chapter - v enzymatic activities chapter -...

CHAPTER - V ENZYMATIC ACTIVITIES

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Chapter - V


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Acetylcholinesterase (AChE)

Cholinesterases (ChEs) are a ubiquitous class of serine hydrolases which

physiologically remove acetylcholine from the synaptic cleft. ChEs are widely distributed

among vertebrate and invertebrate animals (Bocquene et al., 1997). Acetylcholinesterase is an

enzyme that regulates the amount of neurotransmitter acetylcholine at neuron junctions. It is a

serine protease that hydrolyzes the neurotransmitter acetylcholine. AChE is found mainly at

neuromuscular junctions and cholinergic brain synapses, where its activity serves to terminate

synaptic transmission. It belongs to carboxylesterase family of enzymes.

AChE has very high catalytic activity - each molecule of AChE degrades about 25000

molecules of acetylcholine (ACh) per second, approaching the limit allowed by diffusion of

the substrate (Quinn, 1987; Taylor and Radic, 1994). The active site of AChE comprises 2

subsites - the anionic site and the esteratic subsite. The structure and mechanism of action of

AChE have been elucidated from the crystal structure of the enzyme (Sussman et al., 1991;

1993).

During neurotransmission, ACh is released from the nerve into the synaptic cleft and

binds to ACh receptors on the post-synaptic membrane, relaying the signal from the nerve.

AChE, also located on the post-synaptic membrane, terminates the signal transmission by

hydrolyzing ACh. The liberated choline is taken up again by the pre-synaptic nerve and ACh

is synthetized by combining with acetyl-CoA through the action of cholineacetyltransferase

(Whittaker, 1990; Purves et al., 2008; Ohanka, 2012).

AChE is found in many types of conducting tissue: nerve and muscle, central and

peripheral tissues, motor and sensory fibers, and cholinergic and noncholinergic fibers. The

activity of AChE is higher in motor neurons than in sensory neurons (Massoulie et al., 1993;

Chacho and Cerf, 1960; Koelle, 1954). Acetylcholinesterase exists in multiple molecular

forms, which possess similar catalytic properties, but differ in their oligomeric assembly and

mode of attachment to the cell surface. The major form of acetylcholinesterase found in brain,

muscle, and other tissues, known as is the hydrophilic species, which forms disulfide-linked

oligomers with collagenous, or lipid-containing structural subunits.

For a cholinergic neuron to receive another impulse, ACh must be released from the

ACh receptor. This occurs only when the concentration of ACh in the synaptic cleft is very

low. Inhibition of AChE leads to accumulation of ACh in the synaptic cleft and results in

impeded neurotransmission. Irreversible inhibitors of AChE may lead to muscular paralysis,


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convulsions and bronchial constriction.

Organophosphorus (OPs) 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). These inhibit

the action of the cholinesterase by phosphorylating or carbamylating the active centre of the

enzymes. The most important toxic property of organophosphorus pesticide compounds is

inhibiting their target enzyme acetyl cholinesterase activity (O' Brein, 1967; Corbeit, 1974).

Most of the organophosphate compounds are similar with the ester part of acetylocholine and

they react with esterase part of AchE after entering into the exposed animal. The conversion

of acetylcholine into acetic acid and choline catalyzed by AchE is considered to be the key

reaction in synaptic transmission (Bachelard, 1976).

Acetylcholinesterase activity is one of the most frequently used indicators to verify

organophosphate effects (Chuiko, 2000; Aguiar et al., 2004). The inhibition of AChE for

pestcides can affect locomotion and equilibrium in exposed fishes and may impair feeding,

escape, and reproductive behavior (Saglio and Trijasse, 1998; Bretaud et al., 2000). The

inhibitory effects organophosphorus pesticides on cholinesterases are generally considered as

the basis for the biological activity of fishes. Hence AchE estimation in fishes has proved

valuable in detecting the pollution of fresh water and marine waters.

The inhibition of cholinesterase activity serves as a reliable biomarker both of

exposure and of effect of organophosphates (Coppage and Braidech, 1976; Fulton and Key,

2001; Chambers et al., 2002; Vioque-Fernandez et al., 2009). It is well accepted that 20% or

greater inhibition of AChE in birds, fishes and invertebrates indicates exposure to

organophosphate insecticides (Mayer and Ellersiek, 1986).

The inhibitory effects of organophosphorus insecticides are dependent on their

binding capacity to the enzyme active site and by their rate of phosphroylation in relation to

behavior and age (Dutta et al., 1995). Organophosphorus pesticides are converted in vivo to

the corresponding active phosphate ester or oxon (P=O), which is a potent

acetylcholinesterase inhibitor. Studies using animal liver have shown that cytochrome P450

(CYP) enzymes mediate the oxidative desulphuration of the OP parathion to the active

metabolite paraoxon (Neal, 1967). Organophosphorus compounds generally are lipophilic and

therefore cross the blood-brain barrier in most cases (Vale, 1998). Tomokuni et aI., (1985)

observed the accumulation of diazinon in the brain of rats and mice after single interperitoneal


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injection. They also found that brain AChE activity was inhibited markedly after injection.

Inhibition of AChE that is responsible for the degradation of acetylcholine will result

in excessive stimulation of cholinergic nerves. This will result in tremors, convulsion and

finally the death of the aquatic organism (Baxter and Barker, 1998). Several factors seem to

be involved in affecting the AChE activity caused by OPs such as length to time and exposure

concentrations (Uncer et aI., 2006). Inhibition of AChE, impairs cholinergic nerve impulses

and may result in death of organisms (Salles et al., 2006).

Inhibition of AChE was accompanied by an increase in acetylcholine levels

(Brzezinski and Ludwicki, 1973). This condition can lead to increase of catecholamines which

can affect the activity of enzymes involved in glycogenolysis and glycogen synthesis.

Continuous stress may affect the synthesis site of AChE or decrease the levels of excess

AChE. Mortality of fish may be due to inhibition of other enzymes, especially those taking

part in carbohydrate and protein metabolisms. The inhibitory effect on AChE activity

indicates that insecticides might interfere in vital processes like energy metabolism of nerve

cells (Ansari et al., 1987). Consequently, inhibition of AChE leads to paralysis and death.

