Bull. Environ. Contam. Toxicol. (1992) 49:91-97 9 1992Springer-VorlagNew York Inc.
]Enviro~.l
I~,ant,amination and "roxi=ology
Malathion Induced Changes in the Serum Proteins and Hematological Parameters of an Indian Catfish Heteropneustesfossilis(Bloch) H. M. Dutta. ~ J. V. V. Dogra,2 N. K. Singh, 2 P. K. Roy, 2 S. S. T. Nasar, 2 S. Adhikari, 2 J. S. D. Munshif and C. Richmonds'
1Department of Biological Sciences, Kent State University, Kent, Ohio 44242, USA and 2postgraduate Department of Zoology, Bhagalpur University, Bhagalpur, India The i n d i s c r i m i n a t e use o f p e s t i c i d e s in a g r i c u l t u r a l operations a d v e r s e l y a f f e c t s the a q u a t i c environment to a very g r e a t e x t e n t . This p o s e s a g r e a t d a n g e r t o f r e s h w a t e r o r g a n i s m s i n c l u d i n g f i s h e s . Malathion (0,0-dimethyl phosphorothioate S(1,2 d i c a r b e n t h o x y e t h y l ) , an o r g a n o p h o s p h o r u s i n s e c t i c i d e a c t s as a n e u r o t o x i n due t o i t s a b i l i t y t o b l o c k n e u r o t r a n s m i s s i o n by i n h i b i t i n g t h e enzyme, a c e t y l c h o l i n e s t e r a s e ( O ' B r i e n 1960). In a d d i t i o n t o o t h e r c h a n g e s , h e m a t o l o g i c a l a b n o r m a l i t i e s (Mukhopadhyay and Dehadrai 1980; Mishra and S r i v a s t a v a 1983) and changes in b l o o d serum p r o t e i n s (Richmonds 1989) have a l s o been o b s e r v e d in f i s h e s exposed t o m a l a t h i o n . The p u r p o s e o f t h i s s t u d y was t o s t u d y t h e changes in serum p r o t e i n s and hematological parameters of H e t e r o p n e u s t e s f o s s i l i s exposed t o m a l a t h i o n . MATERIALS AND METHODS
Live specimens o f H e t e r o p n e u s t e s fossilis ( 2 8 . 0 - 3 2 . 0 g) were p r o c u r e d from a swamp l o c a t e d in North B i h a r , I n d i a . F i s h were a c c l i m a t e d in t h e l a b o r a t o r y f o r 1 wk. F i s h were f e d w i t h chopped goat liver. B i o a s s a y s t o d e t e r m i n e t h e 9 6 - h r LCs0 were c o n d u c t e d ( i n p l e x i g l a s s a q u a r i a ) employing t h e t e c h n i q u e as d e s c r i b e d by American P u b l i c H e a l t h A s s o c i a t i o n (1981). M a l a t h i o n c o n t a i n i n g 50% a c t i v e i n g r e d i e n t s ( N o r t h e r n M i n e r a l s L t d . , Haryana, I n d i a ) was used in t h i s s t u d y . The w a t e r used in t h e e x p e r i m e n t had t h e f o l l o w i n g mean values f o r the water q u a l i t y c h a r a c t e r i s t i c s : t e m p e r a t u r e 28~C, pH 7 . 3 5 , DO 7.5 mg/L, CO2 4 =g/L, a l k a l i n i t y 115 mg/L as CaCo3, h a r d n e s s 2 1 4 0 mg/L as CaCo3. Groups o f 10 f i s h (30 • 1.0 g) were exposed t o a s u b l e t h a l c o n c e n t r a t i o n , 1.2 mg/L, n e a r l y o n e - t e n t h o f t h e 9 6 - h r LCs0 v a l u e (11.676 mg/L) f o r 24, 48 and 7 2 - h r in p l e x i g l a s s aquaria. C o n t r o l s were t r e a t e d u n d e r i d e n t i c a l c o n d i t i o n s w i t h t h e same number o f f i s h . F i s h were a n e s t h e t i z e d w i t h 100 mg/L t r i c a i n e m e t h a n e s u l f o n a t e (MS222). The c a u d a l p e d u n c l e was c u t o f f w i t h a s h a r p r a z o r b l a d e and f r e e f l o w i n g b l o o d was c o l l e c t e d f o r t h e h e m a t o l o g i c a l s t u d y . H e m a t o l o g i c a l p a r a m e t e r s were e s t i m a t e d by s t a n d a r d methods as d e s c r i b e d by B l a x h a l l and D a i s l e y (1973). The RBC and WBC c o u n t s Send r e p r i n t
r e q u e s t s t o H. D u t t a a t t h e above a d d r e s s 91
were made by Neubauer hemocytometer. performed by Sahli's hemometer.
