o tr
ansf
eras
e (A
AT
) (µ µµµ
mol
es o
f pyr
uvat
e fo
rmed
/mg
prot
ein/
hr) a
nd %
ch
ange
ove
r th
e co
ntro
l in
diff
eren
t tis
sues
of f
ish Cha
nna pu
nctatus
expo
sed
to s
uble
thal
and
leth
al c
once
ntra
tion
s of
qu
inal
phos
tech
nica
l gra
de a
nd 2
5% E
C
Tis
sues
Tec
hnic
al
25%
EC
Con
trol
Su
blet
hal
%
Cha
nge
Let
hal
%
Cha
nge
Subl
etha
l %
C
hang
e L
etha
l %
C
hang
e
Gill
4.56
±0
.16
6.80
±0
.73
+49.
26
7.
22
±1.5
4 +5
8.36
7.05
±0
.29
+54.
75
7.
32
±1.8
4 +6
0.63
Liv
er
4.
16
±1.2
4 5.
94
±1.6
1 +4
2.89
6.08
±0
.91
+46.
31
6.
10
±1.3
3 +4
6.76
6.17
±1
.97
+48.
51
Kid
ney
6.
21
±0.9
8 9.
53
±0.8
2 +5
3.62
10.1
9 ±1
.32
+64.
21
10
.47
±1.6
3 +6
8.62
10.9
5 ±0
.26
+76.
43
Bra
in
3.
18
±1.5
4 4.
19
±1.9
4 +3
1.84
4.74
±1
.61
+49.
08
4.
44
±0.9
1 +3
9.82
4.87
±0
.64
+53.
38
Mus
cle
3.
92
±1.3
4 6.
77
±1.5
1 +7
2.93
7.42
±1
.54
+89.
51
7.
29
±0.5
8 +8
6.11
7.74
±1
.21
+97.
58
Val
ues
are
the
mea
n of
five
obs
erva
tions
St
anda
rd D
evia
tion
is in
dica
ted
as (±
) V
alue
s ar
e si
gnif
ican
t at p
< 0
.05
20
7
Fig
V.4
Cha
nge
in th
e sp
ecifi
c ac
tivity
leve
ls o
f Asp
arta
te a
min
otra
nsfe
rase
( µ
mol
es o
f pyr
uvat
e fo
rmed
/mg
prot
ein/
hr) i
n di
ffere
nt ti
ssue
s of
fish
exp
osed
to s
uble
thal
and
leth
al c
once
ntra
tions
of
quin
alph
os te
chni
cal g
rade
and
25%
EC
024681012
Con
trol
Subl
etha
lle
thal
subl
etha
lle
thal
Tech
nica
l25
%EC
Gill
liver
kidn
eybr
ain
mus
cle
20
8
Tab
le V
.5
Cha
nge
in th
e sp
ecif
ic a
ctiv
ity
leve
ls o
f Ala
nine
am
ino
tran
sfer
ase
(AL
AT
) (µ µµµ
mol
es o
f pyr
uvat
e fo
rmed
/mg
prot
ein/
hr) a
nd %
ch
ange
ove
r th
e co
ntro
l in
diff
eren
t tis
sues
of f
ish Cha
nna pu
nctatus
expo
sed
to s
uble
thal
and
leth
al c
once
ntra
tion
s of
qu
inal
phos
tech
nica
l gra
de a
nd 2
5% E
C
Tis
sues
Tec
hnic
al
25%
EC
Con
trol
Su
blet
hal
% C
hang
e L
etha
l %
Cha
nge
Su
blet
hal
% C
hang
e L
etha
l %
Cha
nge
Gill
13.2
6 ±1
.57
21.1
4 ±1
.62
+59.
49
21
.85
±0.5
7 +6
4.85
21.3
2 ±1
.68
+60.
84
22
.13
±1.8
4 +6
6.95
Liv
er
10
.39
±1.9
4 13
.57
±1.5
4 +3
0.68
13.8
6 ±1
.59
+33.
42
13
.57
±1.2
1 +3
0.67
14.3
5 ±1
.59
+38.
15
Kid
ney
1.
96
±0.5
8 3.
44
±0.2
4 +7
5.95
3.71
±0
.24
+89.
61
3.
64
±0.8
5 +8
6.21
3.76
9 ±0
.81
+92.
34
Bra
in
4.
16
±0.9
2 5.
54
±0.8
4 +3
3.27
5.83
±0
.98
+40.
27
5.
47
±0.6
1 +3
1.64
5.53
±0
.72
+32.
98
Mus
cle
5.
68
±0.3
5 9.
00
±1.5
7 +5
8.61
9.11
±1
.37
+60.
54
8.
47
±1.6
4 +4
9.13
8.96
±0
.63
+57.
84
Val
ues
are
the
mea
n of
five
obs
erva
tions
St
anda
rd D
evia
tion
is in
dica
ted
as (±
) V
alue
s ar
e si
gnif
ican
t at p
< 0
.05
20
9
Fig
V.5 C
hang
e in
the s
pecif
ic ac
tivity
leve
ls of
Alan
ine a
min
otra
nsfe
rase
(µ m
oles
of p
yruv
ate
form
ed/m
g pr
otein
/hr)
in d
iffer
ent t
issue
s of f
ish ex
pose
d to
subl
etha
l and
leth
al co
ncen
tratio
ns o
f qu
inalp
hos t
echn
ical g
rade
and
25%
EC
0510152025
Cont
rol
Subl
etha
llet
hal
subl
etha
llet
hal
Tech
nica
l25
%EC
Gill
liver
kidne
ybr
ainm
usle
210
Increase in aminotransferases activity was reported in Tilapia mossambica, under
different pesticides exposure (Narasimha Murthy, 1983; Siva Prasada Rao and Ramana Rao,
1984; Girija, 1987 and Radhaiah 1988). Samuel and Sastry (1989) reported an elevation of AAT
and ALAT in fish Tilapia mossambica following fenvalerate intoxication. Bashamohideen
(1988) reported an increase of AAT and ALAT activities in fish Cyprinus carpio under
fenvalerate intoxication. The GOT and GPT activities increased under aldicarb, phosphamidon
and endosulfan toxicity on fish tissues (Gill et al., 1990). An elevation in AAT and ALAT
activity levels was reported by Nagendra Reddy et al., (1991), when crab Barytelphusa guerins
exposed to endosulfan 35% EC.
GDH catalyses the reversible deamination of glutamate to α-ketoglutarate and ammonia.
AAT catalyses reversible transamination of glutamate and oxaloacetate to α-ketoglutarate and
asparte, while ALAT catalyses the reversible transamination of glutamate and pyruvate to α-
ketoglutarate and alanine. Thus, the aminotransferases along with GDH contribute some strategic
substances such as α-ketogluterate, pyruvate, oxaloacetate, glutamate etc., to oxidative
metabolism.
The elevation of AAT activity 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 AAT and ALAT in different tissues of
brain, liver, muscle, gill and kidney of the fish Channa punctatus can be considered as a
response to the stress induced by quinalphos to generate ketoacids like α-ketoglutarate and
oxaloacetate for contributing to gluconeogenesis and or energy production necessary to meet the
excess energy demand under the toxic manifestations.
The depletion of proteins under the stress of quinalphos toxicity observed in different
tissues of Channa punctatus indicates the proteolysis, prompting the suggestion that the proteins
were utilized to meet the excess energy demands imposed by the toxic stress. The alterations in
the levels of activity of aminotransferases induced by the pesticide quinalphos clearly indicate
that the stress brings about the metabolic reorientation in the tissues by raising energy resources
through transaminase systems. The increase in activities of aminotransferases as observed in the
present study were in agreement with earlier reports, demonstrating a consistent increase in the
activities of these enzymes under conditions of enhanced gluconeogenesis. The alterations in the
levels of activity of aminotransferases induced by the organophosphate pesticide quinalphos
211
clearly indicate that the stress brings about the metabolic reorientation in the tissues by raising
energy resources through transaminase systems.
Acid phosphatase activity (ACP)
The calculated values of acid phosphatase (ACP) activity along with standard deviation
and percent change over the control are given in Table V.6 and are graphically represented in Fig
V.6. The acid phosphatase activity in the control fish is in the order of:
Kidney > Liver > Gill > Muscle > Brain
Under exposure to sublethal and lethal concentration of quinalphos technical grade the
percent change in acid phosphatase activity is in the order of:
Technical sublethal: Brain > Gill > Muscle > Liver > Kidney
Technical lethal : Gill > Brain > Muscle > Liver > Kidney
Under exposure to sublethal and lethal concentration of quinalphos 25%EC the percent
change in acid phosphatase activity is in the order of:
25%EC sublethal: Gill > Brain > Muscle > Liver > Kidney
25%EC lethal : Gill > Brain > Muscle > Liver > Kidney
In quinalphos technical grade sublethal exposure maximum percentage of elevation in
ACP activity was (79.51%) in brain and minimum elevation was (35.67%) in kidney. But in
technical lethal exposure maximum percentage of elevation was (178.98%) in gill and minimum
percentage of elevation was (49.38%) in kidney. In quinalphos 25% EC sublethal exposure
maximum percentage of elevation in ACP activity was (78.15%) in gill and minimum elevation
was (32.96%) in kidney. In 25% EC lethal exposure maximum percentage of elevation was
(192.58%) in gill and minimum percentage of elevation was (54.18%) in kidney.