Many organophosphates are potent neurotoxins, functioning by inhibiting the action of

AChE in nerve cells. Significant AChE inhibition following organophosphate exposure has

been shown to occur in a number of vertebrate and invertebrate species at concentrations well

below the LC50 value (Day and Scott, 1990; Booth et al., 2001; De Mel and Pathiratne, 2005).

Studies done on the inhibitory effects of organophosphorus compounds on an array of fish

species have produced varied results. Dembele et al. (2000) found that 0.36 ppb of

chlorpyrifos inhibited brain AChE activity in the common carp, Cyprinus carpio by 85%,

while Boone and Chambers (1997) found that 50% inhibition in brain AChE activity in the

mosquitofish, Gambusia affinis, required a 50-fold greater concentration.

Several investigators (Day and Scott, 1990; Boone and Chambers; 1997; Mileson et

al,. 1998; Pan and Dutta, 1998; Chuiko, 1999; Dembele et al., 2000; Booth et al., 2001; Erwin

et al., 2003; De Mel and Pathiratne, 2005; Kristen et al., 2005; Rao; 2006; Venkateswara,

2006; Elif and Demet , 2007; Khalid et al., 2008; Pavlov et al., 2008; Vineet et al., 2008)

reported the inhibition of AChE following organophosphate pesticides exposure in a different

fish species.

The activity of cholinesterases differs from one fish species to another. Chciko et al.


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(2000) observed acetylcholine esterase, butyrylcholinesterase activities in brain and plasma of

fresh water teleosts and reported that brain acetycholinesterase activity varied among fish

species approximately 15-fold ranging from 138 to 2011 µ mol/g/ hr. All cyprinids had higher

brain AChE activity than other fish families.

Individual variation in the enzyme inhibition activity of OP pesticides was also noted.

Varo et al. (2003) have demonstrated an age-dependent correlation in AChE inhibition in

European sea bass, Dicentrarchus labrax exposed to the OP pesticide dichlorvos in his study.

Another important factor in intraspecies variability is the general health of the organism, and

the extent of exposure to other stressors.

The inhibition of AChE activity is regarded as a significant parameter in assessing the

complex toxicogenic effects of pesticides (Bradbury et al., 1986). Great deal of work has been

turned out on these lines with reference to AChE inhibition by organophosphate pesticides,

but no attempt has been made on Cyprinus carpio with reference to acute and chronic toxicity

of phorate. Hence an attempt is made to study the AChE activity in economically important

edible fish Cyprinus carpio on exposure to acute and chronic toxicity of phorate.

RESULTS

The data on the levels of AChE activity in the organs such as gills, liver, muscle,

kidney and brain of the fish cyprinus carpio at 1 and 4 days on exposure to acute toxicity of

phorate and 1, 7, 15 and 30 days on exposure to chronic toxicity of phorate, besides controls,

are presented in the Table-9 and Figure-8. For comparison, the differences obtained in relation

to controls of each organ at the above said exposure periods in acute and chronic toxicity

study of phorate, were converted as percentages of the corresponding controls and those

percent values are also presented in the same table and was plotted a graph of percent changes

against exposure periods in Figure-8.

From the data presented in the Table-9 and Figure-8 it is seen that, relative to controls,

the levels of of AChE activity in all the organs of the fish exposed to phorate gradually

decreased at 1 and 4 days of exposure in acute toxicity in the order of 1>4 and the differences

in the activity between controls and experimental were also found to be statistically significant

(P<0.05). In the fish exposed to chronic toxicity of phorate relative to controls the levels of

AChE activity elevated at day 1 in all the organs. The levels of AChE activity gradually

decreased from day 7 to day 15 followed by an increase at day 30 on exposure to chronic


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toxicity of phorate in all the organs of the fish in the order of 1>7>15<30. The values were

found to be statistically significant (P<0.05). However, based on the percent values obtained

(Table-9 and Figure-8) the decrease in AChE activity was found to be predominantly more in

the organs of the fish exposed to acute toxicity of phorate.

DISCUSSION

Acetylcholinesterase (AChE, EC 3.1.1.7) activity is routinely used as a biomarker of

the exposure to certain groups of contaminants, such as organophosphate insecticides (Grue et

al., 1997). Low concentrations of the compounds can inhibit AChE, which leads to an

accumulation of acetylcholine at central cholinergic synapses and neuromuscular junctions

(Sancho et al., 1997; Varo et al., 2003). The inhibition of the acetylcholinesterase by

pesticides can affect locomotion and equilibrium of exposed organisms (Saglio and Trijasse,

1998; Bretaud et al., 2000).

The AChE activity is vital to normal behaviour and muscular function in animals 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

hyperstimulation of the nerve or muscle fibres, which leads paralysis, and eventual death.

Neurological and behavioral activities of animals can be extremely sensitive to

environmental contamination (Doving, 1991; Scherer, 1992; Silbergeld, 1993; Costa, 1996).

Organophosphate pesticides are competitive inhibitors of acetylecholineesterase (AChE), the

key enzyme in the transmission of nerve impulse. AChE is readily phosphorylated by the

organophosphate pesticides at the active site serine (Aldrige and Reiner, 1972; Taylor, 1990).

organophosphates causes inhibition of AChE and accumulation of acetylecholine at the

synapse (Loskowsky and Dettbam, 1975) which leads to over stimulating the post synaptic

cells (Pope et al., 1995). It is well accepted that 20% or greater inhibition of AChE in birds,

fishes and invertebrates indicates exposure to organophosphate insecticides (Mayer and

Ellersiek, 1986).

In the present investigation the data on AChE activity revealed a decrease in the

enzyme activity in both the nervous (brain) and non-nervous (gill, liver, muscle and kidney)

organs of the fish Cyprinus carpio exposed to acute and chronic toxicity of phorate except at

day 1 in chronic toxicity exposure (Table-9 and Figure-8).


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The decrease in AchE activity was also observed earlier when treated with Roger and

Dimecron in fresh water mussel, L. Marginalis (Vijayaendrababu and Vasudev, 1984). Similar

observations were made in fresh water crab, O.Senex senex by Fenitrothion (Bhagyalakshmi

and Ramamurthi, 1980); in snails exposed to different organophosphorus compounds

(Ramana Rao and Ramurthi, 1979; Singh and Agarwal, 1982) and in fishes ( Anuradha,1993;

Singh and Singh, 2005).