Hemoglobin % determination was
For t h e e l e c t r o p h o r e t i c s t u d y t h e b l o o d samples were a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 1 h r . The b l o o d samples were c e n t r i f u g e d a t 1000 x g f o r 15 min. The serum was removed and s t o r e d a t 4~ I t was used w i t h i n 2 4 - h r f o r e l e c t r o p h o r e t i c a s s a y s . Serum p r o t e i n s were a n a l y z e d by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s ( C l a r k e 1964). Twenty m i c r o l i t e r s o f serum and a few d r o p s o f bromophenol b l u e were a p p l i e d t o each t u b e . The bromophenol b l u e s e r v e d as t h e t r a c k i n g dye. A c o n s t a n t c u r r e n t o f 5 mA/tube was applied. E l e c t r o p h o r e s i s was t e r m i n a t e d in 60 • 5 min, when t h e t r a c k i n g dye was a b o u t 1 cm from t h e bottom o f t h e t u b e . After e l e c t r o p h o r e s i s t h e g e l s were removed from t h e t u b e s . The g e l s were s t a i n e d w i t h 1% aqueous Coomasie B r i l l i a n t Blue and d e s t a i n e d with 7% a c e t i c a c i d u n t i l t h e bands d i s p l a y e d maximum s t a i n i n g e f f e c t w i t h no b a c k g r o u n d s t a i n i n g . Gels were scanned w i t h LKB U l t r a s c a n XL d e n s i t o m e t e r . Blood samples from 10 f i s h were a n a l y z e d f o r each time p e r i o d . RESULTS AND DISCUSSION The e l e c t r o p h e o r o g r a m o f b l o o d serum from c o n t r o l f i s h showed n i n e (I t o IX) d i s t i n c t f r a c t i o n s ( F i g . 1). After 24-hr exposure, c h a n g e s were o b s e r v e d in t h e r e l a t i v e p o s i t i o n , h e i g h t and a r e a o f some p r o t e i n f r a c t i o n s (Table 1). A new f r a c t i o n o f p r o t e i n (IA) was n o t i c e d a f t e r 4 8 - h r e x p o s u r e ( F i g . 3). In a d d i t i o n t o t h i s f r a c t i o n , a n o t h e r new f r a c t i o n o f p r o t e i n (IB) was o b s e r v e d a f t e r 72-hr exposure (Fig. 4). The r e s p e c t i v e p o s i t i o n , h e i g h t and a r e a o f t h e f r a c t i o n s o f p r o t e i n s changed from 24 t o 7 4 - h r e x p o s u r e . All t h e p r o t e i n f r a c t i o n s seemed t o slow down t h e i r m o b i l i t y and s h i f t e d their relative position in a d e c r e a s i n g o r d e r as compared t o c o n t r o l , e x c e p t t h e VII one. Most o f t h e p r o t e i n f r a c t i o n s ( I I , IV, VI, VII and V I I I ) in 2 4 - h r , YI, V I I , V I I I and IX in 4 8 - h r and IV, VI, V I I , V I I I and IX in 7 2 - h r were seen d e c r e a s e d in h e i g h t . Some o f t h e f r a c t i o n s I I I , V and IX in 2 4 - h r , I t o V in 4 8 - h r and I t o I I I and V in 7 2 - h r i n c r e a s e d in h e i g h t as compared t o c o n t r o l . Compared t o c o n t r o l v a l u e s , t h e a r e a o f a l l t h e p r o t e i n f r a c t i o n s d e c r e a s e d e x c e p t I and I I I in 2 4 - h r , and I t o V and IX in 4 8 - h r e x p o s u r e s . A f t e r 7 2 - h r , o n l y t h r e e f r a c t i o n s (I t o I I I ) i n c r e a s e d , whereas, s i x p r o t e i n f r a c t i o n s (IV t o IX) d e c r e a s e d in t h e i r a r e a . The s l o w e s t moving serum p r o t e i n f r a c t i o n from t h e b l o o d o f g o l d e n s h i n e r , Notemigonus c r y s o l e u c a s was i d e n t i f i e d as e u g l o b u l i n by Summerfelt (1964). This a u t h o r d e m o n s t r a t e d a n t i b o d y f u n c t i o n in t h e s l o w e s t moving component o f t h e serum. M e n z e l ' s (1970) e l e c t r o p h o r e t i c study of the blood p r o t e i n s of a f i s h , Notropis c y p r i n i d a e r e s u l t e d in t h e i d e n t i f i c a t i o n o f t h e s l o w e s t m i g r a t i n g f r a c t i o n as a g l o b u l i n f r a c t i o n . Ney and Smith (1976) a n a l y z e d t h e serum proteins of bluegills using polyacrylamide gel e l e c t r o p h o r e s i s , and i d e n t i f i e d t h e s l o w e s t moving f r a c t i o n as a globulin fraction. A c c o r d i n g t o P o s t (1966} who s t u d i e d t h e serum p r o t e i n s and a n t i b o d y p r o d u c t i o n in rainbow t r o u t , Salmo g a i r d n e r i , s p e c i f i c humoral a n t i b o d i e s were found t o be p r e s e n t in t h e 92
electrophoretically least mobile proteins. Based on t h e above f i n d i n g s , serum p r o t e i n f r a c t i o n I , i n t h e p r e s e n t s t u d y was considered to include the globulin fraction. The a p p e a r a n c e o f two new serum p r o t e i n f r a c t i o n s (IA and IB) o f a v e r y low m o b i l i t y may be due t o t h e immune r e s p o n s e o f t h e f i s h . Formation of a new fraction of protein may be due to the breakdown of red blood cells or other cellular components. Findings of our SEM study on respiratory organs (unpublished data) reveal broken microridges, loss of cell boundaries in gill filaments, beaded appearance and fusion of secondary lamellar surface. Richmonds and Dutta (1989) observed necrosis in the gills of bluegills, Lepomis macrochirus exposed to malathion. The differences in position, height and area observed in the sera of exposed fish may be due to the possible changes in the amount of different proteins caused by the necrosis of the cellular components. Previous studies on blood serum proteins have shown that under 93
18.20
50.20
VI
IX
68.60
V
31.20
79.20
IV
25.20
85.60
Ill
VIII
87.20
II
VII
97.0
I
Peak
16 80
24 40
34 80
49 20
57 60
64 80
72 80
86 20
96 60
17.20
24.60
32.20
35.40
49.40
52.80
62.40
71.80
82.40
18.20
25.00
35.40
51.80
56.80
62.20
71.80
78.00
89.40
0.81
0.55
0.67
1.68
0.28
0.12
0.07
0.07
0.01
0.91
0.41
0.24
1.44
0.35
0.06
0.13
0.05
0.01
0.69
0.45
0.38
0.40
1.10
1.16
0.24
0.28
0.19
0.37
0.30
0.25
1.09
0.67
0.10
0.16
0o10
0.06
Height (AU) (AU = absorbance units) N 24-hr 48-hr 72-hr
5.657
3.047
4.105
29.943
2.412
0.665
0.247
0.385
0.036
N
5.491
2.708
1.754
10.677
1.237
0.194
0.921
0.186
0.080
24-hr
5. 937
2.756
1.856
2.481
6.071
6.636
1.480
2.353
1.681
48-hr
Area (AU x mm)
The position, Height and Area of different peaks of serum proteins from control and malathion exposed H. f o s s i l i s
Position (mm) (in mm from tracking dye) Control 24-hr 48-hr 72-hr
Table I.
2 411
2 203
2 360
7 612
2 278
0 397
1 195
0.419
0.438
72-hr
Table 2.