Acid phosphatase is a hydrolytic lysosomal enzyme and release by the lysosomes for the
hydrolysis of foreign materials. It has, hence, a role in certain detoxification function. It is known
as inducible enzyme whose activity in animal tissues goes up when there is a toxic impact and
the enzyme begins to drop either as a result of having partly or fully encountered the toxin (or) as
a result of cell damage. The elevation in alkaline phosphatase suggests an increase in the
lysosomal mobilization and cell necrosis due to pesticide toxicity. Elevation of ACP activity in
brain was reported earlier in stress-exposed Channa punctatus (Sastry and Sharma, 1980) and in
Labeo rohita (Das, 1998). Sub-acute studies with monocrotophos showed increased activities of
ACP content in plasma, which are conventional indicators of liver injury (Jyothi and Narayan,
212
2000). Anabas testudineus was exposed to different sublethal concentrations of monocrotophos
for 1,7,14 and 21 days revealed a significant increase in the activities of acid phosphatase and
concluded that the impact on phosphatase activity was high in the fishes exposed to the highest
of the two sublethal concentrations by Santhakumar et al., (2000a) Dose dependent and
significant increase in the activity of acid phosphatase may be attributed to the hepatic and renal
damage. (Sandhu and Malik, 1988; Janardhan and Sisodia, 1990; Adilaxmamma and Reddy,
1995).
Increase in acid phosphatase can be interpreted as a shift of the tissues emphasis on
energy breakdown pathway from normal ATPase system to phosphatase system. Pesticides are
reported to reduce glycogen levels and increase phosphorylase activities (Mishra and Srivatsava,
1984). In the event of decreased ATPase system, phosphorylation may be proceeded by activated
phosphatases to catalyse the liberation of inorganic phosphatases from phosphate esters. The
phosphatases are active at specific pH and are usually termed phosphomonoesterases. Pesticide
poisoning in the fish altered the activity of acid phosphatase. The ACP is a lysosomal enzyme
and the raise in its activity is probably related to the cellular damage. The increased ACP activity
seems to result form enhanced enzyme turn over under pesticide stress. Joshi and Desai (1981)
examined chronic effects of monocrotophos on the ACP and ALP activities in the liver and
kidney of Tilapia mossambica and found an increased activity in the organs.
21
3
Tab
le V
.6
Cha
nge
in th
e sp
ecif
ic a
ctiv
ity
leve
ls o
f Aci
d P
hosp
hata
se (A
CP
) (m
g pi
/gra
m p
rote
in/h
r) a
nd %
cha
nge
over
the
cont
rol i
n di
ffer
ent t
issu
es o
f fis
h Cha
nna pu
nctatus
expo
sed
to s
uble
thal
and
leth
al c
once
ntra
tion
s of
qui
nalp
hos
tech
nica
l gra
de a
nd
25%
EC
T
issu
es
T
echn
ical
25
% E
C
Con
trol
Su
blet
hal
% C
hang
e L
etha
l %
Cha
nge
Su
blet
hal
% C
hang
e L
etha
l %
Cha
nge
Gill
1.85
±0
.87
3.07
±0
.84
+66.
25
5.
16
±1.4
5 +1
78.9
8
3.29
±1
.22
+78.
15
5.
41
±1.3
7 +1
92.5
8
Liv
er
2.
69
±1.9
4 3.
72
±0.9
3 +3
8.29
4.36
±1
.37
+62.
34
3.
88
±1.8
6 +4
4.52
4.62
±1
.68
+71.
84
Kid
ney
2.
82
±0.5
6 3.
82
±1.8
7 +3
5.67
4.21
±1
.61
+49.
38
3.
749
±0.5
1 +3
2.96
4.34
±1
.45
+54.
18
Bra
in
1.
46
±0.4
8 2.
62
±0.4
8 +7
9.51
2.88
±0
.92
+97.
55
2.
39
±0.9
8 +6
3.74
2.95
±0
.82
+102
.37
Mus
cle
1.
52
±0.5
8 2.
45
±0.4
5 +6
1.33
2.6
±0.3
8 +7
5.48
2.4
±0.8
2 +5
8.26
2.98
±0
.69
+96.
28
Val
ues
are
the
mea
n of
five
obs
erva
tions
St
anda
rd D
evia
tion
is in
dica
ted
as (±
) V
alue
s ar
e si
gnif
ican
t at p
< 0
.05
21
4
Fig
V.6
Cha
nge
in th
e sp
ecifi
c ac
tivity
leve
ls o
f Aci
d P
hosp
hata
se (m
g pi
/gra
m p
rote
in/h
r) in
diff
eren
t tis
sues
of f
ish
expo
sed
to s
uble
thal
and
leth
al c
once
ntra
tions
of q
uina
lpho
s te
chni
cal g
rade
and
25%
EC
0123456
Con
trol
Sub
leth
alle
thal
subl
etha
lle
thal
Tech
nica
l25
%E
C
Gill
liver
kidn
eybr
ain
mus
cle
215
Thus in the present study, the quinalphos intoxication caused elevation in the activity
levels of ACP in all the test tissues. The present results are in agreement with the study of Joshi
and Desai (1981) and Mishra and Srivatsava (1984).
Acetylcholinesterase activity(AChE)
The AChE activity was estimated in different tissues like gill, liver, kidney, brain and
muscle of the fish Channa punctatus exposed to sublethal and lethal concentrations of
quinalphos technical grade and 25% EC after 8 days and the values along with standard
deviation and the percent change over the control are shown in Table V.7 and are graphically
represented in Fig V.7. In the control fish, the acetyl cholinesterase activity levels are in the
following order:
Brain > Gill > Liver > Muscle > Kidney
The variation in activity levels of acetyl cholinesterase in different tissues of fish suggests
the variations in neural activities of those particular organs.
Under exposure to sublethal and lethal concentrations of quinalphos technical the activity
levels of AChE were found to decrease in all the test tissues. The leotropic series in terms of
decrement in AChE activity levels is:
Technical sublethal: Brain > Liver > Gill > Muscle > Kidney
Technical lethal : Brain > Liver > Kidney > Gill > Muscle
Under exposure to sublethal and lethal concentrations of quinalphos 25% EC the activity
levels of AChE were found to decrease in all the test tissues. The leotropic series in terms of
decrement in AChE activity levels is:
25%EC sublethal: Brain > Liver > Gill > Muscle > Kidney
25%EC lethal : Brain > Liver > Muscle > Gill > Kidney
Coppage et al., (1975) reported that inhibition of 87% of the normal activity is necessary
to indicate exposure of fish to anti-AChE-compounds. Inhibition of < 17.7% of normal activity
resulted in 40 to 60% fish mortality. Pinfish exposed to malathion resulted in 40 to 60%
mortality of the fish. The extent of inhibition of AChE was in the range of 72 to 79% (Coppage
et al., 1975).
21
6
Tab
le V
.7
Cha
nge
in th
e sp
ecif
ic a
ctiv
ity
leve
ls o
f Ace
tyl C
holin
este
rase
(AC
hE) (µ µµµ
mol
es o
f ace
tyl t
hioc
holin
e io
dide
hyd
roly
sed/
gr
tiss
ue/m
in) a
nd %
cha
nge
over
the
cont
rol i
n di
ffer
ent t
issu
es o
f fis
h Cha
nna pu
nctatus
expo
sed
to s
uble
thal
and
leth
al
conc
entr
atio
ns o
f qui
nalp
hos
tech
nica
l gra
de a
nd 2
5% E
C
T
issu
es
T
echn
ical
25
% E
C
Con
trol
Su
blet
hal
% C
hang
e L
etha
l %
Cha
nge
Su
blet
hal
% C
hang
e L
etha
l %
Cha
nge
Gill
4.92
±0
.88
3.31
±1
.47
-32.
54
3.
05
±1.4
8 -3
7.84
3.16
±1
.52
-35.
69
2.
46
±0.7
4 -4
9.94
Liv
er
4.
76
±0.0
8 3.
08
±0.1
2 -3
5.29
2.72
±0
.16
-42.
79
2.
95
±0.5
4 -3
7.87
2.25
±0
.41
-52.
67
Kid
ney
4.
42
±0.4
7 3.
31
±1.7
8 -2
4.97
2.67
±0
.71
-39.
37
3.
26
±1.2
1 -2
6.19
2.52
±0
.72
-42.
