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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).
88
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.
Tissue Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 31.10E 35.21C 42.20B 48.34A 45.67B 31.97 D 32.08D 34.88C 34.49C
SD 0.65 0.24 0.29 0.34 0.32 0.22 0.22 0.24 0.24
% Change ---- 13.21 35.69 55.41 46.82 2.78 3.14 12.15 10.90
Muscle 35.42H 37.56F 40.61C 47.55A 44.76B 36.54G 38.94E 39.96D 39.51D
SD 0.50 0.26 0.28 0.33 0.31 0.25 0.27 0.28 0.27
% Change ---- 6.02 14.64 34.21 26.35 3.13 9.92 12.79 11.54
Liver 16.75F 17.29E 18.15B 18.75A 18.28B 17.36E 17.79D 18.07C 17.43D
SD 0.23 0.12 0.12 0.13 0.12 0.12 0.12 0.12 0.12
% Change ---- 3.20 8.34 11.91 9.11 3.62 6.23 7.89 4.08
Table 8: 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.
Tissue Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 4.86A 4.35D 4.19E 3.85F 4.04E 4.59C 4.60B 4.26E 4.49D
SD 0. 031 0.030 0.029 0.027 0.028 0.032 0.032 0.030 0.031
% Change ---- -10.47 -13.71 -20.69 -16.77 -5.48 -5.38 -12.36 -7.62
Muscle 6.57A 6.44B 6.04C 5.36F 5.80E 6.48B 6.03C 5.97D 5.87E
SD 0.09 0.04 0.04 0.03 0.04 0.04 0.04 0.04 0.04
% Change ----- -2.00 -8.10 -18.35 -11.65 -1.30 -8.12 -9.16 -10.55
Liver 2.05A 1.91D 1.62G 1.42H 1.55H 1.88B 1.82C 1.75E 1.67F
SD 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
% Change ----- -6.86 -20.84 -30.44 -24.34 -8.34 -10.95 -14.30 -18.38
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
ecosystem (Eggen, et. al, 2004; Lam and Gray, 2003; Moore, et. al, 2004;
Vasseur and Cossu-Leguille, 2003). For the biomonitoring and management of
the aquatic ecosystems, these biological responses (biomarkers) proposed to
complement and enhance the reliability of the chemical analysis data.
Therefore, much attention paid in last two decades to develop biomarkers as
indicators of chemical exposure and as early signals of pollution (Cormier and
Daniel, 1994; Lagadic, et. al, 1998). Moreover, the use of biomarkers proved a
simple way of providing realistic and relevant data at any level of the
biological organization. Therefore, in risk assessment and environmental
management programmes biomarkers are increasingly used (Adams, et. al,
2001).
Oxidative stress biomarkers though fall in non-specific category, have
provided meaningful indicators of pollution both in the freshwater and
marine ecosystems. Oxidative stress biomarkers though fall in non-specific
category, have provided meaningful indicators of pollution both in the
freshwater and marine ecosystems (Van Der Oost, et. al, 1994; Cossu, et. al,
1997; Yang and Randall, 1997). These biomarkers are indicative of damages to
96
carbohydrates, lipids and proteins by the reactive oxygen species (ROS)
(Miyata, et. al, 1993). In mammalian system including humans that direct
damage to proteins or chemical modification of amino acids in proteins
during oxidative stress can give rise to protein carbonyls (Stadtman and
Berlett, 1998; Zusterzeel, et. al, 2001). The induction of protein carbonyl may
serve as a surrogate biomarker for general oxidative stress (Reznick, et. al,
1992).
In aerobic organisms, oxygen is essential for efficient energy
production but paradoxically, produces chronic toxic stress in cells. Thus,
protective mechanisms must exist for the removal of toxic oxygen byproducts.
Diverse protective systems have evolved to enable adaptation to oxidative
environments. These antioxidant defense systems are critical for survival in
both prokaryotic and eukaryotic organisms.
Animals require molecular oxygen (O2) for the oxidation of food and
the generation of energy. During this process, O2 undergoes tetravalent
reduction to water. However, partial reduction of O2 results in the formation
of reactive oxygen species (ROS), including both radical speciHes such as the
superoxide anion radical (O2-; 1-electron reduction) and hydroxyl radical (OH-
; equivalent to 3-electron reduction) and non-radical species such as H2O2 (2-
electron reduction) (Halliwell and Gutteridge, 1999). The ROS are continually
produced as undesirable toxic bi-products of normal metabolism from
various endogenous processes. ROS can in turn give rise to other ROS such as
peroxyl and alkoxyl radicals (respectively ROO- and RO-) through reaction
97
with other biological molecules. An initial pro-oxidant event can thus give
rise to a spreading web of ROS production within an animal. Any process
which leads to increased ROS production, either directly, or indirectly via
organic radical formation or other mechanisms, can potentially result in
enhanced oxidative stress and biological damage (Halliwell and Gutteridge,
1999). Possible prooxidant agents in the environment are many and varied,
including both natural and man-made sources.
In the normal healthy cell, ROS and pro-oxidant products are
detoxified by antioxidant defenses, including low molecular weight free
radical scavengers and specific antioxidant enzymes (Halliwell and
Gutteridge, 1999). The former comprise both water-soluble (e.g. vitamin C,
reduced glutathione (GSH), carotenoids) and lipid-soluble (e.g. vitamins A
and E) molecules. The antioxidant enzymes include superoxide dismutase
(SOD; EC 1.15.1.1 - converts O2- to H2O2), catalase (EC 1.11.1.6 - converts H2O2
to water) and glutathione peroxidase (GPX; EC 1.11.1.9 - detoxifies H2O2 and
organic hydroperoxides utilising GSH). Thus a balance is thought to exist
between pro-oxidant production and antioxidant defense, although low levels
of oxidative damage, particularly to key biological molecules such as lipid,
protein and DNA, are also always present. However, marked increases in
ROS production can overcome antioxidant defenses, resulting in increased
oxidative damage to macromolecules and alterations in critical cellular
processes. The oxidative damage may be spread far from its point of cellular
origin by the different ROS and other products of oxidation, resulting in a
98
condition of oxidative stress. Exposure to increased ROS production can also
lead to induction of certain antioxidant enzymes via interaction with
antioxidant responsive gene elements and increased transcription.
Defense mechanisms against free radical-induced oxidative damage
include (i) catalytic removal of free radicals and reactive species by factors
such as catalase (CAT), superoxide dismutase (SOD), peroxidase and thiol-
specific antioxidants. (ii) Binding of proteins (e.g., transferrin, metallothionein,
haptoglobins, caeroplasmin) to pro-oxidant metal ions, such as iron and
copper. (iii) Protection against macromolecular damage by proteins such as
stress or heat shock proteins. (iv) Reduction of free radicals by electron donors,
such as GSH, vitamin E (tocopherol), vitamin C (ascorbic acid), bilirubin and
uric acid (Halliwell and Gutteridge, 1999).
Animal catalases are heme-containing enzymes that convert hydrogen
peroxide (H2O2) to water and O2 and they are largely localized in subcellular
organelles such as peroxisomes, mitochondria and the endoplasmic reticulum
contain little CAT. Thus, intracellular H2O2 cannot be eliminated unless it
diffuses to the peroxisomes (Halliwell and Gutteridge, 1999). Glutathione
peroxidases (GSH-Px) remove H2O2 by coupling its reduction with the
oxidation of GSH. GSH-Px can also reduce other peroxides, such as fatty acid
hydroperoxides. These enzymes are present in the cytoplasm at millimolar
concentrations and present in the mitochondrial matrix. Most animal tissues
contain both CAT and GSH-Px activity.
99
Varieties of contaminants enter the aquatic environment and are taken
up from the sediment, water-column and food into the tissues of resident
organisms (Kim, et. al, 2004; Hughes, et. al, 2003; Bhadauria, et. al, 2007). The
contaminants include many chemicals that have been shown to be pro-
oxidants in mammalian systems such as redox cycling compounds, polycyclic
aromatic compounds (PAHs) (benzene, PAH oxidation products),
halogenated hydrocarbons (bromobenzene, dibromomethane,
polychlorobiphenyls (PCBs), lindane), dioxins, pentachlorophenol and metals
(Al, As, Cd, Cr, Hg, Ni, Va). The same general scenario of contaminant-
stimulated ROS production, antioxidant defense and oxidative damage as
seen for mammals is indicated for aquatic organisms, although much less is
known of many of these aspects (Lam, et. al, 2003; Moore, et. al, 2004). The
studies in fish and aquatic invertebrates have largely been carried out on the
major organs of biotransformation and respiration gills, liver of fish, pyloric
caeca of echinoderms, hepatopancreas of crustaceans and digestive gland of
molluscs (Adams, et. al, 2001; Cossu, et. al, 1997; Vasseur, et. al, 2003)
Pyrethroids are hydrophobic than other classes of insecticides
(Michelangeli, et. al, 1990) and this feature indicates that the site of action is in
the biological membrane. In fact, the principal target site for pyrethroids is
defined as the voltage-dependent sodium channel in the neuronal membrane
(Narahashi, 1985; Soderlund and Bloomquist, 1989; Vijverberg and van den
Bercken, 1990). The available data indicate that both Type I, Type II
pyrethroids act potently. Stereo selectively on sodium channels by slowing
100
kinetics of both opening and closing of individual channels. Inhibition of
GABA receptor is an additional mechanism proposed for Type II pyrethroids
(Narahashi, 1992).
It was of interest to investigate the possibility of oxidative stress
induction by pyrethroids, considering the above mentioned data and
considering the followings. (1) There is evidence that excitatory events may
stimulate reactive oxygen species (ROS) production. (2) The induction of
oxidative stress and alteration of antioxidant system by pyrethroids in rats
reported recently (Gupta, et. al, 1989, Kale, et. al, 1999). However the studies
on fishes are meager. Therefore it is pertinent to understand the involvement
of oxidative stress in the pyrethroid action. We investigated the oxidative
stress inducing effects of a Type II pyrethroid, cypermethrin by measuring
indicators of the integrity of the antioxidant defense system such as the
catalase, protease activities and hydrogen peroxide, MDA, protein carbonyls,
free amino acids and protein levels in teleost fish, Labeo rohita. The extent of
lipid peroxidation was also determined since ROS can attack and oxidize the
fatty acid side-chains of phospholipids.
RESULTS
In the present study catalase and protease activity, hydrogen peroxide,
MDA, protein carbonyls, protein content and free amino acids levels
increased in gill, muscle and liver tissues of fish exposed to lethal and
sublethal concentrations of cypermethrin (Tables. 9-15 and Fig. 6-12) which
sowed decline in the levels.
101
Effect on CAT activity
Increase in the CAT activity was observed in L. rohita after exposure to
cypermethrin at both concentrations viz., lethal and sublethal (Table.9). At
lethal concentration, increase in the activity was continuous from 24h to 72 h
in all the tissues; later at 96 h increased activity was reduced in gill and
muscle except liver. The maximum enzyme activity was recorded in liver at
72 h with 48.73% over control and least was recorded in muscle tissue with
18.38% at 24 h. Similar increasing trend was observed at sublethal
concentration also. Increase in CAT activity was continuous with the increase
in exposure periods irrespective of the tissues from day 1 to 10. However the
activity was low at day 15 compared day 1to 10. Maximum and minimum
increase being noted in liver (41.79%) and muscle (16.90%) tissues at day 10
and day 1, respectively (Fig. 6).
Effect on hydrogen peroxide levels
Variations observed in the quantity of hydrogen peroxide (H2O2)
content at both lethal and sublethal exposures (Table 10). At lethal
concentrations H2O2 content increased significantly right from 24 h to 96 hr.