Hematological changes in H e t e r o p n e u s t e s f o s s i l i s exposure t o Malathion (mean v a l u e s ) Treated Blood p ar amet er s C ont r ol 24-hr 48-hr Total RBC count
on 72-hr
4.05
3.01
2.05
2.10
1.57
4.52
4.06
2.40
14.40
13.40
12.20
11.40
(x 106/mm3) Total WBC count
(x 10{/ram3) Hemoglobin (g/dl)
conditions of stress (Bouck 1972) or heavy metal exposure (Qayyum and Gayazuddin 1978; Dutta et al. 1983; Rai 1987) the number of protein fractions either increased or decreased. Rai (1987) observed disappearance of some protein fractions and emergence of a new protein with a very low mobility in fish exposed to mercury. In the present study the protein fractions were not only affected, but also two new protein fractions of very low mobility appeared after exposure to malathion. Noticeable differences were observed in the hematological parameters of exposed H.. fo~silis (Table 2). In control fish the total RBC count was 4.05 x 10~ ~. The total RBC count showed a decreasing trend with increasing exposure time. The reduction in RBC number may be due to microcytic or normocytic anemi~ (T~schiya 1979). In 24-hr exposure the WB~ c~unt increased (4.52 x 10 /mm ) as compared with control (1.57 x 10~/m~), whereas, in 48-hr exposure there was a slight decrease in WBC count compared to 24-hr exposure. ~ This decreasing trend continued after 72-hr exposure (2.4 x 10"/mm~). The hemoglobin percentage in control was 14.40% which decreased to 13.40%, 12.20% and 11.40% during 24, 48 and 72-hr exposures respectively. These findings in H. fossilis are in partial agreement with the results of other researchers (Dalela et al. 1981, Mishra and Srivastava 1983, Ei-Domiaty 1987, Gill et al.
1991.) The increase in WBC count after 24-hr and 48-hr exposures may be attributed to the response of the fish to malathion exposure, where malathion may work as an antigen. A rise in the small lymphocyte count and a fall in the large lymphocyte count in fish exposed to both Cu and Pb have been observed by Gill et al. 1991. The reduction in the WBC count in 72-hr exposure compared to 24-hr and 48-hr exposures may be due to the malfunctioning of the hematopoietic system caused by malathion. Reduction in T-lymphocyte subpopulations following acute exposure to 4 ppm nitrogen dioxide was observed by Damji and Richters (1989). It has also been noted by Harvey et al. (1991) that deoxynivalenol (DON) induces changes in the hematopoietic system of chicks and alters immune responses. WBC are inextricably involved in the regulation of immunological function and a prolonged exposure of H. fossilis to malathion may inflict immunological deficiency. The present study reveals that malathion, an organophosphorus pesticide, randomly used all over the world has profound effect on 95
serum proteins as well as other blood parameters. Acknowledgements. The present work was carried out under the Smithsonian (USA) Project No. 0022570000 Appr. 33 FT 566-00-A20. REFERENCES APHA (1981) S t a n d a r d methods f o r t h e e x a m i n a t i o n o f w a t e r and w a s t e w a t e r , 15th ed. Washington DC B l a x h a l l PC, D a i s l e y KW (1973) R o u t i n e h e m a t o l o g i c a l methods f o r use with f i s h b l o o d . J F i s h B i o l 5:771-781 Bouck GR (1972) E f f e c t s o f d i u r n a l h y p o x i a on e l e c t r o p h o r e t i c p r o t e i n f r a c t i o n and o t h e r h e a l t h p a r a m e t e r s o f r o c k b a s s ( A m b l o p l i t e s rupestris). Trans Amer F i s h Soc 3 : 4 8 8 - 4 9 3 Clarke TT ( 1 9 6 4 ) Simplified " D i s c " (polyacrylamide gel) electrophoresis. Ann NY Acad Sci 124:428-436 Dalela RC, Rani S, Kumar V, Verma SR (1981) Effect of in vivo haematological alterations in a freshwater teleost, Mystus vittatus following subacute exposure to pesticide and their combinations. J Environ Biol 2(2):76-86 Damji K, Richters A (1989) Reduction in T lymphocyte subpopulations following acute exposure to 4 ppm nitrogen dioxide. Environ Res 49 (2):217-224 Dutta HM, Lall SB, Haghighi AZ (1983) Methyl mercury induced changes in the serum proteins of Bluegills Lepomis macrochirus (Teleostei). Ohio J Sci 