81
Bra
in
5.
95
±0.4
5 3.
77
±0.5
7 -3
6.59
2.99
±0
.83
-49.
62
3.
66
±0.2
7 -3
8.32
2.66
±0
.89
-55.
29
Mus
cle
4.
58
±0.8
5 3.
14
±1.7
8 -3
1.24
2.95
±0
.28
-35.
51
3.
03
±1.3
4 -3
3.75
2.26
±0
.84
-50.
55
Val
ues
are
the
mea
n of
five
obs
erva
tions
St
anda
rd D
evia
tion
is in
dica
ted
as (±
) V
alue
s ar
e si
gnif
ican
t at p
< 0
.05
21
7
Fig
V.7
Ch
ang
e in
the
spec
ific
activ
ity le
vels
of A
cety
l Ch
olin
este
rase
(µ m
ole
s o
f ace
tyl t
hio
cho
line
iod
ide
hyd
roly
sed
/gr
tissu
e/m
in) i
n d
iffer
ent t
issu
es o
f fis
h e
xpo
sed
to s
ub
leth
al a
nd
leth
al
con
cen
trat
ion
s o
f qu
inal
ph
os
tech
nic
al g
rad
e an
d 2
5% E
C
01234567
Co
ntr
ol
Su
ble
thal
leth
alsu
ble
thal
leth
al
Tec
hn
ical
25%
EC
Gill
liver
kid
ney
bra
inm
usc
le
218
In quinalphos technical grade sublethal exposure maximum percentage of AChE
inhibition was (-36.59%) in brain and minimum inhibition was (-24.97%) in kidney. But in
technical lethal exposure, maximum percentage of AChE inhibition was (-49.62%) in brain and
minimum inhibition was (-32.51%) in muscle. In quinalphos 25% EC sublethal exposure
maximum percentage of AChE inhibition was (-38.32%) in brain and minimum inhibition was (-
26.19%) in kidney. In 25% EC lethal exposure maximum percentage of AChE inhibition was (-
55.29%) in brain and minimum inhibition was (-42.81%) in kidney.
Responses to OP insecticides by aquatic organisms are broad ranged depending on the
compound, exposure time, water quality and the species (Fisher, 1991; Richmonds and Dutta,
1992). Acetylcholinesterase enzyme is widely used for rapid detection to predict early warning
of pesticide toxicity (Dutta and Arends, 2003). OP insecticides are known to inhibit
acetylcholinesterase, which plays an important role in neurotransmission at cholinergic synapses
by rapid hydrolysis of neurotransmitter acetylcholine to choline and acetate. (Soreq and Zakut,
1993). The inhibitory effects of OP insecticides are dependent on their binding capacity to the
enzyme active site and by their rate of phosphorylation in relation to the behavior and age (Dutta
et al., 1995).
The role of AChE in cholinergic transmission is to regulate nervous transmission by
reducing the concentration of acetylcholine (ACh) in the junction through AChE-catalyzed
hydrolysis of ACh (Kopecka et al., 2004). AChE was identified as the enzyme responsible for
termination of cholinergic transmission by cleavage of ACh to acetate and choline; AChE is
found in cholinergic synapses in the brain as well as in autonomic ganglia, the neuromuscular
junction, and the target tissues of the parasympathetic system (Silman and Sussman, 2005). The
AChE activity is vital to normal behaviour and muscular function and represents a prime target
on which some toxicants can exert a detrimental effect. Inhibition of the AChE activity results in
a build up of acetylcholine causing prolonged excitatory postsynaptic potential. This results in
repeated, uncontrolled firing of neurons leading to hyper stimulation of the nerve/muscle fibres,
which leads paralysis, and eventual death.
Acetylcholinesterase (AChE) is the enzyme that hydrolyzes the neurotransmitter
acetylcholine in cholinergic synapses of both invertebrates and vertebrates. AChE plays an
important role in the maintenance of normal nerve function. AChE is the primary target of
neurotoxic pesticides such as organophosphates and carbamates destined to control pests (Hassal,
219
1990). Inhibition of AChE results in a build-up of acetylcholine, causing a continuous and
excessive stimulation of the nerve/muscle fibers, which leads to tetany, paralysis, and eventual
death. Measurement of AChE inhibition in aquatic organisms, especially in fish, has already
been used as a biomarker of neurotoxic contaminants (Habig and Giulio, 1998; Galgani and
Bocquene, 1990; Payne et al., 1996; Kirby et al., 2000; Wogram et al., 2001) and it was also
reported that AChE represents one of the oldest biomarkers in fish (Sturm et al., 2000). OP
compounds phosphorylate AChE and inhibit its activity causing accumulation of acetylcholine
(Ach) at the nerve synapse, which leads to disruption of the central nervous system and
eventually death of the animal. The properties of AChE differ from species to species and also
show variations in different tissues of the same species. In vitro systems have been suggested as
economical and efficient alternatives to animal testing for OP toxicity (Barber et al., 1999).
Because the specific activities of brain AChE from fish can be affected by many factors
such as environmental temperature, species, sex, age, etc., it is necessary to know the biological
characteristics of AChE. Previous studies showed that specific activity and sensitivity of AChE
to organophosphates varied among different fish species (Chuiko 2000; Chuiko et al., 2003; Li
and Fan 1996; Eder et al., 2004; Silva et al., 2004). AChE inhibition in brain, was observed
earlier, when the fish was exposed to other OP insecticides like chlorpyrifos and profenofos
(Venkateswara Rao et al., 2003; Kumar and Chapman, 2001). The effect of malathion, diazinon,
endosulfan on brain acetylcholinesterase activity in bluegill sunfish and the largemouth bass was
investigated by (Dutta et al., 1992a; Richmonds and Dutta, 1992; Dutta et al., 1995; Guozhong et
al., 1998; Dutta and Arends, 2003) and they found a remarkable reduction in the activity which
influenced the optomotor behavior of the fish that could be detrimental to their existence in the
environment. Similarly, it is reported that monocrotophos inhibited brain AChE in the
Oreochromis niloticus (Nile Tilapia) fish (Thangnipon et al., 1995).
Vellom et al., (1993) showed that one amino acid located at the bottom of the gorge in
AChE might account for the substrate specificity of the enzyme. However, substrate specificity
of AChE in fish brain was related to the structure of substrates. The active site of AChE is made
up of the esterification site and anion site. The quaternary ammonium ion of substrates binds
with the anion site of AChE by electrostatic interaction, and the electrophilic carbon atom of the
carbonyl group reacts with the hydroxyl of serine on the esterification site of AChE (Leng et al.,
1996). Consequently, if the hydrocarbon group combining with the carbon atom of the carbonyl
220
group is bigger, then its spatial structure is bigger, and the reaction between the carbon atom of
the carbonyl group and the hydroxyl of serine on the esterification site of AChE will be weaker.
Our results correspond with the findings of Carr and Chambers (1996) and Monserrat and
Bianchini (1998), which clarified differences in the sensitivity to organophosphate and
carbamate among enzymes of the same tissue from different species. Johnson and Wallace
(1987) reported that species-related differences in enzyme susceptibility to organophosphate
pesticides might primarily be due to degree of inhibitor affinity with cholinesterase. The degree
of inhibitor affinity of insecticides for cholinesterase depends on the toxicity of insecticides and
the toxicity is associated with the molecular structure. Accordingly, the molecular structure of
insecticides plays an important role in the inhibition of AChE. The structural differences in these
insecticides should have had a great effect on the inhibitor affinity to brain AChE, and lead to the
difference in inhibitory capability among them.
It was reported that benthiocarb inhibited in vitro fish brain AChE activity in a
concentration dependent manner (Babu et al., 1989) while metacid – 50 and carbary also
inhibited in vitro brain AChE of Channa punctatus (Ghosh and Bhattacharya, 1992). It was also
reported that chlorfenvinphos, diazinon and carbofuran significantly inhibited in vitro AChE in
carp (Cyprinus carpio) and suggested that carp brain AChE can be a good diagnostic tool for OP
and carbamate pollution (Dembele et al., 2000). The results observed in the current study are in
agreement with the above reports. Our results indicated that the brain tissue was more sensitive
compared to other test tissues. It was also reported that brain AChE activity is a major target of
OP compounds. Its inhibition either directly causes or is an indirect indicator of acute CNS and
PNS symptoms (Bakshi et al., 2000).
