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.

181

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.

182

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.