Pan and Dutta (1998) studied the inhibition of brain acetylcholinesterase activity of

juvenile largemouth bass, Pterus salmoides by sublethal concentrations of diazinon and

reported that juvenile brain acetylcholinesterase activities were significantly inhibited by

sublethal doses of diazinon. Erwin et al. (2003) studied the effects of chronic exposure of

parathion on acetylcholine esterase inhibition and increased food consumption rate in the

zebra fish, Danio rerio and reported that inhibition rate was significant above 0.9 µ g/l after

144 days and above 4.3 µg/l after 250 days of exposure.

Kristen et al. (2005) studied the effects of diazinon exposure in hybrid striped bass on

biochemical and behavioural aspects and reported that the sublethal exposure to diazinon, an

organophosphate pesticide, may lead to feeding behavior abnormalities in hybrid striped bass

through inhibition of brain acetylcholine esterase activity. Venkateswara (2006) studied

sublethal effects of profenofos on locomotive behaviour and gill architecture of the mosquito

fish, Gambusia affinis and reported that the sublethal concentration of profenofos altered

locomotive behaviour such as distance traveled and swimming speed in fish due to inhibition

in the activity of acetylcholine esterase and caused for the deformities in the primary and

secondary lamella of gill.

Khalid et al. (2008) studied the ethological response and haematological and

biochemical profiles of carp, cyprinus carpio exposed to trichlorfos, and reported a significant

reduction in the acetylcholinesterase activity in the brain tissue of the fish exposed to

tricholorfos. Pavlov et al. (2008) studied the effect of DDVP, an organophosphorus

insecticide on feeding behaviour and brain acetyl cholinesterase activity in bream, Abramis

brama (L.) and reported that DDVP exposure resulted in the inhibition of brain

acetylcholinesterase activity. Vineet et al. (2008) studied the behaviour and respiratory

dysfunction as an index of malathion toxicity in the fresh water fish, Labeo rohita (Hamilton)

and reported that the carp in toxic media exhibited irregular, erratic and darting, swimming

movements, hyper excitability, and loss of equilibrium and sinking to the bottom which might

be due to inactivation of AChE activity.


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In the fish exposed to acute toxicity of phorate in the present study the suppression in

AChE activity has increased in all the organs with the increase in exposure period. Jaqueline

et al., (2008) also reported a decrease in AChE activity in the brain and muscle of fingerlings

of the common carp (Cyprinus carpio), grass carp (Ctenopharyngodon idella) and bighead

carp (Aristichthys nobilis) on exposure to lethal concentration (LC50) of diafuran.

The organophosphate pesticides inhibit the cholinesterase activity in almost all animal

tissues (O’ Brein, 1969; Goodman et al., 1979). The AChE activity differs from one species to

the other in fishes. Chuiko (1999) worked on comparative study of acetylcholinesterase and

butyrylcholinesterase in brain and serum of several fresh water fish by DDVP, an

organophosphorus pesticide-reported that brain acetylcholinesterase activity varied among

fish species approximately 10-fold, ranging from 192.6 to 1353.2µ mol/g/h respectively in

perch and white fish. All cyprinids had higher brain acetycholinesterase activity than those

other fish families. Serum acetycholinesterase activity was 100-fold lower than in brain.

Duration of exposure, type of organophosphate, as well as species of fish has an effect on the

extent of AChE activity.

It was well documented that highly purified phosphorothionates (P=S form) are not

direct inhibitors of cholinesterase but when they are metabolized to their corresponding

oxygen analogues (P=O form) become highly potent inhibitors (March et al., 1956 and

Murphy et al., 1968). The susceptibility of animals to poisoining by organophosphorus

insecticides will be dependent upon the rate at which the analogues are made available to

inhibit cholinesterase at critical site in nerve tissue or organs innervated by cholinergic nerves.

In the fish exposed to chronic toxicity of phorate in the present investigation, relative

to controls the levels of AChE activity elevated at day 1 in all the organs. The levels of of

AChE activity gradually decreased at day 7 and at day 15 followed by an increase at day 30 in

all the organs of the fish. Similar results obtained by Sailabala (1988) in major carp, Catla

catla on exposure to an organophosphate insecticide malathion and Prasada Charyulu (1993)

in common carp cyprinus carpio on exposure to Phosphamidan with regarding to AChE

activity.

The AChE activity suddenly activated during 24 hours of exposure. The enhancement

in the activity of AChE might be due to the pesticide stress. But at later periods of exposure

like 7th

day and 15th

day the AChE activity was reduced, with maximal reduction at 15th

day

exposure period of phorate. The inhibition of AChE activity will result in the accumulation of


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acetylcholine (Ach) at different periods of exposure. Heath (1961) and O’ Brein (1967)

reported that OP insecticides react with AChE to form phosphorylated enzyme. This

phosphorylated enzyme inhibits AChE activity for several weeks (Coppage and Duke, 1971)

and such inhibition is observed here in the present study.

However at day 30 the inhibitory activity was increased and came nearer to normal

level. Thus, the fish Cyprinus carpio fairly recovered from the inhibitory activity in the tissues

of fish. The concomitant recovery in AChE activity at day 30 might be due to active

metabolism of phorate which is being removed from the site of action and thus enabling the

enzyme to resume unhindered hydrolysis of Ach. Similar reports were also observed by

Coppage and Duke (1972) in fish brain exposed to malathion.

Maximum decrease in AChE activity in the brain of cyprinus carpio on exposure to

phorate toxicity may indicate disruption in the integratory activity of central nervous system.

The reports of Coppage, (1972) revealed that death occurs in fishes when AChE activity falls

below a critical level and according to Coppage et al., (1975) inhibition of brain AChE to the

level of 70 to 80% is critical to fishes. In the present study this critical situation was observed

in the fish at 4 days of exposure to acute toxicity of phorate. Probably the inhibition of

respiratory centre of the brain, and the inhibitory nature of pesticide may be responsible for

the decrease in this enzyme activity.

