A. ATPases Activity

ATPases represent a colnplex enzynic system which has reqliire~nent For M i ' ,

~ a " , NR', Kt' ions for their activity. Pl~o~pho~~yliitiou is Llic rnosl important stcp i n

several nietabolic pathways particularly i n the energy yielding metabolic processes,

which are catalyzed by the involvement of ATP. ATPases involve in the cleavages of

ATP to ADPIAMP and inorganic phosphate with encrgy relcase. Further tllc ATPasc

being an integrated enzyme of mitochondria, any damage to tlic mitochondria,

ultimately alters its activities, which would interfere with conversion of oxidative

energy to phosphorylated energy. This reaction takes place in the presence of M$

and has an absolute requirement for both ~ a ' ' and K". Among ATPases, the N$-

K' ATPase is mainly p~'esent in the nerve cells, ~ a ' ATPase in the ml~scle cell and

~ g " ATPase in all types of cells (Hnrper el al, 1977). The enzynic ~ a " - K* ATPase

has been shown to play an important role in aquatic organisms, which maintain

haemolymph ion concentrations different from ion concentrations of their environment

(Skou, 1960; Quinn and Lane, 1966). ATPases are not only responsible for

asymmetric distribution of Na" and Kt ions across the cell membrane but d s o in

conjunctiot~ with other transport proteins, mediate the bulk movcment of iol~s ilntl fluitl

in a variety of tissues. The mitocl~ondrial ATPases havc a role in oxidative

pi~osphorylation (Abrams eta!., 1972).

ATPasc is considered as an index to detcrnlinc thc tolcrancc lcvcls of

envirommental contominants like pesticides, which are now a dnys prcvalellt in ttlc

aquatic environlnent due to indiscrimini~tc application against agricultural pests and

vectors. The contanlinants particularly organo phosphorous conipounds in the aquatic

environment, owing to lipophilic nature, easily penetrate into fish tllroogh food, body

surface and gills and alter the lipid level (Subburajn ef nl., 1989). It is evident that thc

lipid moiety, the fatty acid and its fluidity are necessary for the activity of rnenibrane

bound enzyme ATPase (Kinlei berg et 01.. 1974).

N~+-K+ ATPase was found to be l~igll in the brain, while the M ~ ~ ' ATPase was

high in muscle in accordance with their ft~nctionnl roles in nervc conduction and

muscle movements respectively (Riyaz Basha el al., 1997). The membrnne

localization is the key to the physiological function to ATPases, which is coupled with

cations across the membranes from one intra cellular component to mother.

Since the ATPnses seem to be the target enzymes for several pesticides nnd the

efficiency of the organism's metabolic function is also dependent on the ATPases, a

study was performed on the ATPases in the fish, Tilapia nrossanibica exposed to

chlorpyriphos and azadirachtin.

Results

Data presented in Table 2.1.4; Fig. 2 , lA represent thc total ATI1;lscs activity.

The activity levels were expressed as p moles of pi foulled I 111g protein / liour, 111

contl.ol fish total ATPases activity was greater in brein followccl by livcr, gill a~ , t l

muscle.

The total ATPase activity lias shown a significant decreasing t r c ~ ~ d ill all tllc

tissues of fish exposed to chlorpyriphos as well as azadirachtin and it was progressive

through out the timc coilrsc of study. Maximuin inhibition was observed in 15 days

exposure period in all the tissues. In chlorpyriphos exposcd fish, the per cent change

ranged from 15.61 - 39.51% in brain, 29.6 - 44.36% in livcr, 13.67 - 37.47% in gill

and 10.81 - 38.07% in muscle tissues of 7 and 15 days respectively, wllcre ns

azadirachtin exposed fish showed the per cent change ranged from 8.25 - 18.65% in

brain, 8.15 - 31.17 in liver, 9.99 - 25.44 in gill and 1.57 - 7.75 in muscle tissues of 7

and 15 days respectively (Table. 2.1A; Fig. 2.1A).

The decrease in total ATPase activity during different exposure periods is

statistically significant (P<0.001) over control in all the tissues except in muscle tissue

of 7 days exposed to azadirachtin.

Discussion

In the present investigation chlorpyriphos and azadiracl~tin inhibited the activity

of total ATPase and the decrement was more pronounced in 15 days exposed fishes

(Table 2.IA; Fig. 2.1A). The results suggest that the ATPase has a particx~lar

sensitivity to both chlorpyriphos and azadirachtin.