Rahman et al., (2004) revealed that two novel phosphorothionates synthesized by Indian
Institute of Chemical Technology, Hyderbad, designated as RPR – II, RPR – V and
monocrotophos (MCP) have inhibited the target enzyme AChE in brain and liver of fish. MCP
was a more potent inhibitor than RPR – II and RPR – V. However, RPR-II and RPR-V were
equally toxic with regard to RBC AChE, whereas with brain AChE, RPR –V was more potent
inhibitor than RPR-II and the reverse trend was observed for liver AChE. This clearly indicated
that these compounds showed a structure relationship pattern. MCP has a P=O moiety whereas
RPR – II and RPR – V have P=S moieties in their structures. As such MCP was found to be
more toxic than RPR – II and RPR – V. The metabolic conversion of thiophosphoryl (P=S) ester
221
to the corresponding phosphoryl (P=O) ester mediated by mixed function oxidation makes them
highly potent cholinesterase inhibitors. Similar to RPR – II and RPR –V, parathion also contains
a P=S moiety in its structure. It was reported that parathion will be oxidized by monooxygenases
in animals and is thereby changed to a derivative containing the P=O group and this resulting
analogue will be a more powerful inhibitor of cholinesterase than the original thion phosphate
(Hassal, 1982). Similarly, Ma et al., (2003) reported methyl paraoxon was 1,000 fold more
potent inhibitor of in vitro brain AChE in rat than methyl parathion. A considerable AChE
inhibition (80%) was also found in the mosquitofish Gambusia affinis that survived to an
exposure of 72 hr to chlorpyrifos (Carr et al., 1997) Dicentrachus labrax sea bass (76%) after a
96 hr exposure to dichlorvos (Varo´ et al., 2001). In Tilapia, highest levels of AChE inhibition
were noticed in brain followed by muscle, gill and liver (Kabeer Ahammad Sahib and Ramana
Rao, 1980). Species related differences in the sensitivity of brain AChE were noticed by Yamin
et al., (1994).
Brain AChE activity is significantly greater in fish followed by pigeon and rat. The
inhibition of AChE activity by monocrotophos was in the order of rat, pigeon and fish (Yamin et
al., 1994). A significant inhibition of the brain AChE activity in rats was observed by Siddiqui
et al., (1991) with monocrotophos. Parathion was most potent inhibitor of AChE in rat followed
by pigeon, fish and honeybee (Siddiqui et al., 1989). Brain AChE activity was significantly
inhibited when Pimephales promelas (fathead minnow) exposed to chlorpyrifos (Olson and
Christensen, 1980). An inhibition of AChE activity inhibited in Cyprinus carpio exposed to
dimethoate (Manju Tembhre and Santhosh Kumar, 1994), in Oreochromis mossambicus exposed
to phosalone (Devaraj et al., 1991) Catla catla, Labeo rohita and Cirrhinus mrigala exposed to
Chlorpyrifos (Tilak et al., 2005) also support the present work.
The inhibition of AChE results in build up of acetylcholine within the nerve synapses
leading to a variety of neurotoxic effects and decreased cholinergic transmission (Mileson et al.,
1998). Results obtained by different workers independently of tissues and species used are quite
similar in the AChE inhibitory effects. In accordance with earlier observations made Rao (2006)
and Elif and Demet (2007). Depression of AChE activity in the brain is more sensitive to
quinalphos exposure than that in the gill, liver, muscle and kidney. The data reflects that an
inhibition of this magnitude may not be lethal to all species but that it may exercise a deleterious
impact on important neurobehavioral functions such as swimming and motivation. The
222
behavioral changes observed in the intoxicated fish like erratic, darting and burst-swimming can
be directly related to the impaired neuronal dysfunction of central nervous system due to
inhibition of brain AChE activity Caudal bending observed in both the sublethal concentrations
(from studies of behavioural changes, Chapter II) may be a sort of paralysis, which is due to the
inhibition of muscular AChE activity resulting in blockage of neural transmissions. Further
inhibition of AChE activity results in a progressive accumulation of ACh, especially during
periods of repetitive stimulation, leading to desensitization of nAChRs (nicotinic acetylcholine
receptors) and consequent muscular weakness (Giniatullin and Magazanik, 1998). Thus
quinalphos reduced instinctive behavioural responses and affected morphological features by
depression of AChE activity. Quinalphos inhibits AChE activity due to the effects of their active
oxygen analog quinalphos-oxon. The ratio between the toxification /detoxification reactions
determines the degree of enzyme inhibition and can be used to evaluate metabolism processes
(Timchalk et al., 2002).
Decisions to focus resources of an experiment on the most sensitive tissue, or on the most
eco-toxicologically relevant tissue, depend on the nature of the question. Some evidence
indicates that AChE inhibition in muscle is a better predictor of induced mortality than in brain
but that brain inhibition may be a better predictor of behavioral effects (Fulton and Key, 2001).
However, for studies aiming to monitor trends in pesticide contamination of watersheds, it may
be more appropriate to include measurements in the tissue most sensitive to inhibition. Muscle
AChEs were more sensitive to inhibition by diazinon than brain AChEs in Cassia occidentalis.
In four other fish species, muscle has also been identified as the most sensitive tissue (Sturm et
al., 1999 & 2000).
In the present study brain AChE is the most inhibited of all the tissues. This may be due
to the pesticide activity on the brain. Since, the compound is neurotoxic the activity levels of
AChE were inhibited. The studies of the residue analysis (Chapter VII) also reveal the extent of
inhibition of AChE activity. The correlation of the residues and the AChE activity by Coppage
et al., (1975) also supports the present study. The residues of the quinalphos in brain were
maximum where the inhibition of activity was also maximum. As the exposed fish is
continuously bathing in the pesticide medium through out the exposure period, the accumulation
of pesticide residue is a cumulative process; consequently the inhibition is also a cumulative and
is time dependent.
223
Nucleic Acids (DNA and RNA)
The calculated values of nucleic acids along with standard deviation and the percent
change over the control were given in Table V.8 & V.9 and are graphically represented in Fig
V.8 & V.9
The DNA content in control fish Channa punctatus in different tissues are in the order of:
Kidney > Liver > Gill > Brain > Muscle
Under exposure to sublethal and lethal concentrations of quinalphos technical grade and
25% EC the DNA content in gill, liver and kidney increased but was found to decrease in brain
and muscle. The decreasing order of DNA content in different tissues is in the order of:
Technical sublethal: Kidney > Liver > Gill > Brain > Muscle
Technical lethal : Kidney > Liver > Gill > Brain > Muscle
25% EC sublethal : Kidney > Liver > Gill > Brain > Muscle
25% EC lethal : Kidney > Liver > Gill > Brain > Muscle
The RNA content in control fish Channa punctatus in different tissues are in the
order of:
Kidney > Liver > Gill > Brain > Muscle
Under exposure to sublethal and lethal concentrations of quinalphos technical grade
and 25% EC it was found that the gill, liver, kidney and muscle RNA content was decreased but
the brain RNA content was found to increase. The decreasing order of RNA content in different
tissues is in the order of:
Technical sublethal: Kidney > Liver > Gill > Brain > Muscle
Technical lethal : Kidney > Liver > Gill > Brain > Muscle
25%EC sublethal : Kidney > Liver > Gill > Brain > Muscle
25% EC lethal : Kidney > Liver > Gill > Brain > Muscle
The results indicate heterogeneous levels of DNA and RNA in the tissues of brain,
liver, muscle, gill and kidney. The level of DNA in different tissues indicate cell number (Goss,
1966) and it is constant for a species. In the present study, the DNA contents in brain decreased
which may be due to reduction or absence of the essential factors controlling DNA synthesis
which are the substrates (4-Deoxyribonucleoside triphosphates), enzymes (polymerase) templet
activity of deoxyribonucleic-protein and activators like Mg++ and other divalent ions (Altman et
224
al., 1970, Bharitya & Jaimala 1988). According to Holbrook (1980) thymine incorporation into
hepatic DNA is markedly increased after 1-3 days administration of the various toxicants.The
increase of DNA in gill region is due to hypertrophic nature of chloride cells leading to less
transcription supporting the work of Natarajan (1981a), Durairaj and Selvarajan (1992) and Tilak
et al. (2005) which reveal the enlargement of nuclei in the chloride secreting cell in Channa
striatus exposed to metasystox, Oreochromis mossambicus to quinalphos and Catla catla, Labeo
rohita & Cirrhinus mrigala to chlorpyrifos. But according to Das and Mukherjee (2000b), DNA
levels were elevated in the tissues of Indian major carp, Labeo rohita when exposed to
quinalphos for 15, 30 and 45 days. The alterations in DNA levels could be due to the
disturbances in the normal synthesis and turnover rate of DNA besides degenerative changes.
22
5
Tab
le V
.8
Cha
nge
in th
e am
ount
of D
eoxy
rib
onuc
leic
aci
d (D
NA
) (m
g/gr
bod
y w
et w
eigh
t of t
he ti
ssue
) and
% c
hang
e ov
er th
e co
ntro
l in
dif
fere
nt ti
ssue
s of
fish
Cha
nna pu
nctatus
expo
sed
to s
uble
thal
and
leth
al c
once
ntra
tion
s of
qui
nalp
hos
tech
nica
l gra
de a
nd
25%
EC
Tis
sues
Tec
hnic
al
25%
EC
Con
trol
Su
blet
hal
% C
hang
e L
etha
l %
Cha
nge
Subl
etha
l %
Cha
nge
Let
hal
% C
hang
e
Gill
6.46
±0
.15
6.54
±1
.47
+1.2
3
6.86
±1
.27
+6.1
9
6.61
±0
.45
+2.3
2
6.74
±1
.52
+4.3
3
Liv
er
7.
