87 Chapter 6 BIOCHEMICAL CHANGES INTRODUCTION Recent evidence indicates that fish, an extremely valuable resource, are quickly becoming scarce. One consequence of this scarcity is the increasing concern for fish survival and a growing interest in identifying the levels of various chemical pollutants, which are safe for fish and other aquatic life. The acetyl cholinesterase (AChE) activity is vital to normal behavior and muscular function and represents a prime target on which some toxicants exert adverse effects. Inhibition of acetylcholinesterase (AChE), the enzyme involved in terminating the action of neurotransmitter acetylcholine (ACh), is perhaps the most often studied. The two main transmitter substances in vertebrate’s nervous systems are ACh and noradrenaline. Acetylcholine is an ammonium compound. It was the first transmitter substance to be isolated in 1920. Neurons releasing acetylcholine are described as cholinergic neurons and those releasing noradrenaline are described as adrenergic neurons. The arrivals of nerve impulses at the synaptic knob depolarize the presynaptic membrane, causing calcium channels to open. As the calcium ions rush into the synaptic knob they cause synaptic vesicles to fuse with the presynaptic membrane, releasing their level into the synaptic cleft (exocytosis). The vesicles then return to the cytoplasm where they are refilled with the transmitter substance, acetylcholine (Fukuta, 1990).
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87
Chapter 6
BIOCHEMICAL CHANGES
INTRODUCTION
Recent evidence indicates that fish, an extremely valuable resource, are
quickly becoming scarce. One consequence of this scarcity is the increasing
concern for fish survival and a growing interest in identifying the levels of
various chemical pollutants, which are safe for fish and other aquatic life. The
acetyl cholinesterase (AChE) activity is vital to normal behavior and muscular
function and represents a prime target on which some toxicants exert adverse
effects. Inhibition of acetylcholinesterase (AChE), the enzyme involved in
terminating the action of neurotransmitter acetylcholine (ACh), is perhaps the
most often studied. The two main transmitter substances in vertebrate’s
nervous systems are ACh and noradrenaline. Acetylcholine is an ammonium
compound. It was the first transmitter substance to be isolated in 1920.
Neurons releasing acetylcholine are described as cholinergic neurons and
those releasing noradrenaline are described as adrenergic neurons. The
arrivals of nerve impulses at the synaptic knob depolarize the presynaptic
membrane, causing calcium channels to open. As the calcium ions rush into
the synaptic knob they cause synaptic vesicles to fuse with the presynaptic
membrane, releasing their level into the synaptic cleft (exocytosis). The
vesicles then return to the cytoplasm where they are refilled with the
Acetylcholine (ACh) is the only classical neurotransmitter that after
release into the synaptic cleft is inactivated by enzymatic hydrolysis, rather
than by reuptake (consequently, ACh has a turnover rate in vivo that is much
higher than that of any other transmitter, including catecholamines and
amino acids (Haubrich and Chippendale, 1977).
Acetylcholinesterase (AChE, E.C. 3.1.1.7) 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 (Soreq and Seidman, 2001; Silman and
Sussman, 2005). Acetylcholine diffuses across the synaptic cleft, creating a
delay of about 0.5 ms (milliseconds) and attaches to a specific receptor site (a
protein) on the postsynaptic membrane that recognizes the molecular
structure of the acetylcholine molecules. The arrival of the acetylcholine
causes a change in the shape of the receptor site, which results in ion channels
opening up in the postsynaptic membrane. The possible hazard of AChE
inhibiting pesticides in the aquatic environment should not be ignored. Since
these pesticides act as a nerve poison (Coppage and Braidech, 1976). Aquatic
organism exhibit a broad range of inhibitory response to pesticides depending
on the compound, exposure time, water conditions and species (Coppage and
Matehws, 1974)
From the nineteenth century until the 1970s, only pyrethrum mixtures
obtained by solvent extraction of pyrethrum flowers (usually chrysanthemum
89
cineraraefolum) were available for use. However, the development by Martin
Elliott of cheaper and lighter stable synthetic pyrethroids from 1970s led to
their becoming a major pesticide class. Over 1000 pyrethroid structures have
been synthesized and cypermethrin was the most widely used single
pesticide in 2002 globally. The widespread use of the cypermethrin in
agricultural and public health applications is based upon their toxicity to
nontarget organisms. Cypermethrin was used as a chemotherapeutic agent
for the control of ectoparasite infestations (Lepeoptheirus salmonis and Caligus
elongatus) in marine cage culture of Atlantic salmon, Salmo salar (Boxaspen
and Holm, 2001). This resulted in its discharge into the aquatic environment
and consequently several lab studies were conducted, which showed that
cypermethrin was extremely toxic to fish at low concentrations with 96-
hLC50. This is explained due to the poor ability of fish to rapidly degrade and
metabolize this pyrethroid (David, et. al, 2004).