ATPase regulates the sodium metaholis~n and tlic activc c;ltioii Ll.al~sport

through the mernbranc and maintains the ion gradients requirctl in the propagation of

the nemc impulse. ATPasc is responsible for thc integrity ol' cell ~nc~nbrii~ies a i d is

implicated in the direct modulation of synaptic action (Swcacincr nnd Guldin, 1980;

Sweadner, 1979). The inhibition of ATPese activity could change tlic gradients of

sodiun~ and potassiu~n across the cell membrane and disturb the several nctions of

nerve cells. According to Price (1978) the inhibition is due to phosphorylation of

active site of the enzyme as in the case of acetylcholincstertlse iniiibitioo. Since

ATPase is considered as a marker enzyme to understand thc physiological impairment

of the cell (Campbell el a[., 1974), the it~hibition reveals the disruption of ionic

movement in neuronat cclls. Such alterations in. ionic balance dcpolar~ze the nerve tlnd

due to depolarization the nerve cells increase in tile releasing of neurolransniitters,

which in turn inhibit Wa+-Kt ATPase activity (Stojanovic el a!., 1980). Further the

inhibition of ATPase cnzyrne may prcvent the hydrolysis of ATP (Manchando, 1996),

the ATP stimulates the release of neurotransmitter in sytnpathetic end plate. In

mammals, organochlorine compounds are reported to bring their toxic effects by

inhibiting ATPases (Sing et 01. 1998). DDT blocks the activity of mitochondrial M?

ATPase more effectively than the N~+-K' ATPase (Cut kolnp el al. 1982). Aldrin

inhibited the three ATPases in the tissues of liver, kidney of clarins bntrachus and

cirrllinn mrigclla (Gupta, 1989). Babu el al. (1990) reported inhibition of ~ a " ATPase

by Propoxur in rat brain. Nat-Kt ATPase inhibition was seen in fish exposed to

endosulfan (Sastry et a1.1984). Vani (1991) reported the inhibition of ATPases by

chlordane in hepatic tissues of albino rat.

50

Although it is well docilmentcd that orgatiochlo~~inc compountls i l l l ~ i l > i t

ATPases activity. It is evident from the recent report8 that Kl'Pi~scs nrc illflllellcerl

adversely by several other pesticides. Tlic study of Natarajan (1985) rcvunls that Op

pesticides inhibit branchial N~'-K' ATPusc and M ~ " ATPase activity in C / ~ u t ~ t ~ n

striatiis. Methyl parathion and diazinon also suppress [he ATPasc activity ill diffelerlt

lissues of fish (Shiva prflsad Rao and RAO, 1984; Sastry snd Siiarma, 1980). Ih11.ai liaj

ei 01. (1996) reported the decrement of ATPnses eclivity in brain tissue of Quinlphos

and penthoate-exposed fish, o~eocl~roa~is nlossotribicus. Similar inliibition was

observed in different tissues of Heteroptte~cstes fossilis when cxposed to Dinocap

(Icumar and Sharma, 1994). Similar trend was observed by Swarnalntliu and

Rarnalnurthi (1996) in liver and kidney tissues of fenvalerate administered albino rat.

Several authors reported an alteration in the activity of ATPase by heavy

metals. Jagnnnadha Rao (1990) reported that nluminum trifluoridc inhibits ATPase

activity in rat brain. Shoenmakers et nl. (1992) reported the inhibition of NU'-K'

ATPase in the fish Oreochrotr~is mosso~iibicus by cadmium chloride. Jayesh Thakcr el

crl. (1999) reported the inhibition of ~a'-K' ATPase in vital organs of mudskipper,

periopkthnlttzus dipes by chromium. Sushma (1999) reported tl~at aluminurn acetate

inhibits ATPase activity in different tissues of albino mice.

The results of the present study also support the above findings. In this study

total ATPase activity was inhibited differentially in different tissues inferring thc

differential sensitivity of tissues to the insecticides. In tlie present investigation both

the compounds chlorpyriphos and azadirachtin suppressed the total ATPase activity in

all the tissues of experimental fish and the dccrcmcnt was mole sig~iificini~ ill 15 tliiys

exposed animals than 7 days exposed animals.