The rate of inhibition of AChE activity in the organs of animals exposed to pollutants

can be correlated to the concentration of pollutant and length of exposure (Coppage, 1972). It

was reported that, in general, inhibition of brain AChE activity is directly proportional to the

concentration of the pollutant (Coppage, 1971; Coppage, 1972; Coppage and Duke, 1972;

Macek et al., 1972). In the present study, suppression in AChE activity is more in the organs

of the fish exposed to acute toxicity of phorate than to chronic toxicity. Further within acute

toxicity, the suppression in the enzyme activity is more at 4 days of exposure than at 1 day and

it may be due to the availability of more pesticide for enzyme inhibition. This ultimately leads

to the suppression in nervous activity, osmo and ion regulatory activity as well as cellular

enzyme metabolic activity.

Thus the results obtained in the present study shows that the pesticide phorate is

interfering with the nervous system of the fish Cyprinus carpio by inhibiting the enzyme

acetylcholinesterase.


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Table-9

Acetylcholinesterase activity (µ moles of Ach hydrolysed /mg protein/hr) in different organs

of the fish Cyprinus carpio at different periods of exposure to acute and chronic toxicity of

phorate. The values below the mean are percent changes over the respective control.


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ORGAN

EXPOSURE PERIOD IN DAYS

ACUTE TOXICITY CHRONIC TOXICITY

CONTROL 1 4 CONTROL 1 7 15 30

GILL

Mean

S.D. ±

% change

328.760c

0.490

168.430b

0.397

-48.76

85.556a

0.490

-73.97

328.760d

0.490

386.775e

0.249

+17.64

265.407c

0.216

-19.27

220.532a

0.353

-32.92

257.221b

0.401

-21.76

LIVER

Mean

S.D. ±

% change

152.083c

0.610

91.468b

0.434

-39.85

46.055a

0.275

-69.71

152.083d

0.610

188.461e

0.296

+23.92

116.206b

0.165

-23.59

88.968a

0.289

-41.50

122.548c

0.194

-19.42

MUSCLE

Mean

S.D. ±

% change

706.148c

0.517

359.803b

0.324

-49.04

118.081a

0.508

-83.27

706.148d

0.517

846.530e

0.415

+19.88

583.560c

0.102

-17.36

513.440a

0.310

-27.29

561.175b

0.050

-20.53

KIDNEY

Mean

S.D. ±

% change

204.950c

0.469

123.750b

0.531

-39.61

68.106a

0.590

-66.76

204.950d

0.469

236.122e

0.328

+15.21

175.929c

0.244

-14.16

133.094a

0.205

-35.06

156.130b

0.247

-23.82

BRAIN

Mean

S.D. ±

% change

1080.418c

1.246

408.850b

0.526

-62.15

117.435a

0.451

-89.13

1080.418d

1.246

1398.060e

0.295

+29.40

807.720b

0.356

-25.24

567.651a

0.279

-47.46

928.403c

0.465

-14.07

All the values are mean ± SD of six individual observations. Values with different superscripts

with in the column are significantly different from each other at P<0.05 according to Duncan’s

Multiple Range Test (DMR) test.

Figure-8

Acetylcholinesterase activity (µ moles of Ach hydrolysed /mg protein/hr) in different organs

of the fish Cyprinus carpio at different periods of exposure to acute and chronic toxicity of

phorate.


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All the values are mean ± SD of six individual observations.


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Succinate dehydrogenase (SDH), Malate dehydrogenase (MDH) and Lactateate

dehydrogenase (LDH)

Enzymes are protein catalysts that accelerate the rates of biochemical reactions and

regulate metabolic pathways. The study of the various factors that influence the rate of

enzyme-catalyzed reactions is referred to as enzyme kinetics. Much of the pioneering work on

enzyme kinetics was done in the early 1900's by German biochemists Leonor Michaelis and

Maude L. Menten, who formulated a theory to explain the major events in enzyme catalysis.

The Michaelis-Menten theory assumes that the enzyme (E) and substrate (S) combine

reversibly to form an enzyme-substrate (ES) complex. The ES complex then breaks apart to

form free enzyme and the product (P).

Glycolysis is the metabolic pathway that converts glucose into pyruvate. The free

energy released in this process is used to form the high energy compounds of ATP and

NADH. It occurs, with variations, in nearly all organisms, both aerobic and anaerobic. It

occurs in the cytosol of the cell. The most common type of glycolysis is the Embden-

Meyerhof-Parnas (EMP pathway), which was first discovered by Gustav Embden, Otto

Meyerhof, and Jakub Karol Parnas. The citric acid cycle also known as the tricarboxylic acid

cycle (TCA cycle), is a series of chemical reactions used by all aerobic organisms to generate

energy through the oxidization of acetate derived from carbohydrates, fats and proteins into

carbon dioxide. In eukaryotic cells, the citric acid cycle occurs in the matrix of the

mitochondrion. The components and reactions of the citric acid cycle were established in the

1930s by the Nobel laureates Albert Szent-Gyorgyi and Hans Adolf Krebs.

The mitochondria contain the biochemical machinery for aerobic cellular respiration,

the process by which sugars, fatty acids, and amino acids are broken down to carbon dioxide

and water, with some of their chemical energy captured as adenosine triphosphate (ATP). A

key series of reactions in cell respiration is the Krebs (citric acid) cycle, a complex pathway

involving some enzymes and numerous metabolic intermediates. One of the best studied

enzymes in the Krebs cycle is succinate dehydrogenase (SDH).

Succinate dehydrogenase(SDH), a flevin linked enzyme of TCA cycle , catalyses the

reversible oxidation of succinate to fumerate and serves as a link between electron transport

and oxidative phosphorylation (Singer et al., 1973). It is the only enzyme that participates in

both the citric acid cycle and the electron transport chain. Since the activity of SDH in

mitochondria is greater than the other enzymes of TCA cycle, an insight into the alterations of

this enzyme activity may be taken as index to assess the function of TCA cycle in different


112

organs of the fresh water animals (Madanamohan Das and Venkatachari, 1984). SDH is

unique among the Krebs cycle enzymes in that it is tightly bound to the inner mitochondrial

membrane. Any alterations in its activity indicate changes in the structure and function of

mitochondria.

Malate dehydrogenase (MDH) is an enzyme present in the mitochondrial matrix and

cytosol catalyses the inter-conversion of malate-pyruvate as well as oxaloacetate-pyruvate.

MDH is found in all eukaryotic cells as two isozymes, mitochondrial (m-MDH) and

cytoplasmic (soluble, s-MDH). MDH belongs to the NAD-dependent dehydrogenases, which

are one of the largest and best-studied families of nucleotide-binding proteins, over 100

different members have already been identified.

