Bull. Environm. Contam. Toxicol. 20,769-777 (1978)

In vivo Effect on ATPase in Certain Tissues of Labeo rohita and Saccobranchus fossilis, Following Chronic Chlordane Intoxication S. R. Verma,S. K. Bansal,A. K. Gupta,and R. C. Dalela Pollution Relevant Research Lab., Department of Zoology, D.A.V. (P.G.) College, Muzaffarnagar-251001 India

Voluminous i n f o r m a t i o n i s a v a i l a b l e on the r e s i d u a l t o x i c i t y and the o t h e r environmental impacts of the p e s t i cides, b u t t h e r e i s l i t t l e i n f o r m a t i o n a v a i l a b l e on the mechanism of t o x i c a c t i o n os the o r g a n o c h l o r i n e p e s t i c i d e s e s p e c i a l l y on studies concerning a c t i v e t r a n s p o r t across c e l l u l a r membranes. One of the i n i t i a l e f f o r t s to explain the a c t i o n of DDT i n a c t i v e t r a n s p o r t was made by MATSUMURA et al. (1969). By using differential centrifugatlon techniques, they isolated various nerve components of rat brain and localized the source of ATPase sensitive to DDT. Their results indicated that (Na+, K +, Mg++) dependent ATPase in the rat brain is specifically sensitive to DDT. They suggested that DDT is casually related to disruption of ion transport mechanisms in the nervous system in vivo. Using similar methods, JANICKI AND KINTER (1971) c~c[ds~rely showed that DDT inhibits (Na+, K +, M g ~ dependent ATPases engaged in active sodium transport functioning to maintain tissue osmolarity. In long term exposure of o r g a n o c h l o r i n e compounds i n v i y o , (Na+, K+, M ~ 9 dependent ATPases were i n h i b i t e d ~-n several f i s h species (KOCH et a l . 1972, DESAIAH et a l . 1975). They also noted t h a t i n some cases, n o t a b l y at the lower c o n c e n t r a t i o n s of DDT, e r r a t i c s t i m u l a t i o n occurred. However, s t i m u l a t i o n was most pronounced i n kidney and liver tissues. Therefore, the purpose of t h i s study was t o d e t e r mine the e f f e c t of v a r i o u s c o n c e n t r a t i o n s of chlordane upon the ATPase system i n the b r a i n , g i l l , l i v e r and kidney o f two f r e s h water t e l e o s t s , Labeo r o h i t a and Saccobranchus f o s s i l i s f o l l o w i n g c h r o - ~ c ~ r 6 ~ n e intox i c a t t o n a f t e r 30 and 60 d a y s ' t r e a t m e n t .

Materials and Methods The fishes, Labeo rohita and Saccobranchus fossili~ ! known as ! Rohu , and-~Sing i ' ~! , respectively in vernacular Hindi language belong to the order Cypriniformes and family Cyprinidae and ~accobranchidae respectively. These are easily available in the rivers and ponds of this m

Communication No. 48. 0007-4861/78/0020-0769 $01.80 9 1978 Springer-Verlag New York Inc.

region. The size of Saccobranchus fossili$ selected varies from 150-220 mm !average 190 mm) and weight from 45-72 gm (average 60 gm) while in Labeo rohita, the size varies from 130-165 mm (average lSO mm) an--~--~ight varies from I00-II0 gm (average 105 gm). The pesticide chlordane (1,2,4,5,6,7~8,8-octachloro-2,3,3a, 4, 7,7a-hexahydro4,7-methanoindene) made by M/s Rallis India Ltd., Bombay, and available in 20~ emulsifiable concentration was used for the present study. A f t e r the normal process of acclimatization and washing with I~ light KMnO 4 solution so as to avoid the possibility of any infection, the fishes were transferred into the experimental tanks. The concentrations of experimental.pesticide were made by adopting the dilution technique (STANDARD MHTHODS, 13th edition, 1971). The sublethal concentrations of I/2, i/3 and !/6 of the T ~ O values for Labeo rohita and Saccobranchus fossilis as obtained b y ' ~ e-~--a~. (19~7, 1978)'were taken for long term exposure of both the fishes. The fishes were fed with an artificial diet and the water was renewed at every five days'inte~r

