chapter 6 biochemical changes -...

87

Chapter 6

BIOCHEMICAL CHANGES

INTRODUCTION

Recent evidence indicates that fish, an extremely valuable resource, are

quickly becoming scarce. One consequence of this scarcity is the increasing

concern for fish survival and a growing interest in identifying the levels of

various chemical pollutants, which are safe for fish and other aquatic life. The

acetyl cholinesterase (AChE) activity is vital to normal behavior and muscular

function and represents a prime target on which some toxicants exert adverse

effects. Inhibition of acetylcholinesterase (AChE), the enzyme involved in

terminating the action of neurotransmitter acetylcholine (ACh), is perhaps the

most often studied. The two main transmitter substances in vertebrate’s

nervous systems are ACh and noradrenaline. Acetylcholine is an ammonium

compound. It was the first transmitter substance to be isolated in 1920.

Neurons releasing acetylcholine are described as cholinergic neurons and

those releasing noradrenaline are described as adrenergic neurons. The

arrivals of nerve impulses at the synaptic knob depolarize the presynaptic

membrane, causing calcium channels to open. As the calcium ions rush into

the synaptic knob they cause synaptic vesicles to fuse with the presynaptic

membrane, releasing their level into the synaptic cleft (exocytosis). The

vesicles then return to the cytoplasm where they are refilled with the

transmitter substance, acetylcholine (Fukuta, 1990).

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


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


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


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



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


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



Table 12: Protein carbonyls (nmol of DNPH incorporated/mg protein) in the tissues of Labeo rohita following exposure to


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



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


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



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



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



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



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



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


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


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


Gill Liver Muscle

Fig 9: Percent change over control in protein carbonyl levels in the tissues of Labeo rohita following exposure to lethal and


0

5

10

15

20

25

30

35

40

24 48 72 96 1 5 10 15

Perc

en

t ch

an

ge


Gill Liver Muscle

Fig 10: Percent change over control in total protein levels in the tissues of Labeo rohita following exposure to lethal and


-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


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


0

10

20

30

40

50

60

70

80

24 48 72 96 1 5 10 15

Perc

en

t ch

an

ge


Gill Liver Muscle

Fig 12: Percent change over control in protease activity in the tissues of Labeo rohita following exposure to lethal and sublethal


0

10

20

30

40

50

60

70

24 48 72 96 1 5 10 15

Perc

en

t ch

an

ge


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


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


Organs

Control

Exposure periods


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


Organs

Control

Exposure periods


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


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


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


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


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


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


Table 21: Ca2+ ATPase activity (M of Pi formed / mg protein / h ) in the organs of fish, Labeo rohita on exposure to


Organs

Control

Exposure periods


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


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


Fig. 13. Percent change in sodium ion levels in the organs of Labeo rohita on exposure to the lethal and sublethal


-60

-50

-40

-30

-20

-10

0

24 48 72 96 1 5 10 15

Perc

en

t ch

an

ge


Gill Liver Muscle

Fig. 14. Percent change in potassium ion levels in the organs of Labeo rohita on exposure to the lethal and sublethal


-60

-50

-40

-30

-20

-10

0

24 48 72 96 1 5 10 15

Perc

en

t ch

an

ge


Gill Liver Muscle

Fig. 15. Percent change in calcium ion levels in the organs of Labeo rohita on exposure to the lethal and sublethal


-70

-60

-50

-40

-30

-20

-10

0

24 48 72 96 1 5 10 15

Perc

en

t ch

an

ge


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


-70

-60

-50

-40

-30

-20

-10

0

24 48 72 96 1 5 10 15

Perc

en

t ch

an

ge


Gill Liver Muscle

Fig. 17. Percent change in Mg2+ ATPase activity in the organs of Labeo rohita on exposure to the lethal and sublethal


-70

-60

-50

-40

-30

-20

-10

0

24 48 72 96 1 5 10 15

perc

en

t ch

an

ge


Gill Liver Muscle

Fig. 18. Percent change in Ca2+ ATPase activity in the organs of Labeo rohita on exposure to the lethal and sublethal


-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


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


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


Table 22: Soluble protein content (mg/g wet wt.) in the organs of fish, Labeo rohita on exposure to the lethal and


Organs

Control

Exposure period in 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


Table 23: Structural protein content (mg/g wet wt.) in the organs of fish, Labeo rohita on exposure to the lethal and


Organs

Control

Exposure periods


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


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


Table 24: Free amino acid levels (mg amino acid nitrogen / g wet wt.) in the organs of fish, Labeo rohita on exposure to


Organs

Control

Exposure periods


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


Table 25: Protease activity (M amino acid nitrogen / mg protein / h) in the organs of fish, Labeo rohita on exposure to


Organs

Control

Exposure periods


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


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


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


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


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


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


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


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


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


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


-60

-50

-40

-30

-20

-10

0

24 48 72 96 1 5 10 15

Pe

rce

nt

chan

ge


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


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


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


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


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


Gill Liver Muscle

chapter 6 biochemical changes -...

Documents