Malate dehydrogenase (MDH) catalyzes the interconversion of L-malate and

oxaloacetate using nicotinamide adenine dinucleotide (NAD) as a coenzyme. The TCA cycle

is completed when the oxidation of L-Malic acid to oxaloacetic acid is accomplished by the

enzyme malate dehydrogenase. MDH is also involved in gluconeogenesis, the synthesis of

glucose from smaller molecules. Pyruvate in the mitochondria is acted upon by pyruvate

carboxylase to form oxaloacetate, a citric acid cycle intermediate. In order to get the

oxaloacetate out of the mitochondria, malate dehydrogenase reduces it to malate, and it then

traverses the inner mitochondrial membrane. Once in the cytosol, the malate is oxidized back

to oxaloacetate by cytosolic malate dehydrogenase. Finally, phosphoenolpyruvate

carboxykinase converts oxaloacetate to phosphoenolpyruvate.

Lactate dehydrogenase (LDH) is an enzyme present in a wide variety of organisms,

including animals. The glycolytic enzymes in the cell breakdown glucose resulting in the

formation of pyruvate. Further oxidation of pyruvate depends on the availability of oxygen. If

the availability of oxygen is less, the anaerobic condition leads to the conversion of pyruvate

to lactate. This reaction is catalysed by the enzyme lactateate dehydrogenase (LDH). It 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 fluctuation in the cellular

environment alters the activity of this enzyme. LDH is considered to be the most important

enzyme of the glycolytic pathway in animals including fishes.

Reports available on the effects of pesticides on different freshwater animals indicating

noticeable changes in the activities of SDH (Bergen, 1971; Koundinya and Ramamurthi,

1978; Siva Prasada Rao and Ramana Rao, 1979; Bhagyalakshmi et al., 1982; Guruprasad Rao


113

and Nanda Kumar, 1982; Satya Prasad, 1983; Swami et al., 1983; Sambasiva Rao et al., 1984;

Suneetha, 2012); MDH (Thakore et al., 1981; Kabeer Ahmed et al., 1983; Srinivas, 1993;

Mishra and Shukla, 2003; Suneetha, 2012); LDH (Harold Philip, 1984; Naidu, 1985; Prasad et

al., 1987; Ghosh,1989; Ravinder, 1989, David, 1995, Muralimohan, 2000; Hymavathi, 2001;

Suneetha, 2012); SDH and MDH (Srinivas Moorthy et al., 1986); SDH and LDH (Sastry and

Siddiqui, 1982); MDH and LDH (Tripathi and Shukla, 1990) and SDH, MDH and LDH

(Suneetha, 2012) .

Pesticides alter the mitochondrial structure and decrease the SDH activity in the organs

of many animals (Siva Prasada Rao and Ramana Rao, 1979; Bhagyalakshmi et al., 1982;

Swami et al., 1983). It has been reported that there was an increase in LDH activity

accompanied with the decrease in SDH in the organs of the freshwater animals exposed to

pesticide toxicity (Koundinya and Ramamurthi, 1978; Bhagyalakshmi et al., 1982). Sastry and

Siddiqui (1982) reported increase in LDH activity of liver and brain and decreases in succinic

dehydrogenase (SDH) activity of liver and brain of Channa punctatus exposed to sublethal

concnentrations of sevin.

Mishra and Shukla, (2003) reported that endosulfan treatment reduced the activity of

cytoplasmic malate dehydrogenase (cMDH) and mitochondrial malate dehydrogenase

(mMDH) in the muscle of the fish. Suneetha, (2012) observed a decrease in the SDH and

MDH activities in the brain, gill, kidney, liver and muscle of the freshwater fish Labeo rohita

after exposing to lethal and sublethal concentrations of two pesticides, endosulfan and

fenvalerate for 24 hrs and 15 days.

RESULTS

The data on the activities of succinate dehydrogenase (SDH), malate dehydrogenase

(MDH) and lactateate dehydrogenase (LDH) in the organs such as gills, liver, muscle, kidney

and brain of the fish cyprinus carpio at 1 and 4 days on exposure to acute toxicity of phorate

and 1, 7, 15 and 30 days on exposure to chronic toxicity of phorate, besides controls, are

presented in the Tables 10, 11 and 12. For comparison, the differences obtained in relation to

the controls of each parameter in each organ of the fish at the above said exposure periods in

acute and chronic toxicity study of phorate, were converted as percentages of the

corresponding controls and those percent values are also presented in the same tables and was

plotted a graph of percent changes against exposure periods in figures 9, 10 and 11.


114

Activity of Succinate dehydrogenase

From the data presented in the Table-10 and Figure-9 relative to controls, the activity

of SDH in all the organs of the fish exposed to phorate decreased at 1 and 4 days of exposure

in acute toxicity in the order of 1>4 and the differences in the activity between controls and

experimental were also found to be statistically significant (P<0.05). The decrease was more

at day 4 than at day 1 in all the organs of the fish. Suppression of this enzyme progressed from

day 1 to day 4 in all the organs of the fish exposed to acute toxicity of phorate. In the fish

exposed to chronic toxicity of phorate, in the activity of SDH from day 1 to day 7 there was

increase in the decrement but from day 7 to day 30 it was regressed in all the organs of the

fish in the order of 1>7<15<30.

Activity of Malate dehydrogenase

From the data presented in the Table-11 and Figure-10 relative to controls, the activity

of MDH in all the organs of the fish exposed to phorate decreased at 1 and 4 days of exposure

in acute toxicity in the order of 1>4 and the differences in the activity between controls and

experimental were also found to be statistically significant (P<0.05). The decrease was more

at day 4 than at day 1 in all the organs of the fish. Suppression of this enzyme progressed from

day 1 to day 4 in all the organs of the fish exposed to acute toxicity of phorate. In the fish

exposed to chronic toxicity of phorate, in the activity of SDH from day 1 to day 7 there was

increase in the decrement but from day 7 to day 30 it was regressed in all the organs of the

fish in the order of 1>7<15<30. All the values were found to be statistically significant

(P<0.05).