The literature available put forth by several researchers (Rainsford,
1978; Kabeer Ahammad Sahib and Ramana Rao, 1980; Shashikala, 1992;
Manju Singh and Santhakumar, 2000; Parma, et. al., 2002) explain the
inhibition of acetylcholinesterase during the pesticide exposure. The
relationship between the concentration of organophosphates and the
biochemical effects on the acetylcholine (ACh) and acetylcholinesterase
(AChE) are well documented.
A few experiments were carried out earlier to determine the effects of
cypermethrin on AChE and ATPase systems and certain biochemical
90
parameters in Cyprinus carpio (David, et. al, 2004). Aysel and Karasu (2005)
also studied the effect of cypermethrin on glycogen and lipid level of
freshwater fish, L. thermalis. Recently, Marigoudar, et. al., (2009) shown that
cypermethrin inhibits AChE activity at sublethal concentration in functionally
different organs of Labeo rohita. Contamination of aquatic ecosystems with
sublethal levels of cypermethrin is common and had serious impacts on
nontarget fish, Labeo rohita. AChE activity is a biomarker used in aquatic
ecotoxicology studies (Kirby, et. al, 2000) and sensitive enzyme to low
environmental contaminants exposure.
In view of this, the objective of the present investigation was to
determine the acute and subacute effects of cypermethrin on AChE activity
and ACh level of gill, liver and muscle in L. rohita at lethal and sublethal
concentration and related effects from this exposure as a way to establish
toxicity risk of cypermethrin exposure in this test species.
RESULTS
ACh accumulation
In the control fish tissue, maximum quantity of ACh was observed in
brain followed by muscle, gill and liver (Table and figure). The accumulation
of ACh under the median lethal concentration of cypermethrin increased
gradually up to 96 h in all the tissues namely gill, muscle and liver. Liver
recorded the lowest concentration 18.28 µM/g wet wt., which is 9.11 percent
over control at 96 h. A maximum increase of 55.41% was noted in the gill
tissue at 72 h of exposure. ACh level recorded decrease in all the tissues at 96
91
h under lethal concentration. During the median lethal concentration an
overall maximum increase was observed in gill and a minimum was noted in
liver.
In the experimental fish under sublethal exposure very high quantity
of ACh in muscle on 10th day of exposure (12.15%) and lowest increase over
control on day 1 in muscle (3.1381%). ACh level showed a continuous increase
in gill, muscle and liver up to 10th day while the subsequent, day 15 recorded
a low per cent increase. In the whole experiment liver showed minimum
change, while brain showed maximum ACh level.
AChE activity
The decrease in AChE activity was more pronounced in the liver tissue
followed by gill and muscle in the fish exposed to lethal concentrations of
cypermethrin (Table 7 and figure 4). Maximum percent inhibition in the
AChE activity was noted in liver at 72 h (-30.44%) and minimum percent
inhibition was observed in muscle as compared to control at 24 h (-1.98%).
While gill, muscle and liver exhibited continuous decrease in activity up to 72
h, while at 96 h witnessed decrease in the inhibitory activity in the AChE. In
sublethal concentrations the data presented in table 8 and figure 5 revealed
maximum percent inhibition of AChE activity in liver (-18.3862%) followed by
gill and muscle on day 15 in the whole experiment.