Maximum increase was noted in liver with 55.68% at 72 h and minimum was
recorded in muscle tissue at 24 h (24.77%) was. While all the tissues recorded
increase in hydrogen peroxide content at lethal concentration. Sublethal
concentrations shown gradual increase from day 1 to day 10, later at day 15
content was low. Liver recorded the maximum percent increase (48.43%) fish
102
and minimum increase of (23.21%) was noticed in the muscle tissues at day 10
and day 1 respectively.
Effect on levels of MDA
The MDA levels were significantly augmented at lethal and sublethal
concentration in comparison to control. At lethal concentrations levels were
increased in all tissues with increase in exposure periods (Table. 11).
Maximum increase in the level was observed in liver (44.46%) followed by gill
(42.31%) and muscle (38.89%) at 96 h of exposure. At sublethal concentration
MDA levels were increased irrespective of the tissues from day 1 to day 10,
later at day 15 levels were reduced. Maximum increase in the level was
observed in liver with 31.47% at day 10 and minimum in muscle at day 1 with
6.89% change over control respectively (fig.8).
Effect on protein carbonyls
Protein carbonyl measured at lethal and sublethal concentrations
showed significant augmentation over control. Increase in the levels was
gradual with increase in the exposure periods (Table. 12). Maximum increase
was noticed in liver (33.84%) at 72 h and on 10th day (23.68%) of exposure at
lethal and sublethal concentration respectively. Minimum increase noticed in
gill (3.69%) at 24 h and on 1st day of exposure (3.21%) at lethal and sublethal
concentration, respectively (fig. 9)
Effect on protein levels
103
The decline in the protein levels of fish exposed to lethal and sublethal
concentration were observed in gill, muscle and liver. At lethal concentration
gradual decrease was with increase in the exposure periods irrespective of the
tissues (Table. 13). Liver (46.12%) was recorded maximum decline followed
by gill (44.3887%) and muscle (42.4558%) at 96 h of exposure. At sublethal
concentration protein levels were diminished in all tissues from day 1 to 5,
however decline was low at 10th and 15th day of exposure. The maximum
decrease was recorded in the gill (19.68%) followed by muscle (18.17%) and
liver (15.53%), on 10th day of exposure. The lowest decrease was noticed in
liver (1.35%), followed by muscle (3.07%) and gill (2.98%) on 15th day of
exposure (fig. 10).
Effect on free amino acids (FAA)
FAA levels increased at lethal and sublethal concentrations in all
tissues at all periods of exposure regimes over control (Table. 14). At lethal
concentration increase in the levels was gradual with increase in exposure
periods. Gill (69.99%) recorded maximum increase, followed by muscle
(69.66%) and liver (48.19%) at 96 h. FAA levels in all the tissues increased At
sublethal concentration, maximum recorded in muscle (44.83%) followed by
liver (31.87%) and gill (25.60%) on day 5 (fig. 11).
Effect on protease activity
Compared to the control, induction of protease activity was observed
in lethal and sublethal concentration of cypermethrin (Table. 15). At lethal
concentration all the studied tissues exhibited increase from 24 h to 96 h.
Maximum was witnessed in muscle (64.02%) followed by gill (60.09%) and
104
liver (57.50%) at 96 h. While at sublethal concentration activity increased in all
exposure periods from day 1 to day 15. The maximum induction was noticed
in the muscle (41.52%), followed by gill (27.50%) and liver (20.28%) on day 1
and minimum induction was in liver (2.40%) followed by muscle (8.09%) and
gill (8.55%) on 15th day of exposure (Fig. 12).
DISCUSSION
Pyrethroid group of pesticides are the most commonly used in
agriculture today and are efficiently absorbed and rapidly redistributed to
various organs as part of their disposal mechanism (Mehaboob Khan, et. al,
2005). Recent evidences implicate the involvement of oxidative stress
mechanisms under conditions of pyrethroid induced toxic effects (Giray, et. al,
2001; Kale, et. al, 1999). However, studies on the pattern of in vivo
susceptibility of various tissues to cypermethrin induced oxidative stress are
limited.
The present study evidenced time and concentration dependent
induction/reduction of the above parameters by lethal and sublethal
concentrations of cypermethrin in the tissues (gill, muscle and liver) of L.
rohita. Thus the results clearly evoke an imbalance in the cellular oxidative
status by means of oxidative damage and decline in antioxidant defense due
to cypermethrin induced oxidative stress.
The activity of antioxidant may be increased or inhibited under
chemical stress depending on the intensity and the duration of the stress
applied as well as susceptibility of the exposed species. In the presence of
105
xenobiotic, an initial induction response in the antioxidant system may be
followed by a reduction. Thus the existence of an inducible antioxidant
system may reflect an adaptation of organism (Doyotte, et. al, 1997). The
response of antioxidant system to oxidative stress in various tissues shows
differences from one species to another due to the differences in antioxidant
potential of these tissues (Ahmad, et. al, 2000).
It is now clear that oxidative damage may be the primary cause of
subcellular effects of cypermethrin toxicity. Several studies of varying
duration of exposure with organophosphorus pesticides or cypermethrin
have postulated a putative role for the generation of free radicals during the
process (Altuntas, et. al, 2002). In vitro studies have also reported that
chlorpyriphos and cypermethrin cause degradation of hepatocytes and renal
cells Sohn, et. al, (2004). Liver plays a central role in the detoxification process
and faces the threat of maximum exposure to xenobiotics and their metabolic
by-products. The susceptibility of liver and gill (being primarily in contact
with medium) tissues to this stress due to exposure to these pesticides is a
function of the overall balance between the degree of oxidative stress and the
antioxidant capability.
Increase in the CAT activity was observed in L. rohita after exposure to
cypermethrin at both concentrations viz., lethal and sublethal. The highest
CAT activity was determined in liver (25.57% at 96 h; 15.07% on 15th day)
tissue compared to other tissues. Antioxidant enzymes play important role in
the detoxification of cypermethrin or its metabolite. Akthar, et. al, (1994)
106
indicated that deltamethrin is detoxified in the liver, while its metabolites are
detoxified in the kidney. Similar study reported by Sayeed, et. al, (2003) in
catfish on exposure to deltamethrin, stimulated CAT activity and induced
lipid peroxidation in liver, kidney and gill. Results of the present study
suggest that, cypermethrin and its metobolites may be detoxified in liver
tissue, probably due to its characters and route of exposure. The main route
for the detoxification of cypermethrin is hydroxylation and eliminated as
glucoronide conjugates through the ballast (Edwards and Millburn, 1985).
The liver was found to be stronger into the face of oxidative stress than the
other tissues. This could be related to the fact that the liver is the site of
multiple oxidative reactions and maximal free radical generation (Gül, et. al,
2004; Avci, et. al, 2005).
CAT activity was increased in gill tissues than the muscle, as gills
being the primary organs of fish exposed to surrounding medium and
probably indicates an effective antioxidant response. In addition a higher
renovation of gill epithelium and recruitment of chloride cells to increase the
capability to uptake ions from water and may help the animals to prevent the
entry of toxicants by maintaining cation concentration gradient (Fu, et. al,
1990). Moreover, slow elimination of the cypermethrin from the tissue might
be the possible reason for the up regulation of CAT system.
Almost similar effects were observed in muscle tissues on par with the
gill. Induction of CAT activity in both the concentration could be attributed to
higher affinity of cypermethrin towards lipids and possibly reduces the levels
107
of total lipid, unesterified cholesterol, phospholipids and gets accumulated
within the adipose tissues blocking the lipid metabolism. Moreover all the cell
membranes are made of phospholipids; hence it could also be viewed as
sequestration of cypermethrin and its effects at storage organs.
The study of the deleterious effects produced by H2O2 in cells is
important in view of the fact that H2O2 itself is a normal highly reactive
metabolite of aerobic organisms, the production of which can be stimulated
by the metabolism of many carcinogenic or antitumor agents (Subrahmanyam,
et. al, 1987), as well as in a variety of pathological circumstances (Fantone and
Ward 1982; Freeman and Crapo, 1982; Cerutti, 1985). The primarily
mechanism of H2O2 toxicity is the formation of highly reactive oxygen species
(hydroxyl radicals) in the presence of transition metal ions or other various
mechanisms (Halliwell, et. al, 1992). The formation of hydroxyl radicals and
other ROS initiates lipid peroxidation and causes DNA damage. The increase
in H2O2 concentration observed in the present study could induce hydroxyl
radical formation and therefore may induct the deleterious effects leading to
oxidative damage of biomolecules including DNA through lipid peroxidation.
Since, lipid peroxidation is one of the major mechanisms of cellular injury
caused by H2O2 (Yang, et. al, 1999). H2O2 is a genotoxic agent, known to
induce oxidative DNA damage including DNA strand breakage and base
modification (Halliwell and Aruoma, 1991). Moreover, catalase activity
increased during experimental periods, probably a response to toxicant stress
and serves to neutralize the impact of increased ROS generation (John, et. al,
108
2001). Zhi-Hua, et. al, (2010) in brain of rainbow trout (Oncorhynchus mykiss)
made similar observation after chronic carbamazepine treatment. Verlecara, et.
al, (2008) recorded similar increase in hydrogen peroxide levels in digestive
gland of Perna viridis due to mercury exposure.
Lipid peroxidation is a process, which is determined by the extent of
the peroxide-deforming free radical mechanism on the highly
polyunsaturated fatty acids and is particularly important for aquatic animals
since they normally contain greater amounts of highly unsaturated fatty acids
(HUFA) than other species (Huang, et. al, 2003). Lipid peroxidation (LPO) is
major contributor to the loss of cell function under oxidative stress (Storey,
1996) and has usually been indicated by TBARS in fish (Oakes and Van der
Kraak, 2003). In the present study, the extent of gill, muscle and liver LPO
was evidenced by the increase in their respective TBARS levels as well as
inhibition of the endogenous antioxidant enzyme (catalase) after
cypermethrin exposure.
Elevation of lipid peroxidation in tissues after exposure to lethal and
sublethal concentrations of cypermethrin in acute and subacute durations, as
evidenced by increased MDA production in the present study, suggests
participation of free-radical induced oxidative cell injury in mediating the
toxicity of cypermethrin. It is known that cypermethrin could induce
oxidative stress and as a hydrophobic compound may accumulate in cell
membranes and disturbs membrane structure (Gajendra, et. al, 2004). Jin, et. al,
(2011) reported that, cypermethrin has the potential to induce hepatic
109
oxidative stress, DNA damage and the alteration in gene expression related to
apoptosis in adult zebrafish. In the current study, cypermethrin increased
MDA concentrations, indicating the induction of lipid peroxidation, which
can lead to loss of membrane structure and function and implicate a role of
oxidative stress and free radical formation in these effects. Similarly some of
the earlier studies have documented dose and time dependent oxidative
stress in mammalian models with administration (Kale, et. al, 1999; Kanbur, et.
al, 2008; Atessahi, et. al, 2005).
Comparative in vivo and in vitro metabolic studies have shown that
fish have a lower capacity to metabolize and eliminate pyrethroid insecticides
(Glickman and Lech, 1981; Glickman, et. al, 1982). The current results may
suggest the possibility of a redistribution occurring following a rapid initial
penetration of highly lipophilic cypermethrin into the tissues. This is reflected
in the present investigation, where cypermethrin induced peroxidative
damage in all the tissues. Higher elevation of TBARS was noted in the liver a
principle metabolic organ at both acute and subacute exposure regimes
suggesting the production of oxidative metabolites and free radicals possibly
continues during the intensive hepatic metabolism and this may be due to the
progressive nature of the free radical chain reactions.