The low total ATPase activity suggests thc low synthesis of A'I'P, which migllt

be due to the inhibition of dehydrogeneses. Earlicr reports of Shiva prosatl Rao and

Rao (1979), Tripnthi and Shukla (1988), Radhaial~ and Rao (1990) and Santha~nrna

(1999) show that pesticides inhibit the tictivitics of del~ydrogcnases due to tllc

cytoarchitectural damage, particularly thnt of mitochondria. Kanungo (1993) reportcd

that neem oil reduced ATP content i ~ r vitro in rat liver mitochondria, which has il

possible explanation that the nceiu oil uncouples mitochondrial oxidative

phosphorylation and inhibits respiratory chain. This might bc the reason for reduced

ATPase activity in the azadirnchtit~ treated animals. Recently, Rnhaman et (11. (1999)

and Reddy et al. (2001a, b) reported the azadiraction inhibited ATPases activity in the

cockroach, Periplanein Anlerica~in.

B. Acid a n d Alkalinc phosphatases

The phosphatase systein consists of acid and alkaline phosphatases, which are

lysosomal in origin (Allison and Malucci,1964; Hirschorn el al., 19G5; McWhinnie

and Piecchowski, 1976). The phosphatase system comes into operation when the

tissue is facing energy crisis. Their activities indicate lysosomal function. The

continuity in their action will certainly indicate dysfunction of the cellular system and

this is more so incases of severe pathologioal damage as seen in necrosis, dystrophy

etc (Novikoff, 1961; De Duve, 1963). The phosphatase system norn~ally acts to

compensate the energy crisis under conditions where the ATPase system is either

inhibited or not so efficient in functioning.

52

Normally, the alkaline phosphatase is involved in mineral metabolisti~. I t

provides phosphates for bone lnaterial (Robinson, I932), but Further studies link this

enzyme to the elaboration of fibrous proteins (Sobcl and I-Ianok, 1952),

mucopolysaccharides (Moog and Wenger, 1952). Though alkaline pliosphatnse is

ubiquitously distributed in most of the tissues, sites of high activity are those

exhibiting active transport. Allcaline phosphatases are considered non-specific

hydrolyses (Stadtmani, 1961) and they are known to regulate levels of P-

glycerophosphate available for triglyceride syntl~esis brought about through the

catalysis of transphospliorylation reaction (Wallach atid I-Ioward KO, 1964).

Acid phosphatase is a hydrolytic enzyme, which takes part in the dissolution of

dead cells and serves as a good indicator of stress condition in the biological systems

(Gupta et a[., 1975; vernla et al., 1980). The acid phosphatase has limited range of

activity; optimal p1-I being 5.0 (oser, 1965). Variety of isozytnes have been found in

prostate, liver, spleen, kidney, serum and other tissues. Total ACP may be increased

in malignant tumors involving bone, renal disease, hepatobiliary disease, diseases of

the reticuloendothelial system and thromboembolistn (Murray et al., 1995).

Results and Discussion

Alkaline and Acid phosphatases activities were determined in different tissues

of control, chlorpyriphos and azadiraclitin exposed fish. The results are presented in

tables 2.1B and 2.2B; Fig's 2 , lB and 2.2B. In control animals, the activity of

alkaline and acid phosphatases is greater in liver followed by gill, brain and muscle.

In clilorpyriphos exposure, a significant increase in the activity lcvels of

cnzy~nes was observed in all tissues of fish. The elevation of cnzynle activities was

increased with period of exposure, where as in azadirachtin trcattncnt a significant

increase in 7 days was obsclved followed by a significant decrease io 15 days in all the

tissues of fish.

In cl~lorpyripl~os exposed fish, increase in alkaline and acid phospliatase

activities can be interpreted as a shift of the tissues emphasis on energy breakdown

pathway from normal ATPase system to phosphatase system, Pesticides are yeported

to reduce glycogen levels and increase phospl~orylase activities (Mishra and

Srivasthava, 1984). In the event of decreased ATPase system phosphorylation may be

preceded by activated phosphatases to catalyse the liberation of inorganic phosphates

from phosphate esters.

The elevation in the enzyme activity could be attributed to the damage of cell

membranes and subsequent leakage of the enzymes (Srinivason et al., 1991). Similar

increase in phosphatase activity of Raiza hcxadaclyla exposed to a cyclodien

compound aldrin was reported (Vijay joseph et a[., 1993) Venkata Krishna Reddy

(1996) reported increased activity of phosphatases in different brain regions of rat

under the influence of cadmiuln toxicity, Recently Samson Raju (2000) also reported

the increased activity of pl~ospl~atases in different tissues of albino rat in response to

sodium selenite.

The illcreased phosphatase activity in 15 days of chlorpyriphos indicated that it

had toxic effect and strong impact on the degradation of cells.