Discussion
92
In the present study, the results showed a time- and concentration-
dependent inhibition of AChE activity by cypermethrin in the tissues of the
fish, L. rohita (Table 8 and Figure 5). Inconsonance with the decrease in the
AChE activity there is a corresponding increase in the ACh content of the
tissues (Table 7 and Figure 4) suggesting decrease in the cholinergic
transmission and consequent accumulation of ACh in the tissues. At lethal
and sublethal concentrations, cypermethrin produced greater inhibition of
AChE activity in gill, liver and muscle tissues. Further, these effects are seen
following both acute and sub acute conditions. Inhibition of AChE results in
nerve impulses as nerves become permeable to sodium, allowing sodium to
flow into the nerve. Pyrethroids delay the closing of the gate that allows
sodium flow (Vijverberg and Van den Bercken, 1990) and thus, multiple nerve
impulses rather than the usual single one occur. In turn, these impulses
release the neurotransmitter ACh, which stimulates other nerves (Eells, 1992);
ultimately resulting in buildup of ACh within the nerve synapses leading to a
variety of neurotoxic effects and decreased cholinergic transmission (Mileson,
et. al, 1998). Similar results were obtained in tissues and other fish species
(Rao, 2006; Chawanrat, et. al, 2007; Elif and Demet, 2007). Cypermethrin also
affects the enzyme ATPase involved in cellular energy production, transport
of metal atoms and muscle contraction (El-Toukhy and Girgis, 1993).
A similar corroborative increase in the ACh content consequent to a
decrease in the tissue AChE levels was reported in fish, Tilapia mossambica
exposed to malathion for 48 h (Kabeer Ahammad Sahib and Ramana Rao,
93
1980). Manju and Santosh (2000) reported decrease in acetylcholinesterase
activity subjected to sub chronic and acute exposure to malathion in
freshwater teleost, Catla catla. Parma, et. al, (2002), reported similar decrease in
the AChE activity under acute toxicity of monocrotophos in a Neotropical
fish, Prochilodus lineatus. Rao et al., (2003) and Rao, (2006) observed similar
inhibition of AChE activity in the fish, Tilapia moosambica exposed to
chlorpyrifos and RPR-V respectively.
The pyrethroids are neurotoxic and can affect neurotransmitters.
Pesticides bind with the active site and prevent breakdown of ACh resulting
blocking of synaptic transmission in cholinergic nerves. Neurotransmitters
needed to continue the passage of nerve impulses from one nerve cell to
another across the synaptic gap. AChE functions to deactivate ACh almost
immediately by breaking it down. Nerve impulses cannot be stopped if AChE
is inhibited and ACh accumulates causing prolonged muscle contraction,
consequently paralysis occurs and death may result.
It is also known pyrethroid compound fenvelarate which inhibit AChE
activity were known to disrupt the normal behavioral patterns in the effected
animals (Mushigeri and David, 2005). The behavioral changes observed in the
intoxicated animals like repeated opening and closing of opercular covering,
hyper-extension of all fins, cock-screw swimming, S-jerks, coughing, burst-
swimming is directly related to the inhibition of peripheral and or central
nervous system due to inhibition of cholinesterase activity (Kurtz, 1977).
Guilbault (1972) has demonstrated the inhibitory effect of 19 pesticides on the
94
cholinesterase activity of lake trout. The abnormalities in fish behaviour
observed in this study could be related to the inhibitory action of
cypermethrin on AChE and subsequent accumulation of ACh at the nerve
endings. Results obtained by different workers, independently of tissues,
methodologies and species used are quite similar in the AChE inhibitory
effects.
Inhibition of AChE activity in functionally vital organs like gill, muscle
and liver lead to impaired critical neurphysiological activity and block
sodium channels of nerve filaments, thereby lengthening the depolarization
phase. Further, cypermethrin affects the GABA receptors in the nerve
filaments (Bradbury and Coats, 1989) and other related processes. In addition,
the reduction in AChE activity and ACh levels may be attributed to in vivo
biotransformation of sequestered cypermethrin in the storage organs.
Table 7: ACh level (µM/g wet wt.) in the tissues of the fish, Labeo rohita on exposure to the lethal and sublethal concentrations of cypermethrin.
Means are ± SD (n = 6) for a parameter in a row followed by the same letter are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range test.
Means are ± SD (n = 6) for a parameter in a row followed by the same letter are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range test.
Figure 4: Percent change over control in ACh level (µM/g wet wt.) in the tissues of the fish, Labeo rohita on exposure to the
lethal and sublethal concentrations of cypermethrin
0
10
20
30
40
50
60
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) sublethal (days) Exposure periods
Gill Muscle Liver
Figure 5: Percent change over control in AChE activity (µM of acetylcholine hydrolyzed/mg protein/h) in the tissues of the fish,
Labeo rohita on exposure to the lethal and sublethal concentrations of cypermethrin
-35
-30
-25
-20
-15
-10
-5
0
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Muscle Liver
95
INTRODUCTION
The biological response of an organism to xenobiotics following
absorption and distribution starts with toxicant induced changes at the
cellular and biochemical levels, leading to changes in the structure and
function of the cells, tissues, physiology and behaviour of the organism. These
changes can perhaps ultimately affect the integrity of the population and
Means are SD (n=6) for tissues in a row followed by the same letter are not significantly different (P 0.05) from each other
according to Duncan’s multiple range (DMR) test.