Activity of ATPase was determined in fish tissues by measuring the amount of inorganic phosphate produced when adenosine triphosphate was converted to adenosine diphosphate. The f i s h t i s s u e s were d i s s e c t e d out and weighed. Each t i s s u e sample was t r a n s f e r r e d t o a c o l d s o l u t i o n c o n t a i n i n g 0.25 M sucrose, 0.005 M disodium ethylene diamine t e t r a c e t i c acid, and 0.003 M h i s t i d i n e b u f f e r (pH 7 . 4 t o y i e l d a 5%W/V c o n c e n t r a t i o n ) . Tissue were homogenized i n a ground glass homogenizer i n ice. A c t i v i t y of (Na+, K+, M ~ dependent ATPase was determined at 20-24vC. Glassware was washed immediately a f t e r each d e t e r m i n a t i o n w i t h d i s t i l l e d deionized water and w i t h Fiske and SubbaRow reagents. A f t e r each f i f t h determinati o n , the glassware was washed w i t h hot ION HC1. A 0.2 ml a l i q u o t of the t i s s u e homogenate was added to 4.25 ml of the i n c u b a t i o n media c o n t a i n i n g 20 mM h i s t i d i n e b u f f e r (pH 7 . 4 ) , 100 mM NaC1, and 20 n%M ED1. Samples were assayed in triplicate. The r e a c t i o n was i n i t i a t e d by a d d i t i o n of 50 ~L of 100 mM Nat ATP and 100 mM MgC1 and continued agitation for 30 mzn at 24~ ATPase activity was terminated by the addition of I.O 31 of ice cold 30% trichloroacetic acid. Samplesowere then transferred for 30 min to a refrigerator at 5 C to allow complete precipitation of the homogenate proteins. The precipitate was sedimented in Remi clinical centrifuge at 3,000 ~ m for 3 min. Total ATPase activity was measured with Na , K ~ Mg++in the reaction mixture. Mg++ATPase activity was measured

770

using I mM ouabatn, a cardiac glycocide, t n the r e a c t i o n § m i x t u r e , t h e l a t e r being a s p e c i f i c i n h i b i t o r of Na+, K ATPase (McILWAIN 1963). Na+, K+ ATPase a c t i v i t y i s t o t a l a c t i v i t y minus the Mgm+ATPase a c t i v i t y . I n o r g a n i c phosphate produced as a r e s u l t o f the cleavage os ATP to ADP was measured by the method os FISKE AND SUBBARO# (1925) as modified by BARTLETT (1958). Color development proceeded at room temperature f o r 10 mtn. P r o t e i n was determined by the procedure developed by LO#RY et a l . (1951), using a s p e c t r o n i c - 2 0 c o l o r t m e t e r . Results and Discussion According to the several t h e o r i e s of a c t i v e t r a n s p o r t , ATPase i s s p e c i f i c a l l y r e q u i r e d f o r the t r a n s p o r t of i o n s a g a i n s t c o n c e n t r a t i o n g r a d i e n t and across membranes. The mechanism of the a c t i o n of chlordane on ATPase system may be due to the uncoupling of o x i d a t i v e phosphoz~/lation which causes a d e p l e t i o n i n the p h o s p h o r y l a t i o n product-ATP. This r e d u c t i o n i n a v a i l a b l e f r e e phosphate would be d i r e c t l y p r o p o r t i o n a l t o the r e d u c t i o n of t o t a l ATP produced. The s e n s i t i v i t y of ATPases from b r a i n , 9111, l i v e r and kidney o f Labeo r o h t t a and Saccobranchus f o s s i l t s a f t e r chronic c - ~ d a n - - ~ t o x i c a t i o n was determined i n ~ and the r e s u l t s are t a b u l a t e d i n t a b l e s I t o IV. The i n h i b i t i o n os t o t a l , Mg++and Na+, K+ ATPases by chlordane were q u i t e s i m i l a r to each o t h e r and t h e r e was an increased i n h i b i t i o n w i t h increased c o n c e n t r a t i o n os the t o x i c a n t (Table I - I V ) . DESAIAH~ND KOCH (1975) observed t h a t the i n h i b i t i o n os Na+, K+ ATPase by KeponeR and DCPD was q u i t e s i m i l a r and t h e r e was an increased i n h i b i t i o n w i t h increased c o n c e n t r a t i o n of the t o x i c a n t . CUTKOMP et a l . li971a) also observed similar response with dicofol Kelthane R) on blue gill brain Na , K + ATPase. They also showed that a number of other organochlorines which inhibited Na+, K § ATPase did not show such a progressive response. I n t h e present i n v e s t i g a t i o n t authors observed t h a t at t h e lowest s u b l e t h a l c o n c e n t r a t i o n ( l / 6 t h TLsQ) i n both the f i s h e s , i n s i g n i f i c a n t s t i m u l a t i o n was observed i n a l l the t h r e e ATPases. However, i n 60 days t r e a t e d Saccobranchus f o s s i l i s l i v e r Mg++ ATPase showed an i n s i g n i f i c a n t i n h i b i t i o n . KOCH et al. (1972) further noted stimulation in all the ATPases at the lower concentrations of polychlorinated biphenyls in the brain, liver and kidney of Pimephales promelas following a four month exposure period. DESAIAH et ai.(1975) observed an activation of Na*, K + ATPase and oligomycin insensitive Mg++ATPase in brain tissuehomogenate of Pimephales promelas following chronic DDT administration.