Activity of Lactate dehydrogenase

From the data presented in the Table-12 and Figure-11 relative to controls, an increase

was observed in the activity of LDH in all the organs of the fish exposed to both acute and

chronic toxicity of phorate. The LDH activity in the liver of both controls and experimental

fish is higher than in the other organs of the fish. The percent increase was also more in liver

when compared to the other organs of the fish exposed to both acute and chronic toxicity of

phorate. In the fish exposed to acute toxicity of phorate, in the activity of LDH, the increase

was more at day 4 than at day 1 in all the organs of the fish in the order of 1<4. In the fish

exposed to chronic toxicity of phorate, there was a gradual increase in the activity of LDH

from day 1 to day 7 but from the day 15 to day 30 a decrease in the increment was observed in

the order of 1<7>15>30.


115

DISCUSSION

The edible freshwater fishes constitute one of the major sources of nutritious food for

humans. Fish are largely being used for the assessment of the quality of aquatic environment

and as such can serve as bioindicators of environmental pollution. Among the aquatic species,

the fish are the major targets of toxicants contamination.

Analysis of biochemical parameters could help to identify the level of toxicity to target

organs as well as the general health status of animals. It may also provide an early warning

signal in stressed organism (Folmar, 1993). These parameters are the indicators of the

response of the animal to the environmental effects and can also serve as markers for toxicant

exposure and effect in fish. The biochemical parameters in fish are valid for

physiopathological evaluation and sensitive for detecting potential adverse effects and

relatively early events of pollutant, like pesticide damage (Almeida et al., 2002; Matos et al.,

2007; Osman et al., 2010).

Enzymes are biochemical macromolecules which control metabolic processes of

organisms, thus a slight variation in enzyme activity would affect the organisms (Roy, 2002).

Enzymatic activities also provide quick screening methods for assessing the health of fish and

can be used to determine the incipient lethal concentration of a toxicant. Therefore, by

estimating enzyme activities in an organism, it can be easily identify a disturbance in

metabolism. The studies related to the activities of enzymes in the presence of pollutants like

pesticides in water became a routine practice in clinical medicine to diagnose certain diseases

and the extent of tissue or organ damage (Racicool et al., 1975).

Stress is an energy demanding process and the animal mobilizes energy substrates to

cope with stress metabolically (Vijayan et al., 1997). Changes in the activities of the enzymes

like, succinate dehydrogenase, malate dehydrogenase and lactate dehydrogenase are sensitive

to environmental pollutants like pesticides (Devi, 2003).

In the present investigation the oxidative enzymes like succinate dehydrogenase

(SDH) and malate dehydrogenase (MDH) showed a reduction and elevation in non-oxidative

enzyme like LDH in their activity in all the osmoregulatory (gill and kidney) and non-

osmoregulatory (liver, muscle and brain) tissues of the fish Cyprinus carpio, which indicates

the suppression of oxidative metabolism in the fish exposed to acute and chronic toxicity of

phorate (Akhilender Naidu et al., 1984). The decrease in the MDH activity is in line with the

decreased SDH activity indicating suppressed oxidative metabolism. As SDH and MDH are


116

the oxidative enzymes involved in Kreb’s cycle, any disturbance in these enzyme activities

will affect the Kreb’s cycle. Since this cycle represents a central oxidative pathway for

carbohydrates, fats and amino acids, if there is any disturbance in this cycle the whole

metabolism is likely to be affected

In support of present investigation, several reports are available on a decrease in the

activity of SDH and MDH and increase in the LDH activity after exposingto different

pesticides. It has been reported earlier by several investigators that pesticides alter the

mitochondrial structure and decrease the SDH activity in the organs of many animals (Bergen,

1971; Koundinya and Ramamurthi, 1978; Siva Prasada Rao and Ramana Rao, 1979;

Bhagyalakshmi et al., 1982; Guruprasad Rao and Nanda Kumar, 1982; Swami et al., 1983).

Suneetha, (2012) observed a decrease in the SDH activity in the brain, gill, kidney,

liver and muscle of the freshwater fish Labeo rohita after exposing to lethal and sublethal

concentrations of two pesticides, endosulfan and fenvalerate for 24 hrs and 15 days. Khemani

et al., (1989) reported a significant inhibition in the SDH activity in the tissues of rat treated

with dieldrin and suggested that one of the reasons for the observed inhibition of SDH activity

could be diminished availability of the soluble cofactors within the subcellular structures.

Several reports are available on the decrease in SDH activity with the increase in LDH

activity accompanied in the organs of the freshwater animals exposed to pesticide toxicity

(Koundinya and Ramamurthi, 1978; Siva Prasad Rao and Ramana Rao, 1979; Dayananda

Reddy, 1980; Bhagyalakshmi et al., 1982).

Sastry and Siddiqui (1982) reported increases in LDH activity of liver and brain and

decreases in succinic dehydrogenase (SDH) activity of liver and brain of Channa punctatus

exposed to sublethal concnentrations of sevin. Ghosh (1987) reported that there was an

increased activity of lactic dehydrogenase (LDH) in brain, liver and muscle and decreased

activity in kidney and intestine of Clarias batrachus when exposed to sublethal concentrations

of Tara 909, Suquin and Croton 36.

Decrease in the MDH activity was also reported by several investigators. A decrease in

the activity of MDH was observed by Thakore et al., (1981) in the liver of BHC treated mice.

The MDH activity in the tissues of the fish Tilapia mossambica was decreased on exposure to

malathion (Kabeer Ahmed et al., 1983). Srinivas, (1993) studied the impact of endosulfan on

carbohydrate metabolism in the fresh water fish Clarias batrachus and reported decreased

MDH activity. Mishra and Shukla, (2003) studied endosulfan effects on muscle malate

dehydrogenase of the freshwater catfish Clarias batrachus and reported that endosulfan


117

treatment reduced the activity of cytoplasmic malate dehydrogenase (cMDH) and

mitochondrial malate dehydrogenase (mMDH) in the muscle of the fish. Suneetha, (2012) also

observed a decrease in the MDH activity in the brain, gill, kidney, liver and muscle of the

freshwater fish Labeo rohita after exposing to lethal and sublethal concentrations of two

pesticides, endosulfan and fenvalerate for 24 hrs and 15 days.