Previous studies have shown that cypermethrin induce oxidative stress
in mammalian erythrocytes. It has been shown that, cypermethrin exert their
effects through a lipophilic conjugate, 2[R]-2-(4-chlorophenyl) isovalerate and
has been detected in adrenals, liver and mesentric lymph nodes in rats, mice
110
and some other species (World Health Organisation, 1990). The aldehydes
and other lipophilic conjugates may produce oxidative stress in pyrethroid
toxicity.
Oxidative stress biomarkers are meaningful indicators of pollution
both in the freshwater and marine ecosystems (Van Der Oost, et. al, 1994;
Cossu, et. al, 1997; Yang and Randall, 1997). These biomarkers are indicative
of damages to carbohydrates, lipids and proteins by the reactive oxygen
species (ROS) (Miyata, et. al, 1993). Furthermore, it has been established that
direct damage to proteins or chemical modification of amino acids in proteins
during oxidative stress can give rise to protein carbonyls (Stadtman and
Berlett, 1998; Zusterzeel, et. al, 2001). The formation of carbonyl proteins is
non-reversible, causing conformational changes, decreased catalytic activity
in enzymes and ultimately resulting, owing to increased susceptibility to
protease action, in breakdown of proteins by proteases (Zhang, et. al, 2008). In
the current study protein carbonyl levels in both the lethal and sublethal
concentrations increased, indicating that cypermethrin intoxication induced
disruption in cellular protein metabolism (Table 11 and Figure 8). It has been
suggested that induction of protein carbonyl may serve as a surrogate
biomarker for general oxidative stress (Reznick, et. al, 1992).
Protein is one of the main targets for elucidation of effects by the
pesticides. Oxidative modification of protein may occur in a variety of
physiological and pathological processes, which may be primary or
secondary. Protein depletion in tissues may also constitute a physiological
111
mechanism and may play a role of compensatory mechanism under
cypermethrin stress. Klassan (1991) reported that the depletion of protein
suggests increased proteolysis and possible utilization of the products of their
degradation for metabolic purposes. To provide intermediates for the Kreb’s
cycle or to enhance osmolarity, by retaining free amino acid content in
haemolymph and to compensate osmoregulatory problems encountered due
to the leakage of ions and other essential molecules, during the pesticide
stress (Rafat, 1986; Rajeshwari, 1986). They may be fed into TCA cycle
through amino-transferase system to cope up with excess demand of energy
during the elimination of toxicants from the body. Thus, oxidative
modification of proteins is also one of the many consequences of oxidative
stress (Stadtman, 1986).
Decrease in protein content and increase in the protease activity and
amino acid levels as evidenced from the present study suggests that damage
to proteins thus releasing their monomers due to oxidative damage and
chopping by protease. Protein degradation is in active phase over synthesis in
the gill, muscle and liver of fish during experimental periods in both the lethal
and sublethal concentrations of cypermethrin. Elevation in free amino acid as
observed by Kabeer, et. al, (1984) and Rajamannar and Manohar (1998) studies
suggest intensive proteolysis contribute to the rise in the free amino acid pool,
which becomes a source of tricarboxylic acid cycle (TCA) intermediates by
both the transamination reactions. These views support the findings of the
present investigation and also strengthen the earlier reports of Ganeshan, et. al,
112
(1989) and Jha and Verma (2002) and without doubt suggest the operation of
gluconeogenesis in order to mitigate the toxic stress.
High concentrations of amino acids in tissues can lead to hyper
aminoacidemia, which in turn may alter the physiological conditions of the
cell. The increase in the free amino acids in the tissues of fish exposed to lethal
and sublethal concentrations can be partly due to the increased proteolytic
activity and partly due to certain transaminases reported to be indicators of
protein degradation in salmonoids (Bell, 1968) and liver intoxication in
rainbow trout (Gingerich and Weber, 1976). Higher levels of free amino acid
content may also be attributed to the decreased utilization of amino acids
(Seshagiri, et. al, 1987) and is suggestive of catabolism of protein or
transamination of keto acids (Shakoori, et. al, 1976). Amino acids may be
shunted into the Kreb’s cycle through transamination and oxidative
deamination. The increase in free amino acid content may serve in
maintaining the intracellular osmotic balance during the cypermethrin
induced physiological stress.
In conclusion, the results of this study show that cypermethrin
exposure to Labeo rohita induces significant oxidative stress in gill, muscle and
liver tissues at lethal and sublethal concentration. The induced oxidative
damage may be supported with corroborative changes observed in the
membrane bound enzymes, Na+-K+-ATPase and AChE (See chapter 4.3 and
4.1 respectively). Since, activities of these membrane bound enzymes depend
on the phospholipid environment of the membrane (Rauchova, et. al, 1999).
113
Therefore, any change in the lipid component of the membrane due to
oxidative stress will directly affect the activities of these enzymes. Hence,
cypermethrin toxicity was mediated through the oxidative damage of
biomolecules, thereby affecting the integrity of cellular and subcellular
structures, which were also evident in the present study with ultrastructural
changes in hepatic cell organelles (Chapter 6).
Table 9: Catalase activity (mmol of hydrogen peroxide decomposed/mg protein/min) in the tissues of Labeo rohita following
exposure to lethal and sublethal concentrations of cypermethrin.
Organ
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 3.64E 4.36D 4.81B 5.02A 4.84B 4.32D 4.69C 5.04A 4.59C
SD 0.02 0.03 0.03 0.05 0.03 0.03 0.03 0.03 0.03
% Change --- 19.47 31.86 37.66 32.63 18.53 28.63 38.16 25.78
Liver 4.47G 5.42F 6.07C 6.65A 6.35B 5.34F 5.93D 6.34B 5.70E
SD 0.03 0.03 0.04 0.04 0.04 0.03 0.04 0.04 0.04
% Change --- 21.19 35.69 48.73 41.96 19.47 32.60 41.79 27.40
Muscle 3.90F 4.62D 4.91C 5.35A 5.08B 4.56E 4.93C 5.30A 4.79D
SD 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03
% Change --- 18.38 25.95 37.08 30.27 16.90 26.33 35.90 22.78
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 10: Hydrogen peroxide levels (nmol of hydrogen peroxide/mg protein) in the tissues of Labeo rohita fingerlings
following exposure to sublethal concentrations of cypermethrin.
Organ
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 3.83G 4.84
F 5.34
C 5.58
A 5.37
C 4.80
F 5.21
D 5.60
B 5.10
E
SD 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03
% Change --- 26.39 39.50 45.64 40.31 25.40 36.09 46.17 33.07
Liver 4.75G 6.02
E 6.74
C 7.39
A 7.06
B 5.94
F 6.59
C 7.05
A 6.33
D
0.03 0.04 0.04 0.05 0.04 0.04 0.04 0.04 0.04
% Change --- 26.86 42.04 55.68 48.60 25.06 38.80 48.43 33.36
Muscle 4.11H 5.13
G 5.46
D 5.94
A 5.64
B 5.06
G 5.47
E 5.89
C 5.32
F
SD 0.02 0.03 0.03 0.04 0.03 0.03 0.03 0.04 0.03
% Change --- 24.77 32.76 44.49 37.30 23.21 33.15 43.24 29.41
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 11: MDA levels (nmol of TBARS formed/mg of protein) in the tissues of Labeo rohita fingerlings following exposure to
sublethal concentrations of cypermethrin.
Organ
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 4.21G 4.76E 5.46C 5.57B 5.99A 4.64F 5.10D 5.47C 4.77E
SD 0.29 0.33 0.38 0.39 0.42 0.32 0.36 0.38 0.33
% Change --- 13.00 29.80 32.44 42.31 10.31 21.26 30.07 13.35
Liver 3.99H 4.59G 5.19D 5.50B 5.76A 4.50G 5.03E 5.24C 4.71F
SD 0.28 0.32 0.36 0.38 0.40 0.31 0.35 0.37 0.33
% Change --- 15.00 30.08 37.85 44.46 12.89 26.05 31.47 18.17
Muscle 3.65G 3.96F 4.35D 4.75B 5.07A 3.90E 4.25D 4.62C 4.10E
SD 0.25 0.28 0.30 0.33 0.35 0.27 0.30 0.32 0.29
% Change --- 8.50 19.05 30.08 38.89 6.89 16.40 26.43 12.33
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 12: Protein carbonyls (nmol of DNPH incorporated/mg protein) in the tissues of Labeo rohita following exposure to
sublethal concentrations of cypermethrin.
Organ
Control
Exposure periods
Lethal (h) Sub lethal (days)
24 48 72 96 1 5 10 15
Gill 0.90G 0.93E 1.02C 1.13B 1.16A 0.93F 1.01C 1.03C 0.96D
SD 0.06 0.06 0.07 0.08 0.08 0.06 0.07 0.07 0.06
% Change --- 3.69 13.56 25.89 29.59 3.218 12.33 14.92 7.39
Liver 0.65I 0.70H 0.82C 0.87A 0.85B 0.74G 0.80E 0.81D 0.75F
SD 0.04 0.04 0.05 0.06 0.06 0.05 0.05 0.05 0.05
% Change --- 6.76 25.38 33.84 30.45 13.53 21.99 23.68 15.22
Muscle 0.81H 0.87F 0.96B 0.99A 0.96B 0.85G 0.94D 0.95C 0.91E
SD 0.05 0.06 0.06 0.03 0.04 0.01 0.02 0.03 0.03
% Change --- 8.20 19.15 22.02 19.15 5.47 16.41 17.51 12.31
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 13: Total protein content (mg/g wet wt) in the organs of fish, Labeo rohita on exposure to the lethal and sublethal
concentrations of cypermethrin.
Organ
Control Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 99.58A 86.80E 81.40F 71.62G 55.38H 91.15D 79.98F 95.28C 96.61B
SD 2.11 0.61 0.57 0.50 0.39 0.64 0.56 0.67 0.68
% Change --- -12.83 -18.25 -28.07 -44.38 -8.46 -19.68 -4.32 -2.98
Muscle 135.30A 117.30E 106.26G 88.21H 77.85I 121.62D 110.70F 128.14C 131.14B
SD 1.91 0.82 0.75 0.62 0.55 0.86 0.78 0.90 0.92
% Change ---- -13.30 -21.46 -34.80 -42.45 -10.11 -18.17 -5.29 -3.07
Liver 184.28A 158.19D 139.62F 120.78G 99.28H 166.48C 155.65E 167.09C 181.78B
SD 2.60 1.11 0.98 0.85 0.70 1.17 1.10 1.18 1.28
% Change ---- -14.159 -24.23 -34.45 -46.12 -9.66 -15.53 -9.32 -1.35
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 14: Free amino acid levels (mg amino acid nitrogen / g wet wt.) in the organs of fish, Labeo rohita on exposure to the
lethal and sub lethal concentrations of cypermethrin.
Organ
Control Exposure periods
Lethal (h) Sub lethal (days)
24 48 72 96 1 5 10 15
Gill 11.78H 13.93F 15.24C 16.64B 20.03A 14.16E 14.80D 13.20F 12.72G
SD 0.25 0.09 0.10 0.11 0.14 0.10 0.10 0.09 0.08
% Change ---- 18.24 29.38 41.26 69.99 20.22 25.60 12.03 7.93
Muscle 15.02I 16.38G 20.68D 23.42B 25.49A 19.83E 21.76C 18.05F 15.91H
SD 0.21 0.11 0.14 0.16 0.18 0.14 0.15 0.12 0.11
% Change ---- 9.02 37.68 55.88 69.66 31.99 44.83 20.14 5.88
Liver 21.00G 23.00E 23.98E 27.62B 31.12A 25.88C 27.69B 24.88D 22.15F
SD 0.29 0.16 0.16 0.19 0.22 0.18 0.19 0.17 0.15
% Change ----- 9.54 14.20 31.52 48.19 23.28 31.87 18.47 5.47
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.