771

h~

-d

* a b +

5.91+1.68

(59798)*

14.39 + 1.48

(57.E6)*

0.09

11.43 § 1.86 (51.~9)*

22.11 + 1.95

0.09

0.06

0.03

23.56 + 2.20 ( -)

None

13.30+1.20

(+??12)

(+8.~3)

(+4T98)

13.04+1.12

6.15+0.32 (5OT42)*

25.50 + 1.71

(6.15)

(30716) 15.98+1.20 (+8T12)

(10.~0) 37.84 + 0.95 (+10T55)

0.03

12.42+1.51 ( -- )

10.32+1.42

30.50 + 1.42

14.78+1.72( -- )

34.23( -+ 1"95a )b

0.06

60 days

't'

(+15.77)

26.63+1.38

(22T67)

18.59+1.95

10.20+1.52 (57T57)*

24.04+1.95 ( - )

(35.E6)* 36.62 + 1.88 (+12T46)

20.85 + 1.90

(60.01)*

13.02 + 2.02

32.56( -) + 2.12

(+9?20)

14.78+1.62

(23T52)

10.35+1.14

5,39+0.72 (60T12)*

13.54+1.52 ( - )

(35.54)* 16.31+1.54 (+1~. 54)

9,16+0.95

(62T45)*

5.34+0.31

14.24+2.04( - )

t e s t at 95~ l e v e l of confidence.

(+9754)

12.20+1.32

(18T54)

9.07+0.98

5.28+0.71 (52~59)*

11.14+1.31 ( -- )

(+3_78) 21.86+0.75 (+1~. 42)

20.18+0.92

(56T40)*

8.48+1.95

19i -45+1'51.

(+1~.88)

11.85+1.71

(21?48)

8.24+1.12

4.81+0.96 (54~18)*

10.50+1.90 ( -- )

(36.20) 20.31+1.95 (+15.88)

11.69+1.82

(58705)*

7.68+0.51

18.32+2.25( -- )

Na+, K+ATPase

= p moles of inorganic phosphate l i b e r a t e d / m g of p r o t e i n / h r .

(Na*, *,Mg*+ATPase) MATPase Na*,K*Aase (Na*,K*,MATPase) Mg* * ATPase

30 days

activity

os Labeo r o h i t a a f t e r chronic chlordane treatment.

None

mg/1

Specific

i n b r a i n and g i l l

Values are s i g n i f i d a n t when determined by F i s h e r ' s Values expressed as mean + mean standard e r r o r . Values i n par~nthes~s i n d i c a t e percent i n h i b i t i o n , Indicate ~ stimulation.

Gill

Brain

Tissue

Concentration

I n v i v 9 e f f e c t on ATPase

TABLE I

* a b +

-)b

40.75 + 2.02 ( +14708 )

0.06

O. 03

7.81+O.92

(48.'~O)* 30.42 + 1.44 (14.84)

18.54 + 1.72

0.09

(51~78)* 13.84+1.32 (14754) 13+1.71 19 i+1~. 12 )

(+1~. 04)

14.31+1.01

(+10788)

(+5724)

13.45+0.32

(42782~*

7.31+0, 52

(---)

12.78+1.84

0.03

(40.14)*

I0.72 + I. 71

(

18.00 + 2 . O ~

17.61+ 0.98 (2. ZE) 19.96 + 0.95

0.06

0.09

None

i

(45702)* 16.58+1.52 (15705) 21.62+1.98 (~L0778)

10.73+1o42

(+8724)

(20--14) 5.65+0.71

(34754) * 4.16+0.52

3.41+0.22

(-)

5.22+O.31

(50.53)* 27.10 + 1.98 (22.~3)* 38.74+ 2.12 (+15. 74)

17.38 + 1.90

9,68~0.4"

(+674o)

(29754) ~ 6.46eO.7!

6.07( _'tO.~ 3.86+C).2. (36"~.42) ~ 4.28+O.~

(5C28)* (49745) ~ 13.40+1.32 13.70+O.91 (15728) (28--48)~ 17.78+1.42 20.96+1,8r (+13. 42 ) (e9742)

7.70~O.48

(+1~.o9)

6.72+0.72 (48720)* 8.82eO.75 (32708)* 14.28+1.71

10.58 + 0.95 (44. 46)* 13.10 + 1,20 (31.33)* 20.74 + 1,52

(*s.~7)

12i98+0. ~2_

19.C~,_--)+1.82

Specific a c t i v i t y = p moles of inorganic phosphate liberated/mg os p r o t e i n / h r . '" 60 days 30 days "' (Na+, K+,Mg++ATPase) Mg++ATPase Na+, K+ATPase (Na+, K+,Mg++ATPase) Mg+eATPase Na+, K+ATP,

Values are significant when determined by Fisher's '%' test at 95~ level of confidence. Values expressed as mean + m e a n standard error. Values in .parenthesis indicate percent inhibition~ Indicate % stimulation.