83(3):119-122 E1-DomiatyNA (1987) Stress response of juvenile Clarias larva elicited by copper. Comp Biochem Physiol 88C:259-262 Gill TS, Tewari H, Pant JC (1991) Effect of water-copper and lead on the peripheral blood in rosy bard, Barbus (Puntius~ conchonius Hamilton. Bull Environ Contam Toxicol 46:606-612 Harvey RB, Kubena LF, Huff WE, Elissalda MH, Phillips TD (1991) Hematologic and immunologic toxicity of deoxynivalenol (DON) contaminated diets to growing chickens. Bull Environ Contam Toxicol 46:410-416 Menzel BW (1970) An electrophoretic analysis of the blood proteins of the subgenus Luxilus (Notropis cyprinidae). Ph.D. Thesis, Cornell University, Ithaca, New York Mishra J, Srivastava AK (1983) Malathion induced haematological and biochemical changes in the Indian catfish Heteropensutes f o s s i l i s . Environ Res 30:399-411 Mukhopadhyay PK, Dehadrai PV (1980) Biochemical changes in the airbreathing catfish Clarias batrachus exposed to malathion. Environ Pollut Set Ecol Biol 22:149-158 Ney JJ, Smith Jr LL (1976) Serum protein variability in geographically defined bluegill Lepomis macrochirus populations. Trans AmFish Soc 2:281-289 O'Brien RD (1960) Toxic phosphorus esters. Academic Press New York. Post G (1966) Serum protein fractions and antibody production in rainbow trout (Salmo gairdneri). J Fish Res Bd Can 23(12) 1957-1963 Qayyum MA, Gayazuddin M (1978) Early diagnosis of water pollution (heavy metal intoxication) by zone electrophoresis. Ind J Zool 6: 99-107 Rai R (1987) Responses of serum protein in a freshwater fish to 96
experimental mercury poisoning. J. Environ. Biol. 8(2):225-228 Richmonds C (1989) E f f e c t s o f m a l a t h i o n on some p h y s i o l o g i c a l , histological and b e h a v i o r a l a s p e c t s o f b l u e g i l l s u n f i s h Lepomis m a c r o c h i r u s . P h l } D i s s e r t a t i o n , Kent S t a t e U n i v e r s i t y , Kent, Ohio pp 156 Richmonds C, Dutta rIM (1989) Histopathological changes induced by malathion in the gills of bluegill Lepomis macrochirus. Bull Environ Contam Toxicol 43:123-130 Summerfelt RC (1964) Blood proteins, their pattern, variations, and functions in the golden shiner, Notemigonus crysoleucas, cryprinidae. PhD Thesis. Univ of Southern Illinois, Carbondale, Illinois Tuschiya K (1979) Lead in Friberg L, Nordberg GF, Vouk VB (eds) Handbook on the toxicology of metals. Elsevier/North Holland Biomedical Press, Amsterdam Received August 1, 1991; accepted February 8, 1992.
97
]Enviro~.l
I~,ant,amination and "roxi=ology
Malathion Induced Changes in the Serum Proteins and Hematological Parameters of an Indian Catfish Heteropneustesfossilis(Bloch) H. M. Dutta. ~ J. V. V. Dogra,2 N. K. Singh, 2 P. K. Roy, 2 S. S. T. Nasar, 2 S. Adhikari, 2 J. S. D. Munshif and C. Richmonds'
1Department of Biological Sciences, Kent State University, Kent, Ohio 44242, USA and 2postgraduate Department of Zoology, Bhagalpur University, Bhagalpur, India The i n d i s c r i m i n a t e use o f p e s t i c i d e s in a g r i c u l t u r a l operations a d v e r s e l y a f f e c t s the a q u a t i c environment to a very g r e a t e x t e n t . This p o s e s a g r e a t d a n g e r t o f r e s h w a t e r o r g a n i s m s i n c l u d i n g f i s h e s . Malathion (0,0-dimethyl phosphorothioate S(1,2 d i c a r b e n t h o x y e t h y l ) , an o r g a n o p h o s p h o r u s i n s e c t i c i d e a c t s as a n e u r o t o x i n due t o i t s a b i l i t y t o b l o c k n e u r o t r a n s m i s s i o n by i n h i b i t i n g t h e enzyme, a c e t y l c h o l i n e s t e r a s e ( O ' B r i e n 1960). In a d d i t i o n t o o t h e r c h a n g e s , h e m a t o l o g i c a l a b n o r m a l i t i e s (Mukhopadhyay and Dehadrai 1980; Mishra and S r i v a s t a v a 1983) and changes in b l o o d serum p r o t e i n s (Richmonds 1989) have a l s o been o b s e r v e d in f i s h e s exposed t o m a l a t h i o n . The p u r p o s e o f t h i s s t u d y was t o s t u d y t h e changes in serum p r o t e i n s and hematological parameters of H e t e r o p n e u s t e s f o s s i l i s exposed t o m a l a t h i o n . MATERIALS AND METHODS