Organ
Control Exposure periods
Lethal (h) Sub lethal (days)
24 48 72 96 1 5 10 15
Gill 0.342H 0.405F 0.455C 0.467B 0.548A 0.437D 0.421E 0.392G 0.372G
SD 0.072 0.028 0.003 0.003 0.003 0.003 0.002 0.002 0.002
% Change ---- 18.16 32.92 36.37 60.09 27.50 22.82 14.54 8.55
Muscle 0.332H 0.384F 0.485C 0.512B 0.546A 0.471D 0.402E 0.380F 0.359G
SD 0.047 0.027 0.034 0.036 0.038 0.033 0.028 0.026 0.025
% Change ---- 15.49 45.861 54.021 64.00 41.52 21.01 14.34 8.09
Liver 0.428I 0.497E 0.543C 0.561B 0.674A 0.515D 0.484F 0.460G 0.438H
SD 0.006 0.003 0.003 0.003 0.004 0.003 0.003 0.003 0.003
% Change ---- 16.13 26.91 31.09 57.50 20.28 13.14 7.410 2.409
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;
Shaffi, et. al, 2000; Moore, et. al, 2003). The well-known membrane bound
transport ATPases are Ca2+-activated ATPase (Ca2+-ATPase: EC 3.6.3.8), and
Na+/K+-activated ATPase (Na+/K+-ATPase: EC 3.6.3.9). Na+/K+-ATPase
transports Na+ and K+ and plays a central role in whole-body osmoregulation
process (Sancho, et. al, 2003). Ca2+ is essential to the integrity of the cellular
membrane, the intracellular cements, and to the stabilization of branchial
permeability (Torre, et. al, 2000).
The inorganic ions play an important role in osmotic phenomena and
in the regulation of cellular metabolism. These are required by all animals to
provide suitable medium for protoplasmic activity. Any imbalance in the
levels of these ions in animals will lead to impairment in various
115
physiological activities (Leone and Ochs, 1987; Baskin, et. al, 1981). Freshwater
fishes are hyper osmotic to their medium. They gain water osmotically and
tend to loose solutes by diffusion. In the regulation of osmolarity of system
sodium, potassium and calcium ions play significant role to keep the
hyperosmotic properties of these animals (Narasimhan, et. al, 1983).
Sodium (Na+) is the principle cation of extra cellular fluids of most
animals. It maintains electro-neutrality and internal sodium concentrations
(Maetz and Romu, 1964). It also plays an important role in the osmotic
regulation of body fluids and serves as an essential activating ion for specific
enzyme system. The increased sodium ion content may cause a shift in ionic
symmetry with a consequent change in membrane permeability and
functional efficiency of Na+-K+ pumps.
Potassium ion (K+) is the prominent intracellular cation of animals. It is
an important co-factor in the regulation of osmotic pressure and acid-base
balance (Saxena, 1957). It plays a role in protein biosynthesis by ribosome and
is critical for the maintenance of normal membrane excitability. Many
enzymes require potassium for their maximum activity (Lehninger, 1990).
Potassium ions activate certain enzymes (transferases) and are essential for
the maintenance of normal membrane excitability. The consistency of
intracellular potassium, even with varying total osmotic concentration of
habit, may represent a very old cellular chamber (Prosser, 1973). It plays an
important role as an osmotic inorganic effecter in animals.
Calcium ion (Ca2+) is another important osmotic effectors and is
involved in conferring stability to the cell membrane. It is also a co-factor for
116
several oxidoreductases, proteases and ATPases. Calcium couples the
oxidation with contraction in muscle, for the maintenance of structural
integrity of mitochondria, sarcoplasmic reticulum and rate of enzyme
catalysis. Calcium content of tissues is an important factor (Harper, 1985).
Calcium is a general regulator of permeability of cell membrane to water and
other ions. High calcium level generally decreases permeability and low
calcium increases it. Hence, calcium level can be taken as index of
mitochondrial integrity and cellular metabolism. Any change in calcium level
can alter the mitochondrial function, protein synthesis and steady state of
enzymatic reactions (Narasimha Reddy, et. al, 1979). All these ions exist in
bound as well as in free forms. Bound ionic forms involve in metabolic
functions and free ions involve in osmoregularity in order contributing to
homeostasis of the cell system.
Adenosine triphosphatase (ATPase) enzymes are vital for regulating
oxidative phosphorylation, ionic transport, muscle function and several other
membrane transport dependent phenomena. Na+-K+ adenosine
triphosphatase (ATPase) has a central role in branchial transepithelial ion
transportation in fishes (Epstien, et. al, 1980). This enzyme is present in the cell
membrane of virtually all vertebrates (Skou, 1975) and is particularly
abundant in tissues associated with ionic and osmotic regulation (Boonkoom
and Alvarado, 1971). Mg2+ ATPase is a mitochondrial enzyme involved not
only in the lysis of ATP but also have a significant role in the initiation of ATP
synthesis (Lehninger, 1979). Mg2+ ATPase is found in association with both
Na+-K+ and Na+-NH4+ ATPase in fishes and it is related to the transport of
Mg2+ across the gill epithelium (Isaia and Masoni, 1976). This enzyme is also
117
essential for the integrity of the cellular membrane, intracellular cements and
for the stabilization of branchial permeability (Potts and Fleming, 1971; Isaia
and Masoni, 1976). Na+-K+ ATPase is a membrane bound sulfhydryl
containing enzyme whose function is critical for the maintenance of cell
viability (Ozcan, et. al, 2002). Na+-K+ ATPase is a biochemical expression of
active transport of Na+ and K+ in the cells (Skou, 1961). This enzyme carries
out the transport of sodium and potassium ions against concentration
gradient, resulting in the translocation of net charge. The enzyme acts as a
current generator and contributes to the membrane potential of the nerve cells
(Vizi and Oberfrank, 1992). This enzyme is known to be an early target for
oxygen radical induced damage to intact cell (Kako, et. al, 1998). It is an
energy dependent enzyme, which maintains ionic gradients crucial to
metabolite transport and osmotic gradients required for the maintenance of
cell volume. The transport of Na+ and K+ is vital for a number of cellular
processes such as maintenance of electro-physicochemical gradients across
the cell membranes, especially in nerve and muscle cells (Thomas, 1972),
transport of nutrients into interstitial cells (Crane, 1987) and uptake of
neurotransmitters in the brain (Iverson and Kelly, 1975).
The information pertaining to the effect of pyrethroids on ATPase
system in animals is scanty. The first report on ATPase inhibition by the
pyrethroid insecticide was given by Desaiah, et. al, (1972) and later supported
by Prasada Rao, et. al., (1984). Clark (1981) demonstrated a difference among
pyrethroids with respect to their ability to inhibit neural Na+ - K+ ATPase and
Ca2+ and Mg2+ ATPase. Na+ - K+ ATPase are oligomycin sensitive (OS). Mg2+
ATPase of fish and insect brain fractions were sensitive to the pyrethroid
118
compounds (Desaiah, et. al, 1972; Cutkomp, et. al, 1982; Dalela, et. al, 1978).
Janick and Kunter (1971); Campbell, et. al, (1974) and Nagender Reddy (1991)
also recorded similar observations. Several pesticides have been
demonstrated to be inhibitors of ATPase (Mehrotra, et. al, 1982; Desaiah, et. al,
1980; Bansal and Desaiah, 1982). Mitochondrial ATPases (Desaiah and Koch,
1975a) and plasma membrane (Matsumura and Narahashi, 1971) are the
target for their toxic actions. Clark and Matsumura (1982) recorded that the
pyrethroids (cypermethrin and decamethrin) inhibit the ATPase activity in
the squid Loligo. Malla Reddy et al., (1991) stated that the fenvalerate inhibit
the ATPase activity in selected tissues of fish, Cyprinus carpio. Exposure to
lethal and sublethal concentrations of cypermethrin and deltamethrin found
to alter ions and associated ATPases in freshwater fish Cirrhinus mrigala
(Prashant and David, 2010; Naik and David, 2010).
The literature available put forth by several researchers gives an
understanding on the effects of pesticides on ionic composition, associated
ATPase activities of freshwater fish. There is a necessity to understand and
establish relationship between the concentration of cypermethrin and its
responses on ions and associated ATPases. In view of this, an attempt has
been made to study levels of sodium, potassium and calcium ions and Na+-
K+, Mg2+ and Ca2+ ATPase activities in gill, muscle and liver of the freshwater
fish, Labeo rohita at acute and subacute exposure regimes in lethal and
sublethal concentrations of cypermethrin.
RESULTS
119
Changes in the levels of sodium, potassium and calcium ions and
activities of associated Na+ - K+ , Mg2+ and Ca2+ ATPase in acute and subacute
exposure regimes in gill, muscle and liver of fish, Labeo rohita were observed
(Table 16-21 and Figs. 13-19).
Effect on sodium ion levels: Decreases in the levels were observed in lethal
and sublethal concentrations in all the tissues (Table. 16). The maximum
decrease was observed in gill (56.80%) followed by muscle (48.95%) and liver
(41.29%) at 96 h and minimum was noticed in liver (12.10%) followed by
muscle (13.36%) and gill (18.65%) at 24 h. However variations in decrease
were observed at sublethal concentrations. The maximum of 28.79% decrease
was recorded in muscle on day 1 followed liver (23.39%) and gill (21.94%) on
day 5 (Fig. 13).
Effect on potassium ion levels: Potassium ion levels also exhibited similar
tendency of gradual decrement at lethal level and variations at sub lethal level
in all the three organs (Table. 17). The maximum decrease in gill (53.24%),
followed by muscle (50.80%) and liver (48.70%) at 96 h in lethal concentration.
The sublethal concentration depicted maximum (29.87%) in the gill tissue
followed by muscle (20.83%) and liver (12.98%) at day 5 and minimum
(4.28%) was recorded in liver on day 15 (Fig 14).
Effect on calcium ion levels: Reduced levels were observed in lethal and
sublethal concentrations in all the tissues (Table. 18). At lethal concentration
the maximum decrease was observed in muscle (61.52%) followed by gill
(60.45%) and liver (52.26%) at 96 h and minimum was noticed in liver
(12.10%) followed by muscle (13.36%) and gill (18.65%) at 24 h. At sublethal,
120
the maximum (36.29%) decrease was recorded in gill, followed by liver
(12.76%) on day 5 and muscle (15.78%) on day 1. Decline in the levels were
found to be continued from day 5 to 15 in the order of 5>10>15 irrespective of
the tissues (Fig. 15).
Effect on Na+-K ATPase activity: The enzyme activity was inhibited at both
lethal and sublethal concentrations in all the tissues (Table. 19). Lethal
concentration recorded gradual and continuous decrease in all the tissues
right from 24 h to 96 h. The maximum decrease was observed in gill (61.02%)
followed by muscle (49.41%) and liver (22.91%) at 96 h and minimum was
noticed in liver (5.89%) followed by muscle (14.47%) and gill (29.83%) at 24 h.
At sublethal concentration variations in the decreased activity were observed.
Initially from day 1 to 5 reductions were not much pronounced, the activity
was further decreased at day 10 and 15. The maximum (34.64%) decrease was
recorded in liver followed muscle (28.80%) on day 5 and gill (24.32%) on day
1 (Fig. 16).