Table 15: Protease activity (M amino acid nitrogen / mg protein / h) in the organs of fish, Labeo rohita on exposure to the lethal and sub lethal concentrations of cypermethrin.
Means are SD (n=6) for tissues in a row followed by the same letter are not significantly different (P 0.05) from each other
according to Duncan’s multiple range (DMR) test.
Fig 6: Percent change over control in catalase activity in the tissues of Labeo rohita following exposure to lethal and sublethal concentrations of cypermethrin
0
10
20
30
40
50
60
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig 7: Percent change over control in hydrogen peroxide content in the tissues of Labeo rohita following exposure to lethal and
sublethal concentrations of cypermethrin
0
10
20
30
40
50
60
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig 8: Percent change over control in MDA levels in the tissues of Labeo rohita following exposure to lethal and sublethal
concentrations of cypermethrin
0
5
10
15
20
25
30
35
40
45
50
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig 9: Percent change over control in protein carbonyl levels in the tissues of Labeo rohita following exposure to lethal and
sublethal concentrations of cypermethrin
0
5
10
15
20
25
30
35
40
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig 10: Percent change over control in total protein levels in the tissues of Labeo rohita following exposure to lethal and
sublethal concentrations of cypermethrin
-50
-45
-40
-35
-30
-25
-20
-15
-10
-5
0
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig 11: Percent change over control in free amino acid levels in the tissues of Labeo rohita following exposure to lethal and
sublethal concentrations of cypermethrin
0
10
20
30
40
50
60
70
80
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig 12: Percent change over control in protease activity in the tissues of Labeo rohita following exposure to lethal and sublethal
concentrations of cypermethrin.
0
10
20
30
40
50
60
70
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
114
Ions and associated ATPases
INTRODUCTION
There are four possible mechanisms of neurotoxicity of pesticides (1)
interaction with Na+ channels on nerve cell membranes; (2) disruption of K+
membrane permeability in nerve cells; (3) inhibition of Na+, K+-ATPase,
Mg2+- ATPase, Ca2+, Mg+-ATPase, and/or Ca2+-ATPase; and (4) inhibition
of ionic channels. ATPases are enzymes concerned with immediate release of
energy and are responsible for a large part of basic metabolic and
physiological activities. ATPase activity can be taken as meaningful indicator
of cellular activity and forms a useful toxicological tool (Rahman, et. al, 2000).
Several pesticides are known to alter the activities of adenosine
triphosphatases (ATPases), which are integral parts of active transport
mechanisms for cations across the cell membrane (Das and Mukherjee, 2000;
Means are SD (n=6) for a tissue in a row followed by the same letter are not significantly different (P 0.05) from each other according to Duncun's multiple range (DMR) test.
Table 17: Potassium ion levels (M/g wet wt) in the organs of fish, Labeo rohita on exposure to the lethal and
Means are SD (n=6) for a tissue in a row followed by the same letter are not significantly different (P 0.05) from each other according to Duncun's multiple range (DMR) test.
Table 19: Na+-K ATPase activity (M of Pi formed / mg protein / h) in the organs of fish, Labeo rohita on exposure to
the lethal and sublethal concentrations of cypermethrin.
Means are SD (n=6) for a tissue in a row followed by the same letter are not significantly different (P 0.05) from each other according to Duncun's multiple range (DMR) test.
Table 20: Mg2+ ATPase activity (M of Pi formed / mg protein / h) in the organs of fish, Labeo rohita on exposure to the
lethal and sublethal concentrations of cypermethrin.
Means are SD (n=6) for a tissue in a row followed by the same letter are not significantly different (P 0.05) from each other according to Duncun's multiple range (DMR) test.
Table 21: Ca2+ ATPase activity (M of Pi formed / mg protein / h ) in the organs of fish, Labeo rohita on exposure to
the lethal and sublethal concentrations of cypermethrin.