75
±1.2
4 7.
81
±1.9
4 +0
.77
7.
96
±1.8
7 +2
.70
7.
84
±1.4
8 +1
.16
7.
96
±0.2
9 +2
.70
Kid
ney
9.
36
±1.8
4 9.
39
±1.3
7 +0
.32
9.
47
±1.7
5 +1
.17
9.
56
±1.7
6 +2
.13
9.
76
±1.3
4 +4
.27
Bra
in
5.
68
±1.9
1 5.
57
±0.2
6 -1
.93
5.47
±1
.31
-3.6
9 5.
51
±1.8
6 -2
.99
5.46
±1
.49
-3.8
7
Mus
cle
0.
79
±0.3
4 0.
78
±0.2
4 -1
.26
0.77
±0
.24
-2.5
3 0.
77
±0.4
5 -2
.53
0.76
±0
.57
-3.7
9
Val
ues
are
the
mea
n of
five
obs
erva
tions
St
anda
rd D
evia
tion
is in
dica
ted
as (±
) V
alue
s ar
e si
gnif
ican
t at p
< 0
.05
22
6
Fig
V.8
Ch
ang
e in
the
amo
un
t of D
eoxy
rib
o n
ucl
eic
acid
(mg
/gr
bo
dy
wet
wei
gh
t of t
he
tissu
e) in
d
iffer
ent t
issu
es o
f fis
h e
xpo
sed
to s
ub
leth
al a
nd
leth
al c
on
cen
trat
ion
s o
f qu
inal
ph
os
tech
nic
al
gra
de
and
25%
EC
024681012
Co
ntr
ol
Su
ble
thal
leth
alsu
ble
thal
leth
al
Tec
hn
ical
25%
EC
Gill
liver
kid
ney
bra
inm
usc
le
22
7
Tab
le V
.9
Cha
nge
in th
e am
ount
of R
ibo
nucl
eic
acid
(RN
A) (
mg/
gr b
ody
wet
wei
ght o
f the
tiss
ue) a
nd %
cha
nge
over
the
cont
rol i
n di
ffer
ent t
issu
es o
f fis
h Cha
nna pu
nctatus
expo
sed
to s
uble
thal
and
leth
al c
once
ntra
tion
s of
qui
nalp
hos
tech
nica
l gra
de a
nd
25%
EC
Tis
sues
Tec
hnic
al
25%
EC
Con
trol
Su
blet
hal
%C
hang
e L
etha
l %
Cha
nge
Subl
etha
l %
Cha
nge
Let
hal
%C
hang
e
Gill
3.75
±0
.014
3.
74
±0.9
4 -0
.26
3.62
±0
.27
-3.4
6 3.
69
±0.4
8 -1
.6
3.
64
±0.2
8 -2
.93
Liv
er
4.
36
±0.1
5 4.
28
±0.7
2 -1
.83
4.15
±0
.34
-4.8
1 4.
31
±1.6
8 -1
.14
4.27
±1
.20
-2.0
6
Kid
ney
5.
02
±0.1
9 4.
89
±1.9
2 -2
.58
4.95
±1
.69
-1.3
9 4.
87
±1.9
5 -2
.98
4.78
±0
.31
-4.7
8
Bra
in
2.
58
±0.3
7 2.
61
±1.8
2 +1
.16
2.
62
±0.3
5 +1
.55
2.
6 ±0
.54
+0.7
7
2.63
±0
.39
+1.9
37
Mus
cle
1.
92
±0.2
3 1.
65
±0.1
1 -1
4.06
1.74
±0
.21
-9.3
7 1.
89
±0.1
8 -1
.56
1.86
±0
.16
-3.1
2
V
alue
s ar
e th
e m
ean
of fi
ve o
bser
vatio
ns
Stan
dard
Dev
iatio
n is
indi
cate
d as
(±)
Val
ues
are
sign
ific
ant a
t p <
0.0
5
22
8
Fig
V.9
Ch
ang
e in
the
amo
un
t of R
ibo
nu
clei
c ac
id (m
g/g
r b
od
y w
et w
eig
ht o
f th
e tis
sue)
in d
iffer
ent
tissu
es o
f fis
h e
xpo
sed
to s
ub
leth
al a
nd
leth
al c
on
cen
trat
ion
s o
f qu
inal
ph
os
tech
nic
al g
rad
e an
d
25%
EC
0123456
Co
ntr
ol
Su
ble
thal
leth
alsu
ble
thal
leth
al
Tec
hn
ical
25%
EC
Gill
liver
kid
ney
bra
inm
usc
le
229
Nucleic acid content is considered as an index of capacity of an organism for protein
synthesis. Various studies on the effects of toxicants on the nucleic acid content in fishes have
been reported. Significant decrease in RNA and DNA content in the fish, Claria batrachus
exposed to endosulfan was recorded by Asfia Parveen and Vasanta (1986). Quinolphos induced
significant decreases in RNA content of liver muscle and gill and DNA content of brain of fish
Oreochromis mossambicus was observed by Durairaj and Selvarajan (1992). In the present study
decrease in level of RNA was observed in all the tissues of fish exposed to sublethal and lethal
concentrations of both technical grade and 25% EC, whereas RNA increased in brain. The
increase in RNA concentration may be attributed to the increased synthesis of RNA followed by
damage to neuron cells (Mcilwain and Bachelard, 1971) resulting in demyelination (Health,
1961). Increase in RNA content of gill was reported by Brachet (1955) and Ali et al., (1992).
The increased RNA level reflects the intensity of protein synthesis and the metabolic activity of
the tissue (Bulow, 1970). Organophosphorus compounds exhibit strong mutagenic and
clastogenic potentiality (Patankar Nayana & Vaidya, 1980), which may be responsible for the
alteration of DNA level. However the decrease of DNA is not very prominent when compared to
RNA. The decrease may be attributed to the increased activity of DNAase as suggested by
Tayyaba et al., (1981).
The decrease of RNA supports the view of Holbrook (1980) where maximum inhibition
of uridine incorporation occurs after 6-48 hr of toxicant administration in the rat. Gautam et al.,
(2002) reported histo-chemical changes in nucleic acids (RNA & DNA) in the stomach and
intestine of Channa punctatus after the treatment with endosulfan and diazinon pesticides and
significant decrease in nucleic acids of gastrointestine tract was also reported. Significant
decrease in RNA and DNA content in the fish, Clarias bactrachus exposed to endosulfan was
recorded by Asfia Preveen and Vasanta (1986). Quinalphos induced significant decrease in
RNA content of liver, muscle, gill and DNA content in fish Oreochromis mossambicus was
observed by Durairaj and Selvarajan (1992). The RNA levels reflect the intensity to protein
synthesis (Brachet, 1955) and metobolic activity of tissue (Bulow 1970). The depletion of RNA
level suggests increased proteolysis and possible utilisation of the products of their degradation
for metabolic purposes.
The significant decrease in both protein and nucleic acids would suggest that pollutant
impair the process of protein synthesis in the tissues of fishes exposed to pesticides. Since RNA
230
is the biochemical mid wife in the formation of proteins, the diminished RNA content also
affects the cellular protein content. Clark and Eichhorn (1995) have also suggested that the
depression in DNA synthesis is not energy dependent and may be due to the disruption of the
replication process. The RNA and protein concentration decreased in the present study could be
due to increased proteolytic activity necessitated by greater energy demands under toxic stress
(Kabeer Ahmad et al., 1978). A decrease in RNA content results in decreased protein synthesis
in tissues (Brachet, 1955). It is therefore, concluded that quinalphos intoxicantion alters the
DNA, RNA and protein levels.
According to Mukhopadhyay and Dehadrai (1980), the decrease of RNA may also be due
to interference in the incorporation of precursor in the nucleic acid synthesis or inhibiting the
function of RNA polymerase. Dawood (1986) and Benjamin (1990) have suggested that the
decrement of RNA may also be due to the non-coding for the process of protein synthesis,
thereby decrease in the RNA content, which in turn would have reduced the concentration of
RNA.
Maruthanayagam and Sharmila (2004) studied the effect of monocrotophos on Cyprinus
carpio to understand the toxic effects of toxicant on the nucleic acids and concluded that the
pesticide lead to several changes in the biochemical markers like DNA and RNA which may be
due to the increased activity of the enzyme DNAase and the inhibition of RNA polymerase
function. But during recovery period, the DNA and RNA levels increased progressively
indicating a probable from the disruption of internal organs. According to Malla Reddy and
Bashamohideen (1988) the role of nucleic acids particularly RNA/DNA and protein /DNA
rations, which are used as an index of protein synthesis and cell size, are considered to be
important and form an treatment with the pesticides causes variability in the nucleic acid content
in different tissues and the degree of variability or extent of alterations caused by the pesticides
is found to be dose dependent.