Kidney

Liver

Tissue

Conc entration mg/l

In viv9 e f f e c t on ATPase in l i v e r and kidney of Labeo r o h i t a a f t e r chromic chlordane treatment.

TABLE I I

* a b +

22.08 + 1.42 ) (

12.98 + 1.02 (41.51)* 17.71 + 1.12 (19.79) 23.05 + 1.12 (§

0.21

0.07

0.14

(+iiTgi)

None

0.07

27~13 ~ 1.78 (17.~4) 36.52 + 1.95

(52.~4)*

0.14

0.21

32.78 + 2.70a ( )b 15.72 + 1.92

a f t e r chronic chlordane treatment.

)

e.09+0.72 (40T35)* 10.72+0.58 (20T98)* 14.25+0.72 (+4798)

13.57"0.95 ( )

5.67+0.78 (50748)* 9.38%0.72 (18T16) 12.67+0.48 (1o.-b6)

(

Mg++ATPase

't'

_* )

11.41 + 0.98 (51.57)* 18.25 + 1.41 (22.54)* 25.27 + 1.72 (+7.~6)

23. 56 + 1.48 ( -)

12.91 + 0. 98 (57.~6)* 22.33 + 1.95 (27.T2)* 33.46 + 2.04 (+9.~o)

(

(Na+,K§

6.1380.95 (50T62)* 9.39+0.78 (24T42) 13.19+0.68 (+6T20)

12.42+1.10 ( )

(+4T88)

6.09+0.21 (~C42)* 10.01%0.31 (28T42)* 14.6640.95

8.86+0.52 (20744)* 12.08+0.65 (+8~40)

(52T6t)*

5.28+0.42

11.14'0.62 ( - )

(+I~.84)

18.80+1.12

(26T04)*

. 6.82e0.31 ( 59705) * 12.32+0.98

(

Mg+eATPase Na+,K+ATPase

t e s t a t 9 5 ~ l e v e l of confidence.

4.89+0.42 (42T48)* 6.99+0.3"2 (17784) 8.80+0.28 (+3742)

8.51+0.58 ( ) -

I0.05*I.T2 (52786)* 17.75§ (14T72) 23.85+1.02 (+1T.87)

Na+K§

60 days

= p moles of i n o r g a n i c phosphate liberated/mg of p r o t e i n / h r .

30 days

activity

(Na*,K* M ATPa se)

Specis

Values are s i g n i f i c a n t when determined by F i s h e r ' s Values expressed as mean + mean standard e r r o r . Values i n parenthesis i n d i c a t e percent i n h i b i t i o n . Indicate %stimulation.

Gill

Brain

None

mg/Z

Concen%Tissue ration

In vivo e f f e c t on ATPase i n b r a i n and g i l l

TABLE I I I of Saccobranchus f o $ s i l i s

--3

a b +

0.07

0.14

)

17.48 + 1.18 (42.~7)* 26.69 + 1,78 (11.~6) 32.04 + 1,84 (+5.~1)

-

0,21

(

30.28 + 2.12

None

0.07

0.14

0.21

)

7.92+0.90 (44724)* 12.68+1.42 (• 14.9010,56 (+4798)

(

-

)

't'

-

)

-

)

16.74 + 1.30 (40.51)* 24.49 + 1.52 (12.~7) 31.67 + 1.78 (+12T54)

(

28.14 + 1.72

10.41 + 1.00 (42,~9)* 13.14 + 1,02 (27.'~6)* 18.03 + 1,24 (0.0~)

(

1 8 . 0 4 + L, 32

-

)

7.65+0.98 (38T40)* 12.84+1.05 ( +3.42 ) 14.36+1.06 (+15762)

(

12.42+1.05

5.87+0,98 (49.io)* 8.47+0, 52 (26T56) 10.84+0, 8 6 (0(~'~.O8)

17.31+1.16 (+10. T2 )

(25787)

(42_18)* 11.65+0.98

9.09+1.08

15.72+1.42 ( )

4, 54+0, 31 (2oT12)* 4, 67+0, 21 (28~22) 7.19+0.24 (+15, 64)

+ ++ (Na+,K ,Mg ATPase) Mg++ATPase Na+,K+ATPase

60 days

t e s t at 95~ l e v e l os confidence.

9.56+0.95 (40T54)* 14.01+0.78 (12-86) 17.14+0.99 (+6_58)

(

14,20+_1.05 16.08+I,01

I0.08+0,95 7.40"1"0,78 ( - ) ( - ) 6.23+0.72 5.17+0.46 (38~12)* (30T16)* 8.00+0.56 5.76