Means are SD (n=6) for a tissue in a row followed by the same letter are not significantly different (P 0.05) from each other according to Duncun's multiple range (DMR) test.
Fig. 13. Percent change in sodium ion levels in the organs of Labeo rohita on exposure to the lethal and sublethal
concentrations of cypermethrin.
-60
-50
-40
-30
-20
-10
0
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig. 14. Percent change in potassium ion levels in the organs of Labeo rohita on exposure to the lethal and sublethal
concentrations of cypermethrin.
-60
-50
-40
-30
-20
-10
0
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig. 15. Percent change in calcium ion levels in the organs of Labeo rohita on exposure to the lethal and sublethal
concentrations of cypermethrin.
-70
-60
-50
-40
-30
-20
-10
0
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig. 16. Percent change in Na+-K ATPase activity in the organs of Labeo rohita on exposure to the lethal and sublethal
concentrations of cypermethrin.
-70
-60
-50
-40
-30
-20
-10
0
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig. 17. Percent change in Mg2+ ATPase activity in the organs of Labeo rohita on exposure to the lethal and sublethal
concentrations of cypermethrin.
-70
-60
-50
-40
-30
-20
-10
0
24 48 72 96 1 5 10 15
perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig. 18. Percent change in Ca2+ ATPase activity in the organs of Labeo rohita on exposure to the lethal and sublethal
concentrations of cypermethrin.
-60
-50
-40
-30
-20
-10
0
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal Sublethal Exposure periods
Gill Liver Muscle
130
Protein metabolism
INTRODUCTION
Proteins are the most versatile macromolecules in living systems and
serve crucial functions in essentially all biological processes. They function as
catalysts, they transport and store other molecules such as oxygen, they
provide mechanical support and immune protection, they generate
movement, they transmit nerve impulses and they control growth and
differentiation (Berg, et. al, 2005). They are the ubiquitous macromolecules in
a biological system and are the derivatives of high molecular weight
polypeptides. They not only serve as a fuel to yield energy but also play a
vital role in the structural and functional characteristics of the living things.
Functionally, proteins exhibit a great diversity, constitute a heterogeneous
group, having diverse physiological functions and are involved in major
physiological events (Lehninger, 1984). Therefore, the assessment of the
protein content can be considered as a diagnostic tool to determine the
physiological phases of organisms (Kapila and Ragathaman, 1999). The
concentration of proteins in a tissue is a balance between the rate of their
synthesis and degradation or catabolism (Schimke, 1974); the overall protein
turnover in an animal is the dynamic equilibrium between these two (Grainde
and Seglen, 1981; Tavill and Cooksley, 1983).
Hydrolysis of proteins is a quite common phenomenon wherein
proteases split proteins stepwise into amino acids. Among the proteases
described in the literature, some are lysosomal in origin having acidic pH
131
optima. Some are found in association with peroxisomes, lysosomes and
mitochondria possessing neutral pH optima and other proteases with alkaline
pH optima are reported in the cytosolic fraction. Thus, the proteases are of
acidic, neutral and alkaline in nature based on their specificity in action with
reference to optimum hydrogen ion concentration. Amino acids formed by
protein degradation will also be utilized for energy production. Umminger
(1979) suggested that through carbohydrates represent the principle
immediate energy precursors, for fishes subjected to stress, proteins are the
major source during chronic conditions.
Aminotransferases play a principal role in the catabolism of amino
acids and are the key enzymes of nitrogen metabolism. Calabrese, et. al,
(1977) pointed that, aminotransfersaes are important in energy mobilization.
Out of all aminotransferases, aspartate aminotransferase (AAT) catalyses the
inter-conversion of aspartic acid and ketoglutaric acid to oxaloacetic acid and
glutamic acid, while alanine aminotransferase (ALAT) catalyses the inter-
conversion of alanine and ketoglutaric acid to pyruvic acid and glutamic acid.
Aspartate and alanine aminotransferases are present in both mitochondria
and cytosolic fractions of fish cells (Walton and Cowey, 1982). Glutamate
dehydrogenase (GDH) is a regulatory enzyme known to check the
deamination process to minimize the ammonia level and plays a significant
role in the catabolism of amino acids. GDH catalyses the reversible oxidative
deaminiation of glutamate to -ketoglutarate and ammonia with coenzyme
pyridine nucleotide (NAD or NADP. All these enzymes function as a link
between protein and carbohydrate metabolisms and the net outcome is the
incorporation of ketoacids into the TCA cycle. There is much evidence for the
132
shifts in the activities of these enzymes to a variety of environmental and
physiological conditions (Knox and Greengard, 1965).