Effect on Mg2+ATPase activity: Mg2+ATPase enzyme activity was inhibited at
both lethal and sublethal concentrations (Table. 20). The maximum decrease
was in gill (58.47%) followed by liver (49.34%) and muscle (40.85%) at 96 h
and minimum was in gill (17.11%) followed by muscle (20.36%) and liver
(31.31%) at 24 h. At sublethal concentration variations in the decreased
activity were observed. Initially from day 1 to 5 reductions were not much
prominent in comparison to day 10 and 15. The maximum decrease was
recorded in liver (24.95%) followed by gill (22.48%) and muscle (17.93%) on
day 5 (Fig. 17).
121
Effect on Ca2+ATPase activity: Inhibition in the Ca2+ATPase activities of fish
exposed to lethal and sublethal concentrations were observed in gill, muscle
and liver (Table. 21). At lethal concentration decrease was continuous with
increase in the exposure periods irrespective of the tissues. Gill (56.14%) was
recorded maximum reduced followed by muscle (48.15%) and liver (42.25%)
at 96 h of exposure. Similar observations were noted at sublethal
concentration, activity was diminished in all tissues right from day 1 to 15.
The maximum decrease was recorded in the muscle (28.68%) followed by
liver (21.81%) on 5th day and gill (19.92%) on 1st day of exposure. The lowest
decrease was noticed liver (3.47%), followed by gill (4.07%) on 15th day and
muscle (10.98%) on 10th day of exposure (Fig. 18).
DISCUSSION
Freshwater fish take up salts from their ambient medium to
compensate their loss through excretion. This obviously necessitates a high
metabolic demand for the regulation between the energetic and
osmoregulation in aquatic animals (Potts and Parry, 1964). Sodium,
potassium and calcium are not only important for the maintenance of
osmoregulation of body fluids (Baskin, et. al, 1981) but also for the transport of
nutrients from the lumen of the digestive tract into intestinal cells (Crane,
1977) and uptake of neurotransmitters in the brain (Iverson and Kelly, 1975).
ATPase enzyme complex helps in the uptake of ions from the external
medium to the interior of the body of the freshwater fishes. Disturbances in
ion regulation induced by toxicants are manifested by altered ion
concentrations. A number of biochemical studies have revealed that the
functional properties of macromolecules are altered under pesticide stress. To
122
gain an insight into the ion fluxes, the ions of biological importance like Na+-
K+, Ca2+ and Mg2+ were determined in important tissues of fish.
In the present study, the decrease in the levels of Na+ - K+, Ca2+ ions in
the gill, muscle and liver exposed to lethal and sub lethal concentrations of
cypermethrin indicates changes in the permeable properties of the cell
membrane of these organs and of deranged Na+ - K+ and Ca2+ ionic pumps
due to the probable consequences of tissue damage. The findings of present
investigation are in strong agreement with the previous studies under
pesticide stress in fishes (Kabeer Ahmed, et. al, 1981, Walser, 1960; Moorthy,
et. al, 1984; Edwards, 1973; Reddy and Philip, 1991; Siddiqui, et. al, 1993;
David, 1995; Narendra, et. al, 1993; Dave Prakasa Raju, 2000; Durairaj, 2001).
The results in the present study suggest that the sodium content
decreased as a function of time of exposure to cypermethrin. Sodium is the
major component of the cations of the extracellular fluid. It is largely
associated with chloride and bicarbonate in maintenance of acid base balance.
It maintains the osmotic pressure of body fluid and thus protects the body
against excessive fluid loss. It is known that sodium content in tissues mainly
depends on the permeability functional efficiency of bio-membrane and
efficient functional role of Na+ pump, which regulates ionic content of tissues.
The level of Na+ signifies its importance in the mobilization of water
transport, since sodium content in the membrane facilitates the water
movement among the tissues (Wilbur, 1972; Dietz, 1979). From the result, it is
evident that the Na+ loss is higher in the case of gill indicating the
derangement in Na+ transport. Also, the decreased sodium content in the
123
tissues of exposed fish indicates changes in permeable properties of different
bio-membrane systems to different extent by altering the Na+ pump (Kabeer
Ahmed, et. al, 1981; Rafat Yasmeen, 1986) and rupture in the respiratory
epithelium of gill tissue (Radhaiah, 1988; Anand Kumar, 1994).
A continuous decrease of K+ content in the tissue was observed in the
present study. This can be attributed to the fact that, pyrethrins affect nerve
membranes by modifying the sodium and potassium channels, resulting in
depolarization of the membranes. Moreover, the ions are actively taken up
from water via the chloride cells in the gill epithelium. For the ionic
movement, the membrane system in the chloride cells is important as this is
the structure with which Na+ and K+ ATPase is associated (Epstein et al.,
1980). It is known that any remarkable decrease in K+ level might be
accompanied by serious disturbances in muscular irritability, myocardial
function and respiration (Coles, 1967). The decrease in K+ content in the
tissues of Labeo rohita exposed to cypermethrin might be attributed to the
alterations observed in respiration at whole animal as observed in the present
investigation.
The decline of Ca2+ ion levels in the tissues on exposure to
cypermethrin indicating increased decalcification. It is known that Ca2+ plays
an important role in the regulation of cellular metabolism. It is required for
regulation of muscle contraction, transmission of impulses neuromuscular
excitability and regulation of protein binding capacity (Walser, 1960).
Mitochondria and endoplasmic reticulum are the two important subcellular
organelles involved in the maintenance of the calcium homeostasis (Borle,
124
1973). Mitochondrial Ca2+ ATPases and Ca2+ uptake are the two interlinked
processes involved in the maintenance of calcium. It is generally accepted that
many of the calcium’s effect on the cellular processes are regulated by
calmodulin. Calmodulin is responsible for Ca2+ dependent activation of a
variety of enzymes involved in a number of fundamental cellular functions
(Means, et. al, 1982). Lipophilic compounds bind with calmodulin with high
affinity and reduce the stimulatory effect of this protein on several enzymes.
Moreover decreased calcium content during pesticide stress corresponds to
structural changes in mitochondrial integrity (Miroslaw, 1973). Since
mitochondria act as “sinks” for intra cellular Ca2+ (Bygrave, 1978) and
principle storehouses of Ca2+ deposition, it appears that the decreased Ca2+ in
the present study might attribute to the disturbances in mitochondrial
integrity and subsequent respiratory distress. Hoar (1976) suggested that the
levels of amino acids and metabolites like pyruvate and lactate will be
increased under stress conditions to compensate the loss of inorganic ions.
Amino acids and lactate were found increased in the tissue of Cyprinus carpio
and Labeo rohila exposed to sub lethal concentration of fenvalerate (Malla
Reddy et al., 1991; Sridevi, 1991; David, 1995; and Narendra et al., 1997).
The decrease in sodium, potassium and calcium ion levels in the
organs of fish, exposed to cypermethrin could be attributed to the decreased
activities of Na+ - K+ , Mg2+ and Ca2+ ATPase (Renfro, et. al, 1974), since
ATPases have been described as prominent energy linked enzymes in fishes
(Desaiah, et. al, 1975). The decrease in these ions can be attributed to inhibition
of their carriers like ATPases which are found to be inhibited as reported by
125
different authors in fishes exposed to pesticides (Richards and Fromm, 1970;
Dalela, et. al., 1978, Epstein, et. al., 1967; Thebault and Decaris, 1983)
suggesting that the pesticide affects the active transport processes in the
membrane. The reduction in ATPase activities also suggests a drastic decrease
in the prolactin release, which might be particularly responsible for the
hypocalcemia (Roch and Maly, 1979, Giles, 1984; Larsson, et. al, 1981; Koyama
and Itazawa, 1977; Yamawaki, et. al, 1986; Pratap, et. al, 1989; David, 1995;
Dave Prakasa Raju, 2000; Durairaj, 2001). It is evident that the fish, Labeo rohita
under cypermethrin stress affects functional regulation of the ionic transport
and water permeability. The imbalance in bio-chemically changed
components like amino acids could be attributed to imbalance of ionic
composition.
In the present investigation, the activities of Mg2+, Na+ - K+ and Ca2+
ATPases are decreased in gill, muscle, and liver of the fish on exposure to
cypermethrin. The decrease in these activities indicates the demolition of
cellular ionic regulations in the organs of the fish as reported by Renfro, et. al,
(1974) and Schemidt Nelson, (1975). This disruption may be due to the effect
of cypermethrin on passive movement of ions i.e., the permeability
characteristics. In this connection, it is of interest to note that O2 consumption
has decreased in the fish Cyprinus carpio under fenvalerate stress (Malla
Reddy, 1987) and in the prawn Metapenaeus monoceros exposed to fenvalerate
(Reddy, et. al, 1991). The decrease in activities may also be due to interaction
of pesticide with Mg2+ and Na+ - K+ ATPases thereby inducting inhibition
126
(Dikshith, et. al, 1978). According to Price, (1978) the inhibition is due to
phosohorylation of active site of the enzyme.
Pyrethroids have great affinity for ATPase system and interact with the
molecules thereby inhibiting the activity (Desaiah, et. al, 1975; Prasad Rao, et.
al, 1984). The reduction in the activities indicates a general and persistent
derangement of mitochondrial activities under cypermethrin stress. The
inhibition of Mg2+ ATPase by cypermethrin points out the production of ATP
synthesis. Because of this, several energy dependent processes such as neural
Na+ - K+ and Ca2+ pumps result in cellular destruction (Cutkomp, et. al, 1982).
The loss in the ion specific ATPase could be attributed to the loss of sodium
and potassium ions due to cellular leakage into the body fluid.
Na+ - K+ ATPase is considered as a marker enzyme to understand the
physiological impairment of the cell (Campbell, et. al, 1974), the inhibition
reveals the disruption of ionic movement in neuronal and glial cells. Such
alterations in ionic balance depolarize the nerve and due to depolarization the
nerve cells increase in the releasing of neurotransmitter (Kimelberg and
Papahad, 1974) which in turn inhibits Na+ - K+ ATPase activity (Stojanovie, et.
al, 1980). These compounds are known to produce neurotoxic symptoms that
induce aggressive sparring, whole body tremor (Miller and Adams, 1982).
Cutkomp, et. al, (1982) reported that Na+ - K+ATPase are the Oligomycin
Sensitive (OS). Mg2+ ATPase of insect and fish brain fraction are sensitive to
these compounds. Inhibition of Na+ - K+ ATPase in vitro from the cockroach
nerve cord was reported earlier (Desaiah and Cutkomp, 1973). The present
study also demonstrates that cypermethrin acts as a potent inhibitor of
ATPases.
127
Pyrethroid related compounds inhibited Na+ - K+ ATPase
(Matsumura, 1975). Striking similarities exist between the neurotoxic action of
pyrethroids in vertebrates and invertebrates (Matsumura, 1975). Clark and
Matsumura (1982) reported the inhibition of Ca2+, Mg2+ ATPase in squid,
Laligo peales. The inhibition of ATPase activities in the present study and
greater decrease in the levels of ions observed in the gill, muscle and liver of
fish, exposed to lethal concentration of cypermethrin, indicate the effects of
cypermethrin on osmo ion-regulations of this animal. As the ion-regulatory
capacity is energy dependent process, the greater decrease in the energy
releasing pathways in fish subjected to lethal intoxication provides support
for the more decrease in the levels of Na+ - K+ and Ca2+ ions. Further greater
imbalance caused to the gill structures is also one of the probable reasons for
observed perturbations of ATPase activities and ionic levels in the fish. At
cellular level the availability of pesticide to interact with the ATPase might
depend on the cell surface area. However, in the sub lethal concentration,
significant elevations in ion levels and in the ATPase activities in the organs of
fish indicate the some degree of efficiency to resist the sublethal
concentrations of cypermethrin. Reddy et al., (1991), reported recovery of
ATPase in the freshwater crab, Oziotelphusa senex exposed to sub lethal
concentrations of endosulfan. This could be due to their higher protein
synthetic ability.