The effects of cadmium and lead on DNA and RNA contents have been studied in gill,
liver and brain of a common carp, Cyprinus carpio exposed to cadmium chloride and lead
acetate by Muley et al., (2000) and found that both the heavy metals decreased DNA content in
all the tissues along with RNA content in liver and brain, but it was increased in gill due to
cadmium and lead toxicity.
231
The estimation of percentage DNA damage by chemical induction clearly showed high
genotoxicity by the herbicide 2,4-D. Similarly, 2, 4-D has shown higher mitotic index and higher
percentage of aberrations when compared to phosphamidon and sevin. The ring formation,
number variation and gap formations were found predominantly in 2, 4-D and lindane treated
samples than in phosphamidon and sevin compounds 2, 4 – D is reported to have mutagenic and
cytotoxic effects on V 70 cells of Chinese hamster (Pavlica et al., 1991). 2, 4-D was also
reported to cause genotoxicity to freshwater fish Channa punctatus (Abul Farah et al., 2003) and
mouse (Schop et al., 1990). Genotoxicity of lindane to epithelial cells in the rat nasal and gastric
mucosa is known earlier (Pool Zobel et al., 1993).
A number of chemicals, associate with DNA damage, have been tested on live tested on
live aquatic animal, isolated tissues or different cell types. These chemicals were grouped into
four classes: (1) chemicals that act directly on DNA; (2) chemicals whose metabolites cause
DNA damage; (3) chemicals that cause the production of reactive oxygen species that can
damage DNA; (4) chemicals that inhibit DNA synthesis and repair. In addition, many chemical
contaminants damage DNA by multiple mechanisms.
The results observed in the present study reveals that quinalphos caused variability in the
nucleic acid content in different tissues and the degree of variability or extent of alterations
caused by the quinalphos technical was less compared to 25% EC and was found to be dose
dependent.
Protein profile
The relative mobility’s of the proteins fractions in different tissues of the fish Channa
punctatus exposed to quinalphos technical grade and 25% EC in sublethal and lethal
concentrations are given in the Table V.10 to V.14.
The electrophoretogram-1 represents the relative mobility values of proteins fractions
observed in gill of Channa punctatus exposed to quinalphos technical grade and 25% EC in
sublethal and lethal concentrations. In the control tissue a total of 12 protein fractions have been
observed, but after the exposure period decrease in the intensity of protein fractions has been
observed in all the exposures compared to that of control. The protein fractions with Rm 0.80 (20
kDa) and Rm 0.35, & 0.75 (58 & 23 kDa) were absent in technical grade sublethal and lethal
exposure respectively. But in 25% EC the protein fractions with Rm 0.35, 0.41, 0.59 & 0.75 (58,
232
51, 33 & 23 kDa) and Rm 0.22, 0.30, 0.35, 0.41, 0.75 & 0.80 (79, 62, 58, 51, 23 & 20 kDa) were
absent in sublethal and lethal exposure respectively.
The electrophoretogram-2 represents the relative mobility values of proteins fractions
observed in liver of Channa punctatus exposed to quinalphos technical grade and 25% EC in
sublethal and lethal concentrations. In the control tissue a total of 13 protein fractions have been
observed, but after the exposure period decrease in the intensity of protein fractions has been
observed in all the exposures compared to that of control. The protein fractions with Rm 0.28 &
0.30 (68 & 62 kDa) and Rm 0.25, 0.45 & 0.49 (70, 43 & 41 kDa) were absent in technical grade
sublethal and lethal exposure respectively. But in 25% EC the protein fractions with Rm 0.16,
0.18, 0.22, 0.25, 0.30 & 0.36 (90, 84, 79, 70, 62 & 58 kDa) and Rm 0.11, 0.16, 0.18, 0.22, 0.25
,0.30 & 0.36 (100, 90, 84, 79, 70, 62 & 58 kDa) were absent in sublethal and lethal exposure
respectively.
The electrophoretogram-3 represents the relative mobility values of proteins fractions
observed in kidney of Channa punctatus exposed to quinalphos technical grade and 25% EC in
sublethal and lethal concentrations. In the control tissue a total of 11 protein fractions have been
observed, but after the exposure period decrease in the intensity of protein fractions has been
observed in all the exposures compared to that of control. The protein fractions with Rm 0.29 &
0.55 (69 & 36 kDa) and Rm 0.20, 0.55 & 0.56 (80, 36 & 35 kDa) were absent in technical grade
sublethal and lethal exposure respectively. But in 25% EC the protein fractions with Rm 0.20,
0.34, 0.56, 0.66 & 0.92 (80, 59, 35, 29 & 14 kDa) and Rm 0.08, 0.11, 0.20, 0.34, 0.56, 0.66 &
0.92 (110, 100, 80, 59, 35, 29 & 14 kDa were absent in sublethal and lethal exposure
respectively.
The electrophoretogram-4 represents the relative mobility values of proteins fractions
observed in brain of Channa punctatus exposed to quinalphos technical grade and 25% EC in
sublethal and lethal concentrations. In the control tissue a total of 11 protein fractions have been
observed, but after the exposure period decrease in the intensity of protein fractions has been
observed in all the exposures compared to that of control. The protein fractions with Rm 0.59 (31
kDa) and Rm 0.35 (58 kDa) were absent in technical grade sublethal and lethal exposure
respectively. But in 25% EC the protein fractions with Rm 0.67 & 0.93 (28 & 14 kDa) and Rm
0.40, 0.48, 0.59, 0.67 & 0.93 (50, 41, 31, 28 & 14 kDa were absent in sublethal and lethal
233
exposure respectively. In addition to decrease in protein fractions, a new protein fraction with
Rm 0.15 (91 kDa) appeared in the 25% EC sublethal exposure.
The electrophoretogram-5 represents the relative mobility values of proteins fractions
observed in muscle of Channa punctatus exposed to quinalphos technical grade and 25% EC in
sublethal and lethal concentrations. In the control tissue a total of 13 protein fractions have been
observed, but after the exposure period decrease in the intensity of protein fractions has been
observed in all the exposures compared to that of control. The protein fractions with Rm 0.06 &
0.60 (110 & 32 kDa) and Rm 0.54, 0.60, 0.63 & 0.75 (38, 32, 30 & 23 kDa) were absent in
technical grade sublethal and lethal exposure respectively. In addition to decrease in protein
fractions, a new protein fraction with Rm 0.56 (35 kDa) appeared in the technical lethal
exposure. But in 25% EC the protein fractions with Rm 0.45, 0.63, 0.75 & 0.86 (43, 30, 23 & 14
kDa) and Rm 0.45, 0.54, 0.60, 0.63, 0.75 & 0.86 (43, 38, 32, 30, 23 & 14 kDa) were absent in
sublethal and lethal exposure respectively.
23
4
Tab
le V
.10
R
elat
ive
mob
ility
val
ues
of p
rote
in fr
acti
ons
obse
rved
in g
ill o
f fis
h Cha
nna pu
nctatus
expo
sed
to q
uina
lpho
s te
chni
cal
grad
e an
d 25
% E
C in
sub
leth
al a
nd le
thal
con
cent
rati
ons.
Mar
ker
Mol
ecul
ar w
eigh
t of
the
pro
tein
frac
tion
in
(kD
a)
Lan
e- 1
C
ontr
ol
Lan
e-2
Tec
hnic
al
subl
etha
l
Lan
e-3
Tec
hnic
al
leth
al
Lan
e-4
25%
EC
su
blet
hal
Lan
e-5
25%
EC
le
thal
--
110
0.05
0.
05
0.05
0.
05
0.05
0.13
97
.4
0.13
0.
13
0.13
0.
13
0.13
--
94
0.14
0.
14
0.14
0.
14
0.14
--
79
0.22
0.
22
0.22
0.
22
**
0.33
62
0.
30
0.30
0.
30
0.30
**
--
58
0.35
0.
35
**
**
**
--
51
0.41
0.
41
0.41
**
**
0.45
43
--
--
--
--
--
--
41
0.49
0.
49
0.49
0.
49
0.49
--
33
0.59
0.
59
0.59
**
0.
59
0.66
29
--
--
--
--
--
--
23
0.75
0.
75
**
**
**
0.76
20
.1
--
--
--
--
--
--
20
0.80
**
0.
80
0.80
**
0.82
14
.3
--
--
--
--
--
--
14
0.88
0.
88
0.88
0.