The pesticides are found to alter the structural and soluble proteins by
causing histopathological and biochemical changes in the cell (Shakoori, et. al,
1976). Simuel and Sastry (1984) reported an increase in the protein content in
the lethal and sub lethal concentration. Some information is available on the
effects of pesticides on protein metabolism of aquatic animals (Mc Kee and
Knowles, 1986; Saleem and Shakoori, 1987; Ravinder, 1988; Malla Reddy and
Philip, 1991; Jha and Verma, 2002). Pollak and Wendy (1982) reported an
alteration in protein content in the selected tissues of the edible fish on
exposure to pesticide medium.
Many studies have documented involving the toxic effects of pesticides
on proteins in fishes. Sivaprasad Rao, et. al, (1982) studied the impact of
phenthoate on the nitrogen metabolism in Channa punctatus and postulated a
decrease in tissue total protein and an increase in free amino acid levels, with
a decrease in ammonia and urea levels in the muscle and gills with their
increase in the liver. They also reported an increase in the activity of GDH in
the gills and liver, but a decrease in muscle. A decrease in protein content and
an increase in free amino acids, urea levels and GDH activity were observed
by Radhaiah, et. al,(1987) in Tilapia mossambica on exposure to Heptachlor.
Anupam Jyothi, et. al, ., (1999) revealed a significant fall in protein and RNA
contents in the liver, heart and muscle of Channa punctatus on exposure to
malathion. Rajyashree and Neeraja (1989) found that AAT showed maximum
activity in muscle mitochondrial fraction, whereas AlAT showed maximum
activity both in muscle mitochondrial and cytosolic fractions. Ganeshan, et. al,
133
(1989) studied the impact of endosulfan on the protein content in liver tissues
of Oreochromis mossambicus and noticed a decrease in protein level with
increase in the length of exposure to endosulfan. Shiva Prasad Rao, et. al, .,
(1990) confirmed a decrease in the total proteins and increase in the levels of
free amino acids, urea and the activities of AlAT and AAT in Tilapia
mossambica on exposure to chronic sub lethal concentration of heptachlor. In
fry of Cyprinus carpio treated with sub lethal concentration of pyrethroid and
cypermethrin, an increase in the protein content was reported (Piska, et. al,
1992). Hypoproteinemia occurred in Heteropneustes fossilis when it was
exposed to sub lethal concentrations of aidrin (Singh et. al,1993).
Baktavathsalam and Srinivasa Reddy (1988) reported an increase in
aspertate and alanine aminotransferases (AAT, ALAT) in Anàbas testudineus
on exposure to lindane. Narasimha Murthy, et. al, (1987) studied the
decrement of alanine aminotransferase and aspartate amino transferase in
Tilapia mossambica. Reddy and Yellamma (1991) found a decrease in total and
soluble proteins with increase in free amino acids, alanine aminotransferase
(AlAT) and aspertate amino transferase (AAT) in Periplanata americana on
exposure to fenvalerate, Reddy and Philip (1991) registered decrease in total,
structural and soluble proteins and increase in amino acids and protease
activity levels in freshwater fish, Cyprinus carpio on exposure to malathion and
cypermethrin (Rajasree, 1993). The protein content of the liver and muscle got
reduced with the subsequent increase of amino acids, by the effect of lindane
on exposure to Tilapia mossambica (Rajamanickam and Karpagaganapathy,
1988).
134
The above accounts give a brief understanding of the effect of
pesticides on protein metabolism of freshwater fishes. The information of the
above studies is unable to provide a clear concept on the effect of
cypermethrin on protein metabolism of freshwater fish, as it appeared more
or less inconsistent. Hence an attempt was made to study the effect of
cypermethrin on some aspects of protein metabolism in the organs of
freshwater fish, Labeo rohita at lethal and sub lethal concentrations.
RESULTS
The data is presented on the levels of soluble, structural and total
Means are SD (n=6) for a tissue in a row followed by the same letter are not significantly different (P 0.05) from each other according to Duncun's multiple range (DMR) test.