It is known that SDH and MDH acts as indictors of aerobic respiration, their inhibition

and with subsequent elevation of LDH indicates the prevalence of anaerobic conditions

imposed by the stress factor of phorate toxicity. As SDH and MDH are the key enzymes in

TCA cycle, it is logical to assume that with the inhibition of SDH and MDH and elevation of

LDH activities, the metabolic pathway might have turned to anaerobic to meet the increased

energy demands during the phorate exerted toxic stress.

The decrease in SDH and MDH activity also indicates the impairment of oxidative

metabolism in the mitochondria as a consequence of hypoxic conditions under pesticide

exposure, most probably by disrupting the oxygen binding capacity of the respiratory pigment.

The decrease in SDH and MDH activity may be due to the disorganization of mitochondria

affecting enzymes of TCA cycle. The decrease in these enzyme activities might be probably

due to mitochondrial damage and decreased state of respiration (Shomessubra Bag et al.,

1999). The fall in these enzyme activities might be related to the close contact of pesticide

with cell organelle and their subsequent disorganization accompanied by increased

histopathology of gill area and shifting of the aerobic to anaerobic metabolism as reported in

other teleosts (Ramalingam and Reddy, 1982; Shaffi, 1995). Any alteration in the respiratory

area decrease the oxygen absorption capacity of the gill due to its close contact with polluted

water (Hughes and Morgan, 1973; Singh and Sing, 1979). It may also be one of the reasons

for the diminished activity of SDH and MDH.

Nagarathnamma, (1982) and Srinivasulu Reddy and Ramana Rao, (1986) reported that

disruption in gill lamellae caused by the organophosphate pesticides adversely affected the

absorption of oxygen from the surrounding medium in fish and prawn respectively. Decrease

in SDH and MDH activity may be due to damage in the mitochondrial structural integrity in

the organs of the fish (Bergen, 1971; Satya Prasad, 1983). The decrease in the activity of

MDH may suggests the lower level of functioning of TCA cycle due to inadequate supply of

substrate or decreased oxygen uptake at the tissue level during phorate toxicity stress. Another

probable reason for the low level of MDH activity may be the reduced SDH activity which

inturn lowers fumarate-malate conversions.


118

The pesticide induced suppression in the activity of SDH and MDH in all the organs of

the fish can be correlated to the binding of pesticide to the active sites of the oxidative

enzymes and/or impairment in mitochondrial organization (Viarengo, 1985; Hodson, 1988). In

the present study such impairment might have gradually increased with the increase in the

period of exposure in chronic toxicity study upto day 7 due to slow accumulation of pesticide

in the tissues of the fish. Decrease in the activity of SDH and MDH stimulated the fish to

switch over from aerobic to anaerobic glycolysis. Hence an increase in the activity of LDH

was observed. This indicates the fish might have relied more on anaerobic glycolysis in

meeting the energy requirements, as the energetically more efficient oxidative metabolism is

suppressed.

The decreased activities of SDH and MDH observed in the tissues of the fish may be

attributed to the hypoxic conditions or to the inadequate substrate availability or to the

decreased oxygen absorption potential of the tissues. Thus fish have relied more on anaerobic

glycolysis in meeting the increased energy demands during the phorate exerted toxic stress,

thus the elevation of LDH activity was in the tissues of the fish.

May be due to the domination of detoxification over accumulation, the fish slowly

regained the oxidative metabolic activity by activating the SDH and MDH from day 15 to day

30. The suppression in the activities of SDH and MDH and the elevation in the LDH activity

was decreased slowly at day 15 and day 30 in the tissues of the fish on exposure to chronic

toxicity of phorate. The fish might have relied both on energetically more efficient oxidative

metabolism and less efficient anaerobic glycolysis during this period of exposure as more

energy is required either for detoxification or for the degradation of pesticide. So on prolonged

exposure to chronic toxicity of phorate the fish could develop resistance to phorate and could

adapt slowly to the new enivironment (Vijayaram et al., 1989).

More suppression in SDH and MDH activities and subsequent elevation in the activity

of LDH in the liver might be greater interference of pesticide with the activities of oxidative

metabolic enzymes in liver as the liver is important centre for metabolism, inter conversion

and detoxification as it plays a vital role during toxic stress in fishes. There is the possibility

of greater interference of the pesticide in the gill with the activities of oxidative metabolic

enzymes. This is the reason for the greater suppression in SDH and MDH activities with the

elevation in the activity of LDH in the gill. Further, the suppression of SDH and MDH

activities and elevation of LDH activity identified in the muscle, as glycolysis and glycolytic

enzymes are dominant in muscle. The decrease in oxidative metabolism with the suppression


119

of SDH and MDH activities and accumulation of lactate with the elevation of LDH activity in

the muscle of the fish exposed to toxic stress could be one of the reasons for inactive state of

the fish. Stimulation of oxidative metabolism and glycolytic activity in this organ of fish on

prolonged exposure to phorate toxicity indicates the elevation in the muscular activity of this

organ, by which the fish is made possible to adapt to the toxic stress. Kidney is being one of

the important organs of the osmoregulation, suppression of oxidative metabolism leads to a

great set-back in its osmo and ion regulatory function. The suppression of SDH and MDH

activities in the kidney indicates less availability of energy to perform its energetically

expensive oxidative metabolism.

On prolonged exposure the decrease in the suppression of SDH and MDH activities

and also in the elevation of LDH activity in the fish exposed to phorate toxicity might be due

to the gradual restoration of its normal metabolic activities, gradual recovery of the oxidative

metabolic activity, the fish gained tolerance to the sublethal stress of phorate and the increase

in oxidative and glycolytic cycles. This indicate the active role of the fish in controlling and

coordinating the metabolic disturbances occurred during phorate toxic stress on prolonged

exposure. It could be due to the activation of detoxification, degradation and pesticide

eliminating pathways.

Thus on overall assessment the results of this study show that the fish Cyprinus carpio

were stressed after being exposed to acute and chronic toxicity of phorate. The pesticide

phorate altered the activity of the carbohydrate metabolic enzymes significantly thus result in

the instable physiological state of the fish. Therefore, lethal and sublethal concentrations of

phorate have some deleterious effect on the basic activities of enzymes of carbohydrate

metabolism in the gill, liver, muscle, kidney and brain of the experimental fish, Cyprinus

carpio. On comparison, in the activities of enzymes, the suppression in SDH and MDH

activities and the elevation in LDH activity is more in the tissues of the fish exposed to acute

toxicity of phorate due to more pesticidal stress.