The increase in the ionic concentration may be helpful to the fish for
the maintenance of higher osmotic gradient in order to curb the speedy entry
of toxicant. The increase in oxidative metabolism also might have facilitated
128
these animals to elevate the ionic strength by meeting the energy demands.
Further the increase in ion levels may elevate the neuromuscular activity for
the enhancement of their synthetic potentials particularly related to pesticide
detoxification and elimination process. Also the increased ions may help the
easy uptake of the metabolites and the structural rigidity in the cellular
construction.
Greater degree of decrease in Na+ - K+ and Ca2+ levels and the activities
of Na+ - K+, Mg2+ and Ca2+ ATPases in the fish exposed to the lethal
concentration of cypermethrin, indicates severe disruption in the cellular ionic
regulation. High concentration of cypermethrin might have greatly altered the
permeability characteristics of the membranes of the organs by interacting
with the membrane proteins readily to serve alterations in the acute transport
through destabilizing the membrane bound enzymes and related hormonal
and energy producing process. Further, the progressive decrease in the ion
levels and progressive suppression of Na+ - K+, Mg2+ and Ca2+ ATPases
activities in the organs of fish, over time of exposure to the lethal
concentration of cypermethrin indicates the increase in the binding of the
cypermethrin to the active sites of membrane bound enzymes. Since, the
degree of inhibition is dependent on the concentration of cypermethrin
available to the active sites on enzyme molecules.
In sub lethal concentration of cypermethrin the Na+ - K+ and Ca2+
levels significantly decreased with ion competent inhibition of associated Na+
- K+ , Ca2+ and Mg2+ ATPase activities in all the tissues. Possibly the inhibition
of ATPase activity is dependent on the functional groups of the enzyme and
129
the amount of cypermethrin available for the competitive replacement of the
substrate. Further recruitment of chloride cells proposed as a fundamental
and physiologically significant response of freshwater fish to increase the
capability to take up Na+- K+ and Ca2+ from water (Leino, et. al, 1987).
In conclusion cypermethrin causes a decrease in ion levels and
dependent ATPase activities in gill, muscle and liver of Labeo rohita. Possibly,
affecting the cellular integrity and functions by acting at the membrane bound
enzyme system. Moreover these changes can be correlated with the observed
impairment in respiratory responses and altered behavioural anomalies at
both the concentrations, as these are high energy dependent physiological
processes. Moreover these changes can be correlated to the induction of
oxidative damage of bomolecules leading to oxidative stress. Since, activities
of these membrane bound enzymes depend on the phospholipid environment
of the membrane. Therefore, any change in the lipid component of the
membrane due to oxidative stress will directly affect the activities of these
enzymes.
Table 16: Sodium ion levels (M / g wet wt.) in the organs of fish, Labeo rohita on exposure to the lethal and sublethal
concentrations of cypermethrin.
Organs
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 57.26A 46.58E 42.86G 35.32H 24.73I 49.71C 44.70F 47.80D 54.24B
SD 1.21 0.32 0.30 0.24 0.17 0.35 0.31 0.33 0.38
% Change --- -18.65 -25.15 -38.31 -56.80 -13.18 -21.94 -16.51 -5.28
Muscle 46.51A 40.30E 34.48F 27.12H 23.74I 33.12G 42.33D 45.22C 45.30B
SD 0.65 0.28 0.24 0.19 0.16 0.23 0.29 0.31 0.32
% Change --- -13.36 -25.87 -41.68 -48.95 -28.79 -8.98 -2.79 -2.60
Liver 54.99A 48.34D 44.84F 38.69H 32.28I 45.77E 42.12G 51.15C 51.94B
SD 0.77 0.34 0.31 0.27 0.22 0.32 0.29 0.36 0.36
% Change --- -12.10 -18.46 -29.64 -41.29 -16.76 -23.39 -6.99 -5.55
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
sublethal concentrations of cypermethrin.
Organs
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 62.81A 48.79D 46.65E 35.36G 29.37H 52.16C 44.05F 57.19B 57.95B
SD 1.33 0.34 0.32 0.25 0.20 0.36 0.31 0.40 0.40
% Change --- -22.31 -25.72 -43.70 -53.24 -16.95 -29.87 -8.95 -7.74
Muscle 65.95A 58.717D 49.12G 39.99H 32.45I 57.70E 52.21F 59.73C 63.13B
SD 0.93 0.41 0.34 0.28 0.22 0.40 0.36 0.42 0.44
% Change --- -10.97 -25.52 -39.36 -50.8 -12.51 -20.83 -9.43 -4.28
Liver 54.93A 46.66F 42.29G 38.45H 28.18I 49.95D 47.80E 51.59C 52.24B
SD 0.77 0.32 0.29 0.27 0.19 0.35 0.33 0.36 0.36
% Change --- -15.05 -23.00 -30.01 -48.70 -9.07 -12.98 -6.07 -4.90
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 18: Calcium ion levels (M / g wet wt.) in the organs of fish, Labeo rohita on exposure to the lethal and sub
lethal concentrations of cypermethrin.
Organs
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 84.82A 64.13E 51.67G 41.727H 33.54I 74.33D 54.03F 77.33C 80.25B
SD 1.79 0.45 0.36 0.29 0.23 0.52 0.38 0.54 0.56
% Change --- -24.39 -39.07 -50.80 -60.45 -12.37 -36.29 -8.826 -5.38
Muscle 65.57A 53.50D 42.65G 37.04H 25.22I 55.22F 59.19E 62.05C 63.15B
SD 0.92 0.37 0.30 0.26 0.17 0.39 0.41 0.43 0.44
% Change --- -18.40 -34.95 -43.51 -61.52 -15.78 -9.72 -5.37 -3.69
Liver 72.02A 65.54D 56.26G 48.13H 34.38I 66.78E 62.83F 69.27B 67.91C
SD 1.01 0.46 0.39 0.34 0.24 0.47 0.44 0.48 0.48
% Change --- -8.98 -21.87 -33.16 -52.26 -7.26 -12.76 -3.81 -5.69
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.
Organs
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 7.58A 5.32F 4.63G 3.79H 2.95I 5.74E 6.14D 6.90C 7.06B
SD 0.160 0.031 0.032 0.026 0.020 0.040 0.043 0.048 0.049
% Change --- -29.83 -38.91 -50.01 -61.02 -24.32 -18.98 -8.96 -6.91
Muscle 4.91A 4.20D 3.88E 3.57F 2.48H 4.49C 3.50G 4.59B 4.55B
SD 0.069 0.029 0.027 0.025 0.017 0.031 0.024 0.032 0.032
% Change --- -14.47 -21.08 -27.24 -49.41 -8.57 -28.80 -6.59 -7.36
Liver 3.89A 3.6625B 3.46D 3.14E 3.00F 2.78G 2.54H 3.564C 3.65B
SD 0.055 0.025 0.024 0.022 0.021 0.019 0.017 0.025 0.025
% Change --- -5.89 -11.03 -19.30 -22.91 -28.36 -34.64 -8.41 -5.99
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.
Organs
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 5.01A 4.15C 3.62D 3.21E 2.08F 4.12C 3.88D 4.60C 4.78B
SD 0.103 0.029 0.025 0.022 0.014 0.029 0.027 0.032 0.033
% Change --- -17.11 -27.69 -35.94 -58.47 -17.79 -22.48 -8.13 -4.61
Muscle 5.10A 4.06F 3.91G 3.61H 3.02H 4.64D 4.19E 4.95B 4.84C
SD 0.072 0.028 0.027 0.025 0.021 0.032 0.029 0.035 0.034
% Change --- -20.36 -23.43 -29.31 -40.85 -9.12 -17.93 -2.93 -5.22
Liver 7.66A 5.26F 5.19F 4.77G 3.88H 6.36D 5.74E 6.72C 7.32B
SD 0.108 0.037 0.036 0.033 0.027 0.045 0.040 0.047 0.051
% Change --- -31.31 -32.13 -37.64 -49.34 -16.91 -24.95 -12.17 -4.33
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.
Organs
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 8.98A 6.82F 6.61G 5.05H 3.93I 7.19E 7.90D 8.44C 8.61B
SD 0.190 0.048 0.046 0.035 0.027 0.050 0.055 0.059 0.060
% Change --- -24.04 -26.40 -43.72 -56.14 -19.92 -11.96 -6.04 -4.07
Muscle 5.54A 4.05E 3.87G 3.65H 2.87I 4.46D 3.95F 4.79C 4.93B
SD 0.0784 0.0286 0.0274 0.0258 0.0203 0.0315 0.0279 0.0339 0.0349
% Change --- -26.90 -30.11 -34.11 -48.19 -19.56 -28.68 -13.50 -10.98
Liver 2.98A 2.46E 2.23G 1.95H 1.72I 2.64D 2.33F 2.76C 2.87B
SD 0.042 0.017 0.015 0.013 0.012 0.018 0.016 0.019 0.020
% Change --- -17.47 -24.99 -34.40 -42.25 -11.18 -21.81 -7.32 -3.47
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
proteins, free amino acids, protease activities, alanine amino transferase
(AlAT), aspartate amino transferase (AAT), Glutamine dehydrogenase
(GDH), in the organs of the fish Labeo rohita on exposure to lethal 24, 48, 72
and 96 h and 1, 5, 10 and 15 days of sub lethal concentrations of cypermethrin,
besides controls. All results are presented in the tables from 13 and 22-28; fig
10 and 19-25 for comparison.
Soluble, Structural and Total Proteins: From the data presented in tables 13,
22, 23 and figures 10, 19, 20, a significant decrease relative to controls is seen
in the soluble, structural and total proteins of all the organs of fish, Labeo
rohita at all the exposure periods in the lethal concentrations of cypermethrin.
These protein levels also recorded a significant decrease in the organs of fish
on day 1 and 5 on exposure to sublethal concentration but on further
exposure gradual reduction in the increase was observed at 10 and 15 day
(Tables 14, 15, 16 Fig. 10, 11, 12).
135
Among the exposure periods, the levels of soluble, structural and total
proteins significantly decreased in the gill, muscle and liver over control fish.
The minimum decrease was observed at 24 h and maximum at 96 h on
exposure to the lethal concentrations. The decrease was progressive and
found to be in the order of 24 48 72 96 h. The same was not the case at
sublethal concentration, they were found to be in the order of 1 5 10 < 15.
Among the organs of fish, the decrease in protein (Soluble, Structural and
Total) was greater in gill than liver and muscle subjected to the lethal and sub
lethal concentrations (Liver > Gill >Muscle).
Free Amino Acid Levels and Protease Activity: From the data presented in the
table 24 and 25 and figures 21 and 22, corresponding to the decrease in
protein content (Soluble, Structural and Total Proteins) a steep increase in free
amino acid levels and protease activity in all the organs of fish at all the
exposure periods in the lethal concentration of cypermethrin was seen. In sub
lethal concentration also, though free amino acid and protease activity
recorded an reduction magnitude was decreased, it is predominantly more in
the organs of the fish subjected to lethal than the sub lethal concentration.