Table 22: Soluble protein content (mg/g wet wt.) in the organs of fish, Labeo rohita on exposure to the lethal and
Means are SD (n=6) for a tissue in a row followed by the same letter are not significantly different (P 0.05) from each other according to Duncun's multiple range (DMR) test.
Table 23: Structural protein content (mg/g wet wt.) in the organs of fish, Labeo rohita on exposure to the lethal and
Means are SD (n=6) for a tissue in a row followed by the same letter are not significantly different (P 0.05) from each other according to Duncun's multiple range (DMR) test.
Table 24: Free amino acid levels (mg amino acid nitrogen / g wet wt.) in the organs of fish, Labeo rohita on exposure to
the lethal and sublethal concentrations of cypermethrin.
Means are SD (n=6) for a tissue in a row followed by the same letter are not significantly different (P 0.05) from each other according to Duncun's multiple range (DMR) test.
Table 25: Protease activity (M amino acid nitrogen / mg protein / h) in the organs of fish, Labeo rohita on exposure to
the lethal and sublethal concentrations of cypermethrin.
Means are SD (n=6) for a tissue in a row followed by the same letter are not significantly different (P 0.05) from each other according to Duncun's multiple range (DMR) test.
Table 26: The Aspartate amino transferase (AAT) activity (M oxalo acetate / mg protein / h) in the organs of fish,
Labeo rohita on exposure to the lethal and sublethal concentrations of cypermethrin.
Means are SD (n=6) for a tissue in a row followed by the same letter are not significantly different (P 0.05) from each other according to Duncun's multiple range (DMR) test.
Table 27: The alanine aminotransferase (AlAT) activity (M pyruvate formed / mg protein/h) in the organs of fish,
Labeo rohita on exposure to the lethal and sublethal concentrations of cypermethrin.
Means are SD (n=6) for a tissue in a row followed by the same letter are not significantly different (P 0.05) from each other according to Duncun's multiple range (DMR) test.
Table 28: GDH activity (M glutamine / mg protein / h) in the organs of fish, Labeo rohita on exposure to the lethal
Means are SD (n=6) for a tissue in a row followed by the same letter are not significantly different (P 0.05) from each other according to Duncun's multiple range (DMR) test.
Fig 10: Percent change over control in total protein levels in the tissues of Labeo rohita following exposure to lethal and
sublethal concentrations of cypermethrin (presented for ready reference)
-50
-45
-40
-35
-30
-25
-20
-15
-10
-5
0
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig 19: Percent change over control in the soluble protein content (mg/g wet wt.) in the organs of fish, Labeo rohita on exposure
to the lethal and sub lethal concentrations of cypermethrin.
-60
-50
-40
-30
-20
-10
0
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Table 20: Structural protein content (mg/g wet wt.) in the organs of fish, Labeo rohita on exposure to the lethal and sub lethal
concentrations of cypermethrin.
-60
-50
-40
-30
-20
-10
0
24 48 72 96 1 5 10 15
Pe
rce
nt
chan
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig 21: Percent change over control in free amino acid levels in the tissues of Labeo rohita following exposure to lethal and sublethal
concentrations of cypermethrin (presented for ready reference)
0
10
20
30
40
50
60
70
80
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig 22: Percent change over control in protease activity in the tissues of Labeo rohita following exposure to lethal and sublethal
concentrations of cypermethrin (presented for ready reference)
0
10
20
30
40
50
60
70
24 48 72 96 1 5 10 15
Pe
rcen
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig 23: The Aspartate amino transferase (AAT) activity (M oxalo acetate / mg protein / h) in the organs of fish, Labeo rohita on
exposure to the lethal and sub lethal concentrations of cypermethrin.
0
10
20
30
40
50
60
24 48 72 96 1 5 10 15
Perc
en
t ch
an
ge
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig 24: The alanine aminotransferase (ALAT) activity (M pyruvate formed / mg protein/h) in the organs of fish, Labeo rohita
on exposure to the lethal and sub lethal concentrations of cypermethrin.
0
10
20
30
40
50
60
70
80
24 48 72 96 1 5 10 15
Per
cen
t ch
ang
e
Lethal (h) Sublethal (days) Exposure periods
Gill Liver Muscle
Fig 25: Percent change GDH activity (M glutamine / mg protein / h) of the fish organs, Labeo rohita on exposure to the lethal