Live specimens o f H e t e r o p n e u s t e s fossilis ( 2 8 . 0 - 3 2 . 0 g) were p r o c u r e d from a swamp l o c a t e d in North B i h a r , I n d i a . F i s h were a c c l i m a t e d in t h e l a b o r a t o r y f o r 1 wk. F i s h were f e d w i t h chopped goat liver. B i o a s s a y s t o d e t e r m i n e t h e 9 6 - h r LCs0 were c o n d u c t e d ( i n p l e x i g l a s s a q u a r i a ) employing t h e t e c h n i q u e as d e s c r i b e d by American P u b l i c H e a l t h A s s o c i a t i o n (1981). M a l a t h i o n c o n t a i n i n g 50% a c t i v e i n g r e d i e n t s ( N o r t h e r n M i n e r a l s L t d . , Haryana, I n d i a ) was used in t h i s s t u d y . The w a t e r used in t h e e x p e r i m e n t had t h e f o l l o w i n g mean values f o r the water q u a l i t y c h a r a c t e r i s t i c s : t e m p e r a t u r e 28~C, pH 7 . 3 5 , DO 7.5 mg/L, CO2 4 =g/L, a l k a l i n i t y 115 mg/L as CaCo3, h a r d n e s s 2 1 4 0 mg/L as CaCo3. Groups o f 10 f i s h (30 • 1.0 g) were exposed t o a s u b l e t h a l c o n c e n t r a t i o n , 1.2 mg/L, n e a r l y o n e - t e n t h o f t h e 9 6 - h r LCs0 v a l u e (11.676 mg/L) f o r 24, 48 and 7 2 - h r in p l e x i g l a s s aquaria. C o n t r o l s were t r e a t e d u n d e r i d e n t i c a l c o n d i t i o n s w i t h t h e same number o f f i s h . F i s h were a n e s t h e t i z e d w i t h 100 mg/L t r i c a i n e m e t h a n e s u l f o n a t e (MS222). The c a u d a l p e d u n c l e was c u t o f f w i t h a s h a r p r a z o r b l a d e and f r e e f l o w i n g b l o o d was c o l l e c t e d f o r t h e h e m a t o l o g i c a l s t u d y . H e m a t o l o g i c a l p a r a m e t e r s were e s t i m a t e d by s t a n d a r d methods as d e s c r i b e d by B l a x h a l l and D a i s l e y (1973). The RBC and WBC c o u n t s Send r e p r i n t
r e q u e s t s t o H. D u t t a a t t h e above a d d r e s s 91
were made by Neubauer hemocytometer. performed by Sahli's hemometer.
Hemoglobin % determination was
For t h e e l e c t r o p h o r e t i c s t u d y t h e b l o o d samples were a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 1 h r . The b l o o d samples were c e n t r i f u g e d a t 1000 x g f o r 15 min. The serum was removed and s t o r e d a t 4~ I t was used w i t h i n 2 4 - h r f o r e l e c t r o p h o r e t i c a s s a y s . Serum p r o t e i n s were a n a l y z e d by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s ( C l a r k e 1964). Twenty m i c r o l i t e r s o f serum and a few d r o p s o f bromophenol b l u e were a p p l i e d t o each t u b e . The bromophenol b l u e s e r v e d as t h e t r a c k i n g dye. A c o n s t a n t c u r r e n t o f 5 mA/tube was applied. E l e c t r o p h o r e s i s was t e r m i n a t e d in 60 • 5 min, when t h e t r a c k i n g dye was a b o u t 1 cm from t h e bottom o f t h e t u b e . After e l e c t r o p h o r e s i s t h e g e l s were removed from t h e t u b e s . The g e l s were s t a i n e d w i t h 1% aqueous Coomasie B r i l l i a n t Blue and d e s t a i n e d with 7% a c e t i c a c i d u n t i l t h e bands d i s p l a y e d maximum s t a i n i n g e f f e c t w i t h no b a c k g r o u n d s t a i n i n g . Gels were scanned w i t h LKB U l t r a s c a n XL d e n s i t o m e t e r . Blood samples from 10 f i s h were a n a l y z e d f o r each time p e r i o d . RESULTS AND DISCUSSION The e l e c t r o p h e o r o g r a m o f b l o o d serum from c o n t r o l f i s h showed n i n e (I t o IX) d i s t i n c t f r a c t i o n s ( F i g . 1). After 24-hr exposure, c h a n g e s were o b s e r v e d in t h e r e l a t i v e p o s i t i o n , h e i g h t and a r e a o f some p r o t e i n f r a c t i o n s (Table 1). A new f r a c t i o n o f p r o t e i n (IA) was n o t i c e d a f t e r 4 8 - h r e x p o s u r e ( F i g . 