Aspertate Amino transferase (AAT) and Alanine Amino Transferase (ALAT)
Activity: From the data presented in the tables 26 and 27 and figures 23 and
24, it is evident that the AAT and ALAT activities increased in the fish on
exposure to lethal and sub lethal concentrations of cypermethrin. It was
observed that AAT and ALAT activities significantly increased in the gill,
muscle and liver of fish exposed to lethal concentration of cypermethrin and
was in the order 24 < 48 < 72 < 96 h; at sublethal concentration also similar
trend was witnessed. Increased magnitude was less than the lethal
136
concentration in the order 1 < 5 > 10 > 15 days. Among the organs of fish
subjected to lethal concentration the increase in AAT, ALAT activities were
more in the muscle than in the gill and liver, which was in the order Muscle >
Gill > Liver on day 1 and 5. However, further increase in exposure periods
magnitude was found to be reduced.
GDH Activity: From the data presented in the table 28 and fig 25 the activity
of GDH elevated progressively and significantly over control. At lethal
concentration, the GDH activity significantly increased in the gill, muscle and
liver. The level of increase was greater at 96 h (gill 48.34%; muscle 48.32%;
liver 42.13%) and less at day 24 h (liver 12.38%; gill 11.11%; muscle 5.85%).
The increase was progressive and the magnitude of it was in the order 1 < 2<
3 < 4 day. In sub lethal concentration, variations were observed in increase
day 1 to 15. The maximum increase in the activity was noted in gill (21.58%)
tissue, followed by muscle (19.50%) and liver (16.98%) on day 10. The
minimum was noted in liver on day 15 (4.66%). Gill, liver and muscle showed
continuous increase from day 1 to 10 and on 15th day the activity found to be
reduced in the increased activity.
DISCUSSION
In the present study, in vivo effects of cypermethrin on the protein
metabolism of the tissues of the fish, Labeo rohita exhibited tissue-specific and
time-dependent alterations. Total, structural and soluble protein contents
were depleted in all the tissues (gill, liverand muscle) exposed to the lethal
concentration of cypermethrin indicating the breakdown of these proteins due
to the acute pesticide toxic stress. Generally the breakdown of proteins
dominates over synthesis under enhanced proteolytic activity (Harper, et. al,
137
1979). It is evident in the present study that the hypoprotenemia is associated
with the steep elevation in protease activity and free amino acid levels in the
organs of the fish exposed to the lethal concentrations. Estimation of total
proteins and amino acid contents of various internal organs of tissue are
considered as important factors for toxicological studies (Mary Chandravathy
and Reddy, 1996). The maintenance of proteins in a highly organized state
requires an active and continuous supply of energy. If this is impaired the
organ structures breakdown and proteins particularly denature in their
configuration. According to Bradbury, et. al, (1987), pyrethroids are reported
to have profound effects on tissue protein reserves.
Similar observations were recorded with other pesticides in various
fishes, as reported in Cirrhinus mrigala (Swarup, et. al, 1981) exposed to
endosulfan. Acute exposure to Benzenehexachloride (BHC) caused a marked
reduction in structural proteins and soluble proteins in the tissues of Channa
punctatus (Singh and Singh, 1998). Short-term exposure of Ozioteiphusa senex
senex to endosulfan caused a significant reduction in the total proteins of gill
(Rajendra, 1985). In the light of these observations, it would seem logical to
state that pesticide toxicity in short-term exposures stimulates proteolysis in
tissues by activating protease enzymes. Protein depletion in tissues may
constitute a physiological mechanism and may play a role of compensatory
mechanism under pesticidal stress, to provide intermediates to the Kreb’s
cycle or to enhance osmolarity, by retaining free amino acid content in
hemolymph, to compensate osmoregulatory problems encountered due to the
leakage of ions and other essential molecules, during the pesticide stress
(Rafat Yasmeen, 1986; Rajeshwari, 1986). The depletion of protein suggests
138
increased proteolysis and possible utilization of the products of their
degradation for metabolic purposes (Klassan, 1991). The depletion of protein
level induces to diversification of energy to meet the impending energy
demands during the toxic stress (David, 1995).
Increased protease activity, a lysosomal enzyme, in the organs of
exposed fish could be due to the damage caused by the high concentration of
pesticide in lysosomes resulting in the leakage of these enzymes into the
cytosol. In addition, an increase in proteolytic activity can be attributed to the
destruction of organ systems and thereby disturbing the biochemical
functioning of cellular activities (Karel and Saxena, 1975) and also due to the
impairment of protein synthetic potentials (Gary, et. al, 1989; Malla Reddy and
Philip, 1991). High concentrations of metals also decrease the proteolytic
activity (Sreedevi, et. al, 1992; Sreenivasula Reddy and Bhagyalakshmi, 1994;
David, 1995). Thus the severe proteolytic activity, whether due to the
lysosomal instability, cellular destruction or the decreased protein synthetic
potentials might be the reason for the decreased soluble, structural and total
protein content, in the organs of fish exposed to the lethal concentration for a
longer period. Intensity of the proteolytic activity decreased with the increase
in exposure periods. Reddy and Yellamma (1991) also reported a steep
proteolytic activity over time of exposure in the organs of cockroach exposed
to acute concentrations of fenvalerate.
Increase in protease activity in different tissues in the present study is
clearly reflected in the decrease in soluble, structural and total protein levels
of the tissues. Tissue-specific and time-dependent depletion of protein content
accompanied with an enhanced acid, alkalineand neutral protease activity has
139
been reported in freshwater fish, Channa punctatus, (Geethanjali, 1988) during
BHC intoxication. The depletion of protein level induces to diversification of
energy to meet the impending energy demand during toxic stress (Jagadeesan
and Mathivanan, 1999). Under proteolysis, enhanced breakdown dominates
over synthesis while in the case of anabolic process, increased synthesis
dominates the protein breakdown (Harper, 1979). This is further corroborated
through the increased levels of free amino acids in all the tissues. These amino
acids might be fed into the TCA cycle as keto acids by way of transantination,
since transmnases are known to be elevated during pesticide intoxication
(Kabeer Ahmed Sahib 1979; Jha and Verma, 2002). The increased levels of free
amino acids might also be due to increased synthetic potentiality. This
possibility might exist in the tissues of cypermethrin exposed fish.
It appears that protein degradation is in active phase over synthesis in
the gill, muscle and liver of fish at sub lethal concentration of cypermethrin as
evidenced from the decrease in soluble, structural and total proteins with the
significant increase in protease activity and amino acid levels. Similar reports
were observed in Mus boodoja on exposure to BHC (Philip, et. al, 1988). Malla
Reddy and Philip (1991) reported similar effects on the liver of freshwater
fish, Cyprinus carpio treated with malathion. But reduced decrease in soluble,
structural and total proteins along with gradual rise in protease activity and
free amino acid levels in the gill, muscle and liver of fish at day 10 and 15
indicates the onset of acceleratory phase of protein synthesis over breakdown.
The reduced decrease in structural proteins could be helpful to the animal to
fortify its organs for developing resistance to the imposed sub lethal toxic
stress; further the reduced magnitude of decrease in soluble protein fraction
140
could indicate the synthesis of enzymes necessary for detoxification. This
serves as a device to remove the fraction of cypermethrin from the general
intracellular environment and helps the animal to adopt to the imposed toxic
stress. Protein synthesis being an energetically expensive process, the increase
in oxidative metabolism of the fish during sub lethal cypermethrin stress also
strengthens the increase in its protein synthetic potentials. Elevation in free
amino acid as observed by Kabeer, et. al, (1984); Rajamannar and Manohar,
(1998); Deva Prakasa Raju, (2000) reports, suggest intensive proteolysis
contributing to the rise in the free amino acid pool, which becomes a source of
carboxylic acid cycle intermediates by both the transmination reactions. This
view supports the findings of the present investigation and also strengthens
the earlier reports of Shakoori, et. al, (1976); Ganeshan, et. al, (1989); David
(1995); Deva Prakasa Raju (2000) and Jha and Verma (2002) and suggest the
operation of gluconcogenesis in order to mitigate of toxic stress.
Degradation of proteins by proteolytic enzymes results in increased
amino acid pool. Further, prevalence of pathological conditions in the organ
systems of an animal may decrease protein synthetic acid pool. The above two
factors could be responsible for the increase in free amino acid levels in the
organs of fish exposed to the lethal concentration of cypermethrin. The
increased free amino acids might have been fed into TCA cycle as keto acids
by the way of trans-de amination since AAT, ALAT and GDH activity
increased upon exposure. High concentrations of amino acids in tissues can
lead to hyper amino acedemia which inturn can cause a number of side effects
on the physiological conditions of the cell. The increase in the free amino
acids in the organs of fish exposed to sublethal concentrations can be partly
141
due to the increased proteolytic activity and partly due to certain
transaminases reported to be indicators of protein degradation in salmonoids
(Bell, 1968) and liver intoxication in rainbow trout (Gingerich and Weber,
1976). Amino transferases are influenced by a variety of environmental and
physiological conditions (Knox and Greengard, 1965). To have an insight into
the role of these enzymes in the altered metabolism of cypermethrin
intoxicated fish, the activities of both AAT and ALAT were investigated in the
present experiment. Elevated levels of AAT and ALAT indicate the enhanced
transamination of amino acids, which may provide keto acids to serve as
precursors in the synthesis of essential organic elements. These are in
consonance with earlier reports in field crab, Barytelphusa querini (Nagender
Reddy, et. al, 1991), Clarias batrachus (Ravinder, et. al, 1989), Cyprinus carpio
(Malla Reddy, et. al, 1991) during the toxic stress of endosulfan,
phosphomidon, dichlorovos and fenvalerate respectively. It is likely that toxic
stress imposed by cypermethrin might be one of the factors for the observed
activities of AAT and ALAT in the present study.
The activity of aspartate and alanine amino transferases (AAT and
AlAT), which serve as strategic links between protein and carbohydrate
metabolisms, is known to alter under several physiological and pathological
conditions (David, et. al, 2004). GDH, a mitochondrial enzyme, catalysis the
oxidative deamination of glutamate, providing -ketoglutarate to the kerbs
cycle (Reddy and Philip, 1991). This enzyme is having several metabolic
functions with great physiological significance. It is closely associated with
the detoxification mechanisms of tissues. GDH in extra-hepatic tissues could
be utilized for channelling of ammonia released during proteolysis for its
142
detoxification into urea in the liver. Hence, the activities of AAT, AlAT and
GDH are considered as sensitive indicators of stress (Gould, et. al, 1976).
Higher levels of free amino acid content may also be attributed to the
decreased utilization of amino acids (Seshagiri, et. al, 1987) and is also
suggestive of catabolism of protein or transamination of keto acids (Shakoori,
et. al, 1976). The transaminase (AAT, ALAT) elevation in the present study
offers an excellent support to the observed increase, in amino acid levels.
Amino acids may be shunted into the Kreb’s cycle through transamination
and oxidative deamination. The aminotransferases serve as a strategic link
between carbohydrate and protein metabolism under environmental stress
(Knox and Greengard, 1963). Increase in AAT and ALAT levels indicates in
this study shown in the fish under toxic stress, the amino acids appear to be
mobolised to get transmineted to 2-keto acids, for use in the production of
energy rich compounds (Knox and Greengard, 1965; David, 1995; Deva
Prakasa Raju, 2000 and Rajamannar and Manohar, 2000).
The decreased magnitude of increase in free amino acid levels with the
increased exposure periods could indicate the speedy channelling of these
bio-molecules for the synthesis of required proteins and to meet the energy
demands by incorporating into TCA cycle in the form of keto acids through
trans-de-amination reactions (Suresh, et. al, 1991), as evidenced by the
gradual increase in AAT, ALAT and GDH activities. Further, more increase in
the free amino acids level during the initial periods of sublethal exposure can
also act as an osmotic and ionic effector (Jurss, 1980) to bring electrostatic
equilibrium between the external medium and ions of the blood and regulate
ionic and osmotic balance (Schemidt-Nielson, 1975). Hence, there will be
143
constant mobilization of these biomolecules to contribute to various metabolic
pathways and regulate protein synthesis.