In contrast to the above findings azadiraclitin i~~liibited tllc activity of alkalinc

and acid phosphatases. Similar decreasing t re~~ds in alknline and acid phosphatase

activities were reported in all tissues of Notopterlrs riotoptcrrcs exposcd to plienol and

dinitrophenol (Venna et a/., 1980). Decrcase in liver alkaline phosphatase activity

was recorded in Opkiocephnlus punctatus exposed to coppcr (Srivastavn and Pandy,

1982) and Cyprinus carpio exposed to vegetable oil factory effluent (Ramcsh et al.,

1984), Baby shaikila et nl. (1993) have reported that the observed decrease in tlie

activity of these enzymes in liver of Sarotherodorz mossnmbicirs probably facilitates

the increased activity of pliospliorylnse enzyme aud subsequent breakdown of

glycogen for energy releasing during pesticide stress, Hence the reduction in the

activity of these enzymes could be considered adaptive for fish to facilitate tlie

breakdown of glycogen to meet the energy demand. They further inferred that severe

acidosis rnay be responsible for inhibition of alkalille and acid phosphatases in liver of

Sarotherodon rnossantbicus, which in turn could be adaptive for fish to meet the

energy demand in anaerobic breakdown of glycogen.

Alkaline phospllatase a brush border eilzyme is responsible for the

phosphorylation more likely by hydrolysis of sugar phosphate esters in the diet at

alkaline pH and mediates membrane transport (Ramesh et al., 1984). Uncoupling of

Oxidative phosphorylation is the main cause for the inhibition of phosphatases (Verma

et nl,, 1980). Dalela et a/. (1982) suggested that the uncoupling might have also

occurred which promotes the spilling of an energy rich intermediate compound prior to

ATP production, Recently Indm et 01. (1999) have reported the reduced activity of

alkalinc phosphatase in 0reocArolni.s r i~oss~~i~~l~icrrs cxposctl to phenol. Iickha SI1:lrmn

(1996) reported an initial increase and later dcclinc in acid and alkaline phosphstasc

activity in kidney tissue of Ci~nnncc pitrilat~rs exposcd to niercuric clrloridc. Muniya11

and Vceraraghavan (1999) llave reported the rcdoced activity of thcsc cnzynnes in

O~~cochro~t~is ~iiossciii~bicirs cxposcd to etofenprox (Trebon). Kasutri ci (11. (1907)

reported the decreased activity of acid phosphatase in selninal vcsicles and vc~ltral

prostate of albino rats administered to dry Azrrdirnclttn indicn Icaf powdcr. Subash

peter (1993) also reported the reduced activity of Alkaline phosphatase in blooti and

liver of fresh water teleost, Atrnbns iest~rdineus exposed to Nceni kernal extract.

From the foregiven account, it is clear that chlorpyriphos and azadirachtin toxic

stress is lowering the rate of the energy metabolic levels forcing the fish to adilpt to a

sustained metabolic level, by shifting the gears of energy trapping from one segmcnt to

the other. This state of metabolism is obtained by taking advantage of thc diversified

pathways of energetics in physiological functions. But it is certain that either

clilorpyiphos or azadirachtin intoxicated fishes cannot raise to the situation to cope up

with high-energy demands, if necessary as there is limited energy production. Thus

ally process on the part of the fish that require high energy will tend to prove fatal.

Table 2.1A

Effect of chlorpyriphos and slzatlirachtit~ on Total ATPases activity (p moles of inorganic phosphate formeti/ mg protcin/h) in fish, T.nr ossninbicn.

Values in the parenthesis are percent change over control Each value is mean of six individual observations *SD: standard deviation

Liver

Muscle

Brain

Gill

I

Control

13.977 .C 0.381

10.048 f 0.392

20.362 k0.279

11.109 k0.427

One-way Analysis of Variance (ANQYA) Source of Variation

Treatments(between columns) Residuals(within columns)

Total 1119

The P value is < 0.0001, considered extremely significant,

Menn F

1817.70

7 Days

9.839 f0.317 (-29.60)

8.901 i0.305 (-108 1

17.182 f 0.592 (-15.6)

3.590 f0.964 (-13,67)

Degrees of freedom

19

100

94.174

0.2836

15 Days

7.776 10.405 (-44.36)

6.222 +0.502 (-38.07)

12.316 k0.813

(-39.51)

6.946 k0.779 (-37.47)

7 Days -

12.837 k0.279 (-8.15)

9.890 t0.351 (-1.57)

18.682 f0.405 (-8,25)

9.999 f 0.476 ( 9 9 )

Sum of squares

1789.30

28.364

332.02

15 Days

9.538 k0.613 (-31.17)

9.269 t0.3 1 6 (-7.75)

16.564 k0.45 1 (-18.65)

8.282 k0.832 (-25.44)

Effect of chlorpyriphos and azndisscbtin 011 the activity of alknli~~e Phosphatase (p moles of illorgallic phosphate forrnctl/ mg proteinlh) in fish, T,/iiossn~tzbicn.