3). In a d d i t i o n t o t h i s f r a c t i o n , a n o t h e r new f r a c t i o n o f p r o t e i n (IB) was o b s e r v e d a f t e r 72-hr exposure (Fig. 4). The r e s p e c t i v e p o s i t i o n , h e i g h t and a r e a o f t h e f r a c t i o n s o f p r o t e i n s changed from 24 t o 7 4 - h r e x p o s u r e . All t h e p r o t e i n f r a c t i o n s seemed t o slow down t h e i r m o b i l i t y and s h i f t e d their relative position in a d e c r e a s i n g o r d e r as compared t o c o n t r o l , e x c e p t t h e VII one. Most o f t h e p r o t e i n f r a c t i o n s ( I I , IV, VI, VII and V I I I ) in 2 4 - h r , YI, V I I , V I I I and IX in 4 8 - h r and IV, VI, V I I , V I I I and IX in 7 2 - h r were seen d e c r e a s e d in h e i g h t . Some o f t h e f r a c t i o n s I I I , V and IX in 2 4 - h r , I t o V in 4 8 - h r and I t o I I I and V in 7 2 - h r i n c r e a s e d in h e i g h t as compared t o c o n t r o l . Compared t o c o n t r o l v a l u e s , t h e a r e a o f a l l t h e p r o t e i n f r a c t i o n s d e c r e a s e d e x c e p t I and I I I in 2 4 - h r , and I t o V and IX in 4 8 - h r e x p o s u r e s . A f t e r 7 2 - h r , o n l y t h r e e f r a c t i o n s (I t o I I I ) i n c r e a s e d , whereas, s i x p r o t e i n f r a c t i o n s (IV t o IX) d e c r e a s e d in t h e i r a r e a . The s l o w e s t moving serum p r o t e i n f r a c t i o n from t h e b l o o d o f g o l d e n s h i n e r , Notemigonus c r y s o l e u c a s was i d e n t i f i e d as e u g l o b u l i n by Summerfelt (1964). This a u t h o r d e m o n s t r a t e d a n t i b o d y f u n c t i o n in t h e s l o w e s t moving component o f t h e serum. M e n z e l ' s (1970) e l e c t r o p h o r e t i c study of the blood p r o t e i n s of a f i s h , Notropis c y p r i n i d a e r e s u l t e d in t h e i d e n t i f i c a t i o n o f t h e s l o w e s t m i g r a t i n g f r a c t i o n as a g l o b u l i n f r a c t i o n . Ney and Smith (1976) a n a l y z e d t h e serum proteins of bluegills using polyacrylamide gel e l e c t r o p h o r e s i s , and i d e n t i f i e d t h e s l o w e s t moving f r a c t i o n as a globulin fraction. A c c o r d i n g t o P o s t (1966} who s t u d i e d t h e serum p r o t e i n s and a n t i b o d y p r o d u c t i o n in rainbow t r o u t , Salmo g a i r d n e r i , s p e c i f i c humoral a n t i b o d i e s were found t o be p r e s e n t in t h e 92
electrophoretically least mobile proteins. Based on t h e above f i n d i n g s , serum p r o t e i n f r a c t i o n I , i n t h e p r e s e n t s t u d y was considered to include the globulin fraction. The a p p e a r a n c e o f two new serum p r o t e i n f r a c t i o n s (IA and IB) o f a v e r y low m o b i l i t y may be due t o t h e immune r e s p o n s e o f t h e f i s h . Formation of a new fraction of protein may be due to the breakdown of red blood cells or other cellular components. Findings of our SEM study on respiratory organs (unpublished data) reveal broken microridges, loss of cell boundaries in gill filaments, beaded appearance and fusion of secondary lamellar surface. Richmonds and Dutta (1989) observed necrosis in the gills of bluegills, Lepomis macrochirus exposed to malathion. The differences in position, height and area observed in the sera of exposed fish may be due to the possible changes in the amount of different proteins caused by the necrosis of the cellular components. Previous studies on blood serum proteins have shown that under 93
18.20
50.20
VI
IX
68.60
V
31.20
79.20
IV
25.20
85.60
Ill
VIII
87.20
II
VII
97.0
I
Peak
16 80
24 40
34 80
49 20
57 60
64 80
72 80
86 20
96 60
17.20
24.60
32.20
35.40
49.40
52.80
62.40
71.80
82.40
18.20
25.00
35.40
51.80
56.80
62.20
71.80
78.00
89.40
0.81
0.55
0.67
1.68
0.28
0.12
0.07
0.07
0.01
0.91
0.41
0.24
1.44
0.35
0.06
0.13
0.05
0.01
0.69
0.45
0.38
0.40
1.10
1.16
0.24
0.28
0.19
0.37
0.30
0.25
1.09
0.67
0.10
0.16
0o10
0.06
Height (AU) (AU = absorbance units) N 24-hr 48-hr 72-hr
5.657
3.047
4.105
29.943
2.412
0.665
0.247
0.385
0.036
N
5.491
2.708
1.754
10.677
1.237
0.194
0.921
0.186
0.080
24-hr
5. 937
2.756
1.856
2.481
6.071
6.636
1.480
2.353
1.681
48-hr
Area (AU x mm)
The position, Height and Area of different peaks of serum proteins from control and malathion exposed H. f o s s i l i s
Position (mm) (in mm from tracking dye) Control 24-hr 48-hr 72-hr
Table I.