It may be concluded that the phorate induced alterations in the activities of

carbohydrate metabolic enzymes caused significant metabolic effect on the physiological

consequences. The physiological conditions are directly related to the bioavailability of the

pesticide. The alteration in these enzyme activities can be taken as good markers or indicators

of the pesticide induced stress. These biochemical parameters offer a rapid and sensitive

means of monitoring towards the impact of pesticides on aquatic biota and ultimately whole of

the ecosystem.


120

Table-10

Succinate dehydrogenase activity (µ moles of formazone formed/mg protein/hr) in different

organs of the fish Cyprinus carpio at different periods of exposure to acute and chronic

toxicity of phorate. The values below the mean are percent changes over the respective

control.

ORGAN




GILL

Mean

S.D. ±

% change

0.174c

0.0074

0.124b

0.0091

-28.27

0.083a

0.0044

-51.91

0.174d

0.0074

0.096a

0.0013

-44.82

0.092a

0.0016

-47.12

0.122b

0.0018

-29.88

0.155c

0.0029

-10.80

LIVER

Mean

S.D. ±

% change

0.579c

0.0065

0.295b

0.0034

-48.97

0.173a

0.0030

-70.00

0.579e

0.0065

0.248b

0.0025

-57.16

0.216a

0.0013

-62.57

0.312c

0.0021

-46.12

0.403d

0.0047

-30.31

MUSCLE

Mean

S.D. ±

% change

0.152c

0.0047

0.095b

0.0026

-37.38

0.051a

0.0037

-66.36

0.152e

0.0047

0.098c

0.0016

-35.67

0.073a

0.0013

-51.53

0.091b

0.0017

-40.21

0.116d

0.0021

-23.78

KIDNEY

Mean

S.D. ±

% change

0.146c

0.0037

0.120b

0.0051

-17.67

0.073a

0.0032

-49.59

0.146e

0.0037

0.101b

0.0034

-30.82

0.094a

0.0015

-35.47

0.107c

0.0023

-26.57

0.127d

0.0017

-12.86

BRAIN

Mean

S.D. ±

% change

0.189c

0.0034

0.154b

0.0029

-18.60

0.115a

0.0038

-38.79

0.189e

0.0034

0.137c

0.0013

-27.48

0.108a

0.0011

-42.70

0.128b

0.0021

-31.84

0.152d

0.0024

-19.66




Table-11

Malate dehydrogenase activity (µ moles of formazone formed/mg protein/hr) in different



control.

ORGAN




GILL

Mean

S.D. ±

0.253c

0.0029

0.178b

0.0031

0.157a

0.0032

0.253e

0.0029

0.205c

0.0013

0.181a

0.0021

0.198b

0.0016

0.225d

0.0037


121

% change -29.26 -37.78 -18.65 -28.18 -21.70 -10.88

LIVER

Mean

S.D. ±

% change

0.373c

0.0036

0.185b

0.0043

-49.76

0.145a

0.0050

-61.01

0.373e

0.0036

0.281c

0.0021

-18.03

0.188a

0.0018

-49.42

0.277b

0.0025

-25.56

0.315d

0.0043

-15.54

MUSCLE

Mean

S.D. ±

% change

0.128c

0.0041

0.113b

0.0025

-11.70

0.082a

0.0042

-35.85

0.128e

0.0041

0.114c

0.0029

-11.07

0.087a

0.0013

-31.51

0.095b

0.0018

-25.27

0.116d

0.036

-9.51

KIDNEY

Mean

S.D. ±

% change

0.165c

0.0051

0.128b

0.0037

-22.53

0.113a

0.0031

-31.50

0.165d

0.0051

0.133b

0.0015

-19.15

0.121a

0.0013

-26.70

0.148c

0.0021

-10.45

0.150c

0.0017

-9.06

BRAIN

Mean

S.D. ±

% change

0.193c

0.0032

0.140b

0.0053

-27.41

0.132a

0.0043

-31.29

0.193e

0.0032

0.152b

0.0016

-21.36

0.148a

0.0018

-23.27

0.162c

0.0036

-16.19

0.178d

0.0021

-7.81




Table-12

Lactate dehydrogenase activity (µ moles of formazone formed/mg protein/hr) in different



control.

ORGAN




GILL

Mean

S.D. ±

% change

0.183a

0.0026

0.234b

0.0069

+28.15

0.308c

0.0028

+68.61

0.183a

0.0026

0.219c

0.0029

+20.01

0.244e

0.0025

+33.54

0.229d

0.0034

+25.45

0.206b

0.0017

+12.99

LIVER

Mean

S.D. ±

% change

0.348a

0.0344

0.472b

0.0125

+35.91

0.604c

0.0042

+73.65

0.348a

0.0344

0.477d

0.0030

+37.20

0.521e

0.0026

+49.76

0.446c

0.0026

+28.40

0.424b

0.0017

21.95

MUSCLE

Mean

S.D. ±

% change

0.162a

0.0039

0.233b

0.0032

+44.05

0.263c

0.0062

+62.37

0.162a

0.0039

0.203c

0.0034

+25.44

0.223e

0.0028

+38.10

0.212d

0.0021

+31.19

0.193b

0.0020

+19.32

KIDNEY

Mean

S.D. ±

% change

0.121a

0.0027

0.153b

0.0060

+26.54

0.167c

0.0034

+38.55

0.121a

0.0027

0.140c

0.0021

+16.08

0.153e

0.0023

+26.81

0.146d

0.0017

+21.37

0.134b

0.0018

+11.55


122

BRAIN

Mean

S.D. ±

% change

0.212a

0.0063

0.277b

0.0079

+30.89

0.304c

0.0035

+43.54

0.212a

0.0063

0.250c

0.0028

+18.19

0.277e

0.0030

+30.94

0.259d

0.0016

+22.48

0.234b

0.0020

+10.84




Figure-9

Succinate dehydrogenase activity (µ moles of formazone formed/mg protein/hr) in different


toxicity of phorate.


Figure-10

Malate dehydrogenase activity (µ moles of formazone formed/mg protein/hr) in different




123


Figure-11

Lactate dehydrogenase activity (µ moles of formazone formed/mg protein/hr) in different




124


chapter - v enzymatic activities chapter -...

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