Values in the parenthesis are percent change over control Each value is mean of six individual observations kSD: standard deviation

Liver

Muscle

Brain

Gill

One-way Analysis of Variance (ANOVA)

Control

1.907 It 0.044

1.054 rt0.027

1.210 k0.055

1.849 +0.064

The P value is < 0.0001, considered extremely significant.

Treatments(between columns) Residuals(within columns)

Total

-- Chlorpyripl~os

Sum of squares

46.143

0.3239

46.467

Degrees of freedom

19

100

119

7 Dnys

2.033 40,042 (+9.75)

1.149 f0.030 (+9.13)

1.550 40.065

(+28.09)

2.887 20.094

(+56.13)

hacl i racl~l i t~

15 Days

2.611 20.008

(+36.91)

1.225 k0.072

(+29.88)

1.750 k0.104

(-t.44.62)

3.204 20.071

(4-73.28)

7 Days

2.008 t0.028 (-529)

1.106 M.029 (+4.93)

1.341 k0.034 (+10.82)

1.988 20.070 (+7.51)

Mean square

2,423

0.003239

15Dnys

1.628 -1-0.062 (-14.G3)

0.887 k0.03 1 (-15.84)

1.065 40.052 (-1 1.98)

1.528 i0.117 (-17.36)

-

F

749.71

Table 2.21)

Effect of cblorpyriphos and azadiracl~tin on the activity of acid phosphatases ()I. moles of inorganic phosphntc formed I mg pt.otein/lt) in fisli, T.r~iossar~tlbicn.

Values in the parenthesis are percent change over control Each value is mean of six individual observations kSD: standard deviation

Liver

Mr~scle

Brain

Gill

One-way Analysis of Variance (ANOVA)

Colltrol

1.193 +_ 0.055

0.645 k0.027

0.735 k0.030

0.874 t0.054

-

The P value is < 0.0001, considered extremely significant.

Treatments(between columns) Residuals(within columns)

Total

7 Days -----

1.367 3.033 (+14.58)

0.766 k0.023 (t20.54)

0.886 50.030 (-t20.54)

1.265 k0. 122 (t44.73)

Mean square

0.5555

0.003559

15 Days

1.615 k0.055

Ct-35.37)

0.923 39.039

(+43.10)

0.95G k0.047

(t30.06)

1.451 40,115

(+66.01)

7 Days

1.328 a 0 3 6

(-1-1 1.37)

0.750 Y3.00 (t16.27)

0.881 k0.033

(+19.86)

0.979 k0.064

(+12.01)

P

156.08

Degrees of freedom

19

100

119

15 Days

1.082 k0.043 (-930)

0.536 3 . 0 4 7 (-16.87)

0.632 k0,027 (-14.10)

0.658 k0.121 (-24.71)

Sum of squares

10.555

0.3559

10,911

Iiig. 2.1 A: FI'i'i.c.t ol' (:lilorl~yril)ltos r l~ t t l kusdiritcl~li~l ott 'li)t:tl iYl'l?~sc activity (~fi~to'les of'i~~el'j:;t~~ic 11ltospllntc formrtl I tug l11.1)fci11111) ~ I I lislt, ;I~rrr~srsrr~v~bir~ir

-- , Liver M U S C I ~ Braln G ~ I I

OClllorpyrlphos 7 Days Q A ~ d l r s c h N n 7 Days

Liver Musols Brain 011

ClChlarpyrlphos 16 Days BlAzatllraohUn ~~ 16 ~ Days

Llver Muscle Braln 0111

ClChlorpylphos 7 Day8 Mhzadlracl~tln 7 Days

Llvar Musole Braln GI11

I;IChlorpylphos 16 Days MAzedlraohUn 16 Oays

Llver Musolo Braln Gill

CIChlorpplphos 7 Days OAzartlraohUn 7 Days

-- , ~ l v e r Mu~ole Braln Glll

mChlorpplphos 16 Days mAzadfraohtln 16Days