GDH is also known to play a crucial role in ammonia metabolism and
is known to be affected by a variety of effectors (Ramanadikshitulu, et. al,
1976). It has several metabolic functions with great physiological significance
and known to be closely associated with the detoxification mechanisms of
tissues. In the present study the significant elevation in the activities of these
enzymes in the organs of fish, exposed to lethal concentration of cypermethrin
indicates greater association of oligomers of these enzymes in response to
toxic stress, probably the elevation in the trans-de-aniination reaction may
facilitate the fish to reorganise as energetics in order to resist the toxic stress.
This shows that oxidative deamination is contributing towards high ammonia
production. The high levels of ammonia produced is not eliminated but is
salvaged through GDH activity which is utilized for amino acid synthesis
through transminases (Rajyashree and Dabeer, 1994)
Increase in AAT and ALAT levels indicates that there is an active
transamination of amino acids and operation of keto acids. The increase in the
activities of hepatic aminotransferases in the present study is in agreement
with earlier reports demonstrating consistent increases in these activities
under conditions of enhanced gluconeogenesis (Knox and Greengard, 1965).
Dikshith, et. al, (1978) have reported similar effects on the liver transaminase
levels of guinea pigs treated with lindane. Enhanced levels of transaminases
were also observed in Anabas testudineus exposed to lindane (Ghosh and
Chattergie, 2002). Reddy and Yellamma (1991) reported that elevated AAT,
ALAT and GDH activities reveal increased operation of transamination in
144
order to contribute glucogenic amino acids to carbohydrate metabolic
pathways to cope with cypermethrin induced energy crisis. It is also evident
that ALAT representing the anaerobic segment was comparatively greater
than feeding of ketoacids through oxalo acetate into citric acid cycle by
AATand ALAT.
The steady rise in the activities of AAT, ALAT and GDH in the organs
of fish exposed to sub lethal concentration of cypermethrin from 1, 5, 10 and
15 days may be due to the synthesis of these enzymes under the subacute
cypermethrin stress. The slow increase in soluble protein in the fish exposed
to the sub lethal stress could also support the elevation in these enzyme
activities. The increase could be due to the stepwise induction of these
enzymes greater and eater association of their oligomers (Kulkarni and
Kulkarni, 1987). The increase in these enzyme activities could be helpful to the
fish for structural reorganization of proteins and incorporation of keto acids
into the TCA cycle to favour gluconeogenesis or energy production. The
steady increase in these enzyme activities may be helpful in metabolic
compensation and to allow the animal to adapt to the imposed toxic stress.
The elevation in GDH activity in the sub lethal concentration could lead to
increased production of glutamate in order to eliminate ammonia (Harper, et.
al, 1979). Increased amino acid levels could be partly responsible for the GDH
activity. In addition, the increase in AAT and ALAT activities results in the
greater production of glutamate, which in turn favours the elevation of GDH
activity. The increased glutamate partly aids in meeting the energy demands
under toxic stress by entering into the TCA cycle. This links protein
metabolism with energetics.
Table 13: Total protein content (mg/g wet wt) in the organs of fish, Labeo rohita on exposure to the lethal and
sublethal concentrations of cypermethrin (Presented for ready reference)
Organs
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 99.58A 86.80E 81.40F 71.62H 55.38I 91.15D 79.98G 95.28C 96.61B
SD 0.31 0.61 0.57 0.50 0.39 0.64 0.56 0.67 0.68
% Change -12.83 -18.25 -28.078 -44.38 -8.46 -19.68 -4.32 -2.98
Muscle 135.30A 117.30E 106.26G 88.21H 77.85I 121.62D 110.70F 128.14C 131.14B
SD 0.91 0.82 0.75 0.62 0.55 0.86 0.78 0.90 0.92
% Change -13.30 -21.46 -34.80 -42.45 -10.11 -18.17 -5.29 -3.07
Liver 184.28A 158.19E 139.62G 120.78H 99.28I 166.48D 155.65F 167.09C 181.78B
SD 0.60 0.11 0.98 0.85 0.70 0.17 0.10 0.18 0.28
% Change -14.15 -24.23 -34.45 -46.12 -9.66 -15.53 -9.32 -1.35
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
sublethal concentrations of cypermethrin.
Organs
Control
Exposure period in days
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 41.80A 35.68D 32.9535E 29.39F 23.95G 38.29C 34.93D 39.44B 39.34B
SD 0.88 0.25 0.23 0.20 0.16 0.27 0.24 0.27 0.27
% Change -14.64 -21.16 -29.69 -42.70 -8.39 -16.42 -5.64 -5.87
Muscle 59.97A 52.76D 50.62E 43.97F 41.70G 55.36C 50.76E 57.02B 57.71B
SD 0.84 0.37 0.35 0.31 0.29 0.39 0.35 0.40 0.40
% Change -12.02 -15.59 -26.67 -30.46 -7.69 -15.37 -4.92 -3.77
Liver 86.22A 70.30F 65.86G 55.20H 43.89I 82.65D 79.04E 84.05B 83.38C
SD 1.21 0.49 0.46 0.39 0.31 0.58 0.55 0.59 0.58
% Change -18.46 -23.61 -35.97 -49.09 -4.14 -8.32 -2.51 -3.29
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
sublethal concentrations of cypermethrin.
Organs
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 57.89A 51.12E 48.45F 42.23H 31.32I 52.86D 45.04G 54.82C 56.25B
SD 1.22 0.36 0.34 0.29 0.22 0.37 0.31 0.38 0.39
% Change -11.69 -16.31 -27.04 -45.89 -8.69 -22.18 -5.29 -2.83
Muscle 75.31A 63.52E 55.64G 44.23H 36.13I 66.22D 59.94F 70.41C 73.42B
SD 1.06 0.44 0.39 0.31 0.25 0.46 0.42 0.49 0.51
% Change -15.66 -26.12 -41.27 -52.02 -12.06 -20.40 -6.50 -2.51
Liver 98.06A 87.89C 73.76F 65.58G 55.38H 83.83D 76.61E 83.04D 94.70B
SD 1.38 0.62 0.52 0.46 0.39 0.59 0.54 0.58 0.66
% Change -10.37 -24.78 -33.12 --43.51 -14.51 -21.87 -15.31 -3.42
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.
Organs
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 11.78A 13.93E 15.24C 16.64B 20.03A 14.16D 14.80D 13.20F 12.72G
SD 0.25 0.09 0.10 0.11 0.14 0.10 0.10 0.09 0.08
% Change 18.24 29.38 41.26 69.99 20.22 25.60 12.03 7.93
Muscle 15.02A 16.38G 20.68D 23.42B 25.49A 19.83E 21.74C 18.05F 15.91H
SD 0.21 0.11 0.14 0.16 0.18 0.14 0.15 0.12 0.11
% Change 9.02 37.68 55.88 69.66 31.99 44.83 20.14 5.88
Liver 21.00I 23.00F 23.98F 27.62C 31.12A 25.88D 27.6B 24.88E 22.15H
SD 0.29 0.16 0.16 0.19 0.22 0.18 0.19 0.17 0.15
% Change 9.54 14.20 31.52 48.19 23.28 31.87 18.47 5.47
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.
Organs
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 0.3427I 0.4050F 0.4556C 0.4674B 0.5487A 0.4370D 0.4210E 0.3926G 0.3721H
SD 0.0072 0.0028 0.0032 0.0033 0.0038 0.0030 0.0029 0.0027 0.0026
% Change 18.16 32.92 36.37 60.09 27.50 22.82 14.54 8.55
Muscle 0.3329G 0.3845E 0.4856C 0.5128B 0.5460A 0.4712C 0.4029D 0.3807E 0.3599F
SD 0.0047 0.0027 0.0034 0.0036 0.0038 0.0033 0.0028 0.0026 0.0025
% Change 15.49 45.86 54.02 64.00 41.52 21.01 14.34 8.09
Liver 0.4282I 0.4973E 0.5435C 0.5614B 0.6745A 0.5151D 0.4845F 0.4600G 0.4385H
SD 0.0060 0.0035 0.0038 0.0039 0.0047 0.0036 0.0034 0.0032 0.0031
% Change 16.13 26.91 31.09 57.50 20.28 13.14 7.41 2.40
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.
Organs
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 1.2807H 1.5129E 1.6007D 1.7469C 1.9491A 1.6485D 1.8283B 1.4458F 1.3450G
SD 0.027 0.010 0.011 0.012 0.013 0.011 0.012 0.010 0.009
% Change 18.13 24.98 36.40 52.19 28.72 42.76 12.89 5.02
Muscle 1.9163H 2.3439E 2.7133C 2.8278B 2.9639A 2.2273F 2.4101D 2.1942G 2.1153G
SD 0.027 0.016 0.019 0.019 0.020 0.015 0.017 0.015 0.014
% Change 22.31 41.59 47.56 54.66 16.23 25.76 14.50 10.38
Liver 2.2265H 2.7054D 2.9310C 3.0967B 3.3130A 2.6077E 2.7538D 2.4397F 2.3830G
SD 0.031 0.019 0.020 0.021 0.023 0.018 0.019 0.017 0.016
% Change 21.50 31.64 39.08 48.79 17.12 23.68 9.57 7.02
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.
Organs
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 1.3917I 1.7819F 1.8948E 2.0883B 2.3909A 1.6704G 1.9934C 1.9608D 1.4861H
SD 0.029 0.012 0.013 0.014 0.016 0.011 0.014 0.013 0.010
% Change 28.03 36.14 50.05 71.79 20.02 43.23 40.88 6.78
Muscle 4.1811I 4.7954E 5.7707C 6.0528B 6.5803A 4.7493F 5.1162D 4.5110G 4.3554H
SD 0.059 0.033 0.040 0.042 0.046 0.033 0.036 0.031 0.030
% Change 14.69 38.01 44.76 57.38 13.59 22.36 7.88 4.16
Liver 5.8937I 7.0902F 7.8158E 8.8378B 9.9917A 6.5715G 8.0909C 7.9910D 6.1788H
SD 0.083 0.050 0.055 0.062 0.070 0.046 0.057 0.056 0.043
% Change 20.29 32.61 49.95 69.53 11.49 37.27 35.58 4.83
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
and sublethal concentrations of cypermethrin.
Organs
Control
Exposure periods
Lethal (h) Sublethal (days)
24 48 72 96 1 5 10 15
Gill 0.1245H 0.1383F 0.1469D 0.1687B 0.1847A 0.1317G 0.1394E 0.1514C 0.1382F
SD 0.0026 0.0009 0.0010 0.0011 0.0013 0.0009 0.0009 0.0010 0.0009
% Change 11.11 18.01 35.53 48.34 5.766 12.01 21.58 11.04
Muscle 0.1599I 0.1692H 0.1858D 0.2239B 0.2371A 0.1710G 0.1768E 0.1910C 0.1743F
SD 0.0022 0.0059 0.0013 0.0015 0.0016 0.0012 0.0012 0.0013 0.0012
% Change 5.85 16.23 40.05 48.32 6.95 10.61 19.50 9.03
Liver 0.3771H 0.4238E 0.4472C 0.5139B 0.5360A 0.4005F 0.4310D 0.4412C 0.3947G
SD 0.0053 0.0029 0.0031 0.0036 0.0037 0.0028 0.0030 0.0031 0.0027
% Change 12.38 18.59 36.27 42.13 6.19 14.28 16.98 4.66
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
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