88
0.88
23
5
23
6 T
able
-V.1
1
R
elat
ive
mob
ility
of p
rote
in fr
acti
on o
bser
ved
in li
ver
of fi
sh C
hann
a pu
nctatus
expo
sed
to q
uina
lpho
s te
chni
cal
grad
e an
d 25
% E
C in
sub
leth
al a
nd le
thal
con
cent
rati
ons.
M
arke
r M
olec
ular
wei
ght
of th
e pr
otei
n fr
acti
on
in (k
Da)
Lan
e-1
Con
trol
L
ane-
2 T
echn
ical
su
blet
hal
Lan
e-3
Tec
hnic
al
leth
al
Lan
e-4
25%
EC
su
blet
hal
Lan
e-5
25%
EC
le
thal
--
100
0.11
0.
11
0.11
0.
11
**
0.13
97
.4
--
--
--
--
--
--
90
0.16
0.
16
0.16
**
**
--
84
0.
18
0.18
0.
18
**
**
--
79
0.22
0.
22
0.22
**
**
--
70
0.
25
0.25
**
**
**
--
68
0.28
**
0.
28
0.28
0.
28
--
62
0.30
**
0.
30
**
**
0.33
66
--
--
--
--
--
--
58
0.
36
0.36
0.
36
**
**
--
50
0.40
0.
40
0.40
0.
40
0.40
0.
45
43
0.45
0.
45
**
0.45
0.
45
--
41
0.49
0.
49
**
0.49
0.
49
--
36
0.58
0.
58
0.58
0.
58
0.58
0.66
29
--
--
--
--
--
--
25
0.
70
0.70
0.
70
0.70
0.
70
23
7
23
8
Tab
le-V
.12
Rel
ativ
e m
obili
ty o
f pro
tein
frac
tion
s ob
serv
ed in
kid
ney
of fi
sh C
hann
a pu
nctatus
expo
sed
to
quin
alph
os te
chni
cal g
rade
and
25%
EC
in s
uble
thal
and
leth
al c
once
ntra
tion
s.
Mar
ker
Mol
ecul
ar w
eigh
t of
the
prot
ein
frac
tion
in
(kD
a)
Lan
e-1
Con
trol
L
ane-
2 T
echn
ical
s
uble
thal
Lan
e-3
Tec
hnic
al
leth
al
Lan
e-4
25%
EC
su
blet
hal
Lan
e-5
25%
EC
le
thal
--
110
0.08
0.
08
0.08
0.
08
**
--
100
0.11
0.
11
0.11
0.
11
**
0.13
97
.4
--
--
--
--
--
--
80
0.20
0.
20
**
**
**
--
69
0.29
**
0.
29
0.29
0.
29
0.33
66
--
--
--
--
--
--
59
0.
34
0.34
0.
34
**
**
0.45
43
--
--
--
--
--
--
42
0.
46
0.46
0.
46
0.46
0.
46
--
36
0.55
**
**
0.
55
0.55
--
35
0.
56
0.56
**
**
**
0.66
29
0.
66
0.66
0.
66
**
**
0.76
20
.1
--
--
--
--
--
0.82
14
.3
--
--
--
--
--
--
14
0.92
0.
92
0.92
**
**
--
13
0.96
0.
96
0.96
0.
96
0.96
23
9
24
0
Tab
le-V
.13
Rel
ativ
e m
obili
ty o
f pro
tein
frac
tion
s o
bser
ved
in b
rain
of f
ish Cha
nna pu
nctatus
expo
sed
to
quin
alph
os te
chni
cal g
rade
and
25%
EC
in s
uble
thal
and
leth
al c
once
ntra
tion
s.
Mar
ker
Mol
ecul
arw
eigh
t of
the
prot
ein
frac
tion
in
(kD
a)
Lan
e-1
Con
trol
L
ane-
2 T
echn
ical
su
blet
hal
Lan
e-3
Tec
hnic
al
leth
al
Lan
e-4
25%
EC
su
blet
hal
Lan
e-5
25%
EC
le
thal
--
11
1 0.
10
0.10
0.
10
0.10
0.
10
--
100
0.11
0.
11
0.11
0.
11
0.11
0.
13
97.4
--
--
--
--
--
--
91
--
--
--
(0
.15)
--
--
80
0.
20
0.20
0.
20
0.20
0.
20
--
58
0.35
0.
35
**
0.35
0.
35
0.33
66
--
--
--
--
--
--
50
0.
40
0.40
0.
40
0.40
**
0.
45
43
--
--
--
--
--
--
41
0.48
0.
48
0.48
0.
48
**
--
31
0.59
**
0.
59
0.59
**
0.
66
29
--
--
--
--
--
--
28
0.67
0.
67
0.67
**
**
--
25
0.
70
0.70
0.
70
0.70
0.
70
0.76
20
.1
--
--
--
--
--
0.82
14
.3
--
--
--
--
--
--
14
0.93
0.
93
0.93
**
**
--
13
0.
96
0.96
0.
96
0.96
0.
96
24
1
24
2 T
able
-V.1
4 R
elat
ive
mob
ility
of p
rote
in fr
acti
ons
obse
rved
in m
uscl
e of
fish
Cha
nna pu
nctatus
expo
sed
to
quin
alph
os te
chni
cal g
rade
and
25%
EC
in s
uble
thal
and
leth
al c
once
ntra
tion
s.
Mar
ker
Mol
ecul
ar w
eigh
t of
the
prot
ein
frac
tion
in
(kD
a)
Lan
e-1
Con
trol
L
ane-
2 T
echn
ical
su
blet
hal
Lan
e-3
Tec
hnic
al
leth
al
Lan
e-4
25%
EC
su
blet
hal
Lan
e-5
25%
EC
le
thal
--
120
0.03
0.
03
0.03
0.
03
0.03
--
11
0 0.
06
**
0.06
0.
06
0.06
0.
13
97.4
--
--
--
--
--
--
90
0.
16
0.16
0.
16
0.16
0.
16
--
64
0.29
0.
29
0.29
0.
29
0.29
0.
33
66
--
--
--
--
--
--
49
0.42
0.
42
0.42
0.
42
0.42
0.
45
43
0.45
0.
45
0.45
**
**
--
38
0.
54
0.54
**
0.
54
**
--
35
--
--
(0.5
6)
--
--
--
32
0.60
**
**
0.
60
**
--
30
0.63
0.
63
**
**
**
0.66
29
--
--
--
--
--
--
25
0.
70
0.70
0.
70
0.70
0.
70
--
23
0.75
0.
75
**
**
**
0.76
20
.1
--
--
--
--
--
0.82
14
.3
--
--
--
--
--
--
14
0.86
0.
86
0.86
**
**
--
13
0.
95
0.95
0.
95
0.95
0.
95
24
3
244
Fish are one of the major sources of protein for human beings and the nutritional value
fish depends on their biochemical composition like protein, amino acids, vitamins, mineral
contents, etc. The clinical value of the protein analysis by electrophoresis depends upon whether
a given change represents an adaptation to stress conditions or a failure in the supportive
physiological and biochemical mechanisms of the animals.
Muthukumaravel (2007) studied the sublethal toxic effects of the heavy metal cadmium
on the electrophoretic protein fractions of gill and muscle tissues of Oreochromis mossambicus
exposed to 10 % sub lethal concentration (96 hr LC50) of cadmium for a period of 10 days and
observed the protein fractions in the muscle of experimental fishes were increased with respect to
controls. The numbers of protein fractions in the gills of test fishes were found to be lesser than
the control fishes. A study conducted by Kumar et al., (1995) on Heteropneustes fossilis exposed
to malathion, an organophosphate pesticide, showed profound effect on the protein pattern. Some
new electrophoretic protein bands appeared and some others disappeared after the treatment.
Malathion enhanced the plasma protein in its qualitative and quantitative retrospect. The increase
in number of protein bands was gradual and synchronous with increase in malathion
concentration and exposure period. Similarly in the present study the appearance of new protein
fractions in brain and muscle could be stress proteins to over come the toxic effect of quinalphos.
Manna and Mukherjee (1986) made similar findings in Tilapia on exposure to radiation,
malathion and mercuric chloride.
Riji John and Jayabalan (1993) observed protein pattern of the gill varied at different
sampling periods characterised by disappearance of certain fractions and occurrence of
additional fractions in Cyprinus carpio exposed to endosulfan and concluded that the severity
of protein pattern variations seen in the gill was dependent on both the duration of exposure and
the concentrations of endosulfan exposure. Tripathi and Shukla (1990a, 1990b) performed SDS-
PAGE of the cytoplasmic protein fractions of the liver and the skeletal muscle of Clarias
batrachus exposed to endosulfan and methyl parathion for 1 to 28 days and observed appearance
of new protein bands at different time intervals after the exposure of the pesticide demonstrating
alterations in the cytoplasm proteins. These changes in the protein band pattern in response to
exposure to pesticides may be attributed to the changes in the turnover (synthesis/degradation of
various proteins). The pesticides may inhibit the expression of some genes (or) activate the
others to produce specific mRNAs which may subsequently be translated into specific proteins
245
called stress induced proteins (Adam and Rinne, 1982; Pelham, 1985) Alterations of proteins
were observed in fish exposed to various types of environmental stresses like metals and
pesticides. Pesticidal stress cause changes in serum proteins (Jyothirmayee et al., 2006; Loughna
and Goldspink, 1984; Koban et al., 1988).
The significance in the relative areas of protein fractions reported in the present study as
a result of quinalphos stress is mainly due to the polymorphism and disappearance of some
fractions. This explanation was suggested previously by many authors (El-Sharkawi et al., 1978;
Siliem, 1994; Yacoup, 1994 and El-Serafy and Badaway, 1998). The protein electrophoresis
revealed a high difference between control and polluted samples due to the production or
activation of a new sequence of DNA responsible for synthesizing new types of protein as
concluded by El-Bermawy et al., (2000).
Bus et al., (1977) described that paraquat induced a damage of membranes, protein and
DNA. Khud-Bukhsh and Barat (1987) mentioned that polyacrylamide gel electrophoretic bands
of glutamine, albumin, globulin and muscle protein of X radiated tilapia differed significantly
with respect to number, mobility and density of bands than that of the control. Finally,
Marinovich et al., (1994) found that diazinon could induce a dose-related inhibition of protein
synthesis in HL60 cells at 24 hour exposure. The inhibition of protein synthesis may cause
fractions to decrease and proposed that in largemouth bass exposed to diazinon, tissue necrosis
leads to losses of intracellular enzymes or other proteins. This may trigger the cells in the fish
body to compensatorily repair the damaged cell organelles and to regenerate the tissue by
producing greater amount of proteins, possibly resulting in the increase or decrease of certain
fractions. Formation of a new fraction of protein may be due to the breakdown of red blood cells
or other cellular components. According to Orr and Downer (1982) the reduction of proteins
could be due to the impact on the protein synthetic pathway or due to the depletion of reserve
proteins to over come to stress caused by pesticide.
Munshi et al., (1999) studied changes in different serum protein fractions caused by the
action of malathion, to Heteropneustes fossilis exposed to a sublethal doses of malathion for 24,
48, 72, and 96 hr and observed the formation of three low and four high mobility fractions and
the disappearance of some protein fractions at different periods of exposure. The appearance of
low-mobility protein fractions may be due to altered immune responses caused by cellular
damage. The appearance of new high-mobility fractions is possibly due to the breakdown of red
246
blood cells and other cellular components indicating that the high concentration of malathion (4
mg L-1) induced more alterations in serum proteins compared with the low concentration (1.2 mg
L-1).
Jyothirmayee et al., (2005) had done polyacrylamide gel electrophoresis for endosulfan
induced changes in LDH pattern in freshwater fish Anabas testudineus and Clarias batrachus.
The bands showed a steady decreasing trend in intensity of all the fractions throughout the
exposure period demonstrating an inhibitory effect of endosulfan on kidney and muscle LDH.
Jyothirmayee et al., (2006) observed chromium induced changes in the electrophoretic patterns
of esterases in kidney, liver, gill and muscle of two freshwater, edible fishes, Anabas testudineus
and Clarias batrachus and noticed maximum changes were noted in the liver. Jyothirmayee et
al., (2006) studied the impact of chromium and endosulfan, on the serum protein electrophoretic
profile of two important edible fishes Anabas testudineus and Clarias batrachus revealed blood
borne toxicants are cleared from the plasma and stored mainly in the liver, kidney and gills and
then excreted. Thus fish regulate the toxicant concentration either through reduced absorption
and / or increased excretion of these toxicants. Jyothirmayee (2006a) observed a change in the
electrophoretic patterns of esterase in response to endosulfan was noticed in two edible fishes
Anabas testudineus and Clarias batrachus. All the enzyme fractions showed on initial marked
increase in concentration followed by a gradual decrease.
Pan and Dutta (2000) analyzed the major serum protein fractions of control and diazinon
exposed largemouth bass and six major protein fractions were separated by SDS- PAGE.
Fraction 1 and 3 of diazinon exposed fish did not show any significant difference from the
control fish. Fraction 2 of 270 µg L-1 and 450 µg L-1 diazinon exposed groups showed significant
increases from that of the control group Fraction 2. Low mobility protein of diurnal oxygen pulse
stressed largemouth bass in the study of Bouck and Ball (1965) also had a mean value of 26.30
while their control had a mean of 17.12 displaying a significant increase. The general increase in
the low mobility proteins in the organophosphorus pesticides exposed fish serums were also
observed by Dutta et al. (1992) and Datta-Munshi et al. (1999) after 24 hours exposure to
malathion the investigators suggested that the low-mobility proteins including globulin (
antibodies) (Richmonds and Dutta, 1992a; Menzel, 1970) the formation seems to occur as an
immune response in the organophosphorus pesticide exposed fish One of the reasons for the
increase in fraction 2 may be the period binding of pesticides to blood proteins after entering
247
their system (Plack et al., 1979) The binding of the pesticides to the proteins may trigger some
changes in the characteristics of these proteins The changed proteins may be recognized as
foreign bodies by the immune system resulting in the increased quantity of fraction 2 of
immunoglobin ( Richmonds and Dutta, 1992a) . Formation of the new protein may be attributed
to the cellular damages caused by this pesticide. Tissue damage would result in “leakage” from
the plasma membrane of cellular proteins, for instance, intracellular enzymes, into the blood.
Previous studies on blood serum proteins have shown that under conditions of stress
(Bouck, 1972) or heavy metal exposure (Quyyum and Gayazuddin, 1978; Dutta et al., 1983; Rai,
1987) the number of protein fractions either increased or decreased. Rai (1987) observed
disappearance of some protein fractions and emergence of a new protein with a very low
mobility in fish exposed to mercury. Results from studies conducted the Bouck (1966) showed
that changes in the plasma protein were due, in parts, to the loss of protein (enzymes) from the
tissue to blood. Such a change could occur as a result of an increase in the rate of cellular
degeneration or due to the leakage of proteins across the affected cell membranes.
The biochemical studies have been based on the detection of negatively charged protein
fractions by electrophoresis is the integrated part of the present investigation. Bhide et al.,
(2006) reported that due to the intoxication of pesticides most of the developmental stages
showed the gradual decline not only in the number of protein fractions but also showed gradual
decline in the intensities of some protein fractions as reported by Gupta and Bhide (2001 &
2004) in Lymnaea stagnalis after nuvan treatment but in control the successive development
stages showed the gradual increase in the protein fractions indicated the progressive development
of corresponding snails (Goel, 1999) .The alterations in the number of protein fractions were due
to partial or total arrest in the transcription of mRNA and ultimately affecting the translation and
that is why specific fractions were missing in the corresponding developing stages as observed in
both trochophore and veliger larval stages and prior to hatching in Lymnaea stagnalis proved the
larvicidal nature of the pesticide nuvan (Gupta and Bhide, 2001). The decline in the number of
protein fractions could be correlated with the increase in enzymatic activity of proteases prior to
hatching. but increase in free amino acids have not been investigated in the present investigation
while at some stages e.g. in gastrula stage after nuvan treatment and veliger larval stage of
control groups exhibited the increase in number of protein fractions could be correlated with the
248
synthesis of new types of proteins by the combination of different types of free amino acids as
observed by Li-Qi et al., (1998) in the Pacific oyster Crassostrea gigas.
Singh and Agarwal (1996) studied the effect of deltamethrin on the quantitative
extraction of protein in the snail Lymnaea acuminate and reported that exposure to 40% and 60%
of 48 hr LC50 of the synthetic pyrethorid deltamethrin for 24, 48, 72 and 96 hr significantly
reduced the endogenous levels of protein in foot tissue in Lymnaea acuminate while the decline
in protein fractions was observed in most of the developmental stages Lymnaea stagnalis after
treatment with baygon and nuvan.
Richmods and Dutta (1989) observed necrosis in the gills of bluegills, Lepomis
macrochirus exposed to malathion and reported differences in position, height and area in the
sera of exposed fish may be due to the possible changes in the amount of different proteins
caused by the necrosis of the cellular components. Similarly in the present study electrophoretic
changes in the proteins of different organs were may be due to the histopathalogical changes
caused by the toxicant vivid from chapter-VI.
The present study may provide an insight in rate of turnover of various proteins
alterations at cellular and subcellular levels and changes in the biological properties of fish in
reference to quinalphos pesticides at different levels of biological organization. The findings may
further attribute to the toxic effect of quinalphos pesticide in fish on functional alterations which
are often manifested by the impaired tissues such as gill, liver, brain, muscle and kidney. It can
thus be concluded that electrophoretic analysis provides a very useful method for certain aspects
of biology that it can be used as an additional tool to evaluate environmental stress on animals
with success.