2 411
2 203
2 360
7 612
2 278
0 397
1 195
0.419
0.438
72-hr
Table 2.
Hematological changes in H e t e r o p n e u s t e s f o s s i l i s exposure t o Malathion (mean v a l u e s ) Treated Blood p ar amet er s C ont r ol 24-hr 48-hr Total RBC count
on 72-hr
4.05
3.01
2.05
2.10
1.57
4.52
4.06
2.40
14.40
13.40
12.20
11.40
(x 106/mm3) Total WBC count
(x 10{/ram3) Hemoglobin (g/dl)
conditions of stress (Bouck 1972) or heavy metal exposure (Qayyum and Gayazuddin 1978; Dutta et al. 1983; Rai 1987) the number of protein fractions either increased or decreased. Rai (1987) observed disappearance of some protein fractions and emergence of a new protein with a very low mobility in fish exposed to mercury. In the present study the protein fractions were not only affected, but also two new protein fractions of very low mobility appeared after exposure to malathion. Noticeable differences were observed in the hematological parameters of exposed H.. fo~silis (Table 2). In control fish the total RBC count was 4.05 x 10~ ~. The total RBC count showed a decreasing trend with increasing exposure time. The reduction in RBC number may be due to microcytic or normocytic anemi~ (T~schiya 1979). In 24-hr exposure the WB~ c~unt increased (4.52 x 10 /mm ) as compared with control (1.57 x 10~/m~), whereas, in 48-hr exposure there was a slight decrease in WBC count compared to 24-hr exposure. ~ This decreasing trend continued after 72-hr exposure (2.4 x 10"/mm~). The hemoglobin percentage in control was 14.40% which decreased to 13.40%, 12.20% and 11.40% during 24, 48 and 72-hr exposures respectively. These findings in H. fossilis are in partial agreement with the results of other researchers (Dalela et al. 1981, Mishra and Srivastava 1983, Ei-Domiaty 1987, Gill et al.
1991.) The increase in WBC count after 24-hr and 48-hr exposures may be attributed to the response of the fish to malathion exposure, where malathion may work as an antigen. A rise in the small lymphocyte count and a fall in the large lymphocyte count in fish exposed to both Cu and Pb have been observed by Gill et al. 1991. The reduction in the WBC count in 72-hr exposure compared to 24-hr and 48-hr exposures may be due to the malfunctioning of the hematopoietic system caused by malathion. Reduction in T-lymphocyte subpopulations following acute exposure to 4 ppm nitrogen dioxide was observed by Damji and Richters (1989). It has also been noted by Harvey et al. (1991) that deoxynivalenol (DON) induces changes in the hematopoietic system of chicks and alters immune responses. WBC are inextricably involved in the regulation of immunological function and a prolonged exposure of H. fossilis to malathion may inflict immunological deficiency. The present study reveals that malathion, an organophosphorus pesticide, randomly used all over the world has profound effect on 95
serum proteins as well as other blood parameters. Acknowledgements. The present work was carried out under the Smithsonian (USA) Project No. 0022570000 Appr. 33 FT 566-00-A20. REFERENCES APHA (1981) S t a n d a r d methods f o r t h e e x a m i n a t i o n o f w a t e r and w a s t e w a t e r , 15th ed. Washington DC B l a x h a l l PC, D a i s l e y KW (1973) R o u t i n e h e m a t o l o g i c a l methods f o r use with f i s h b l o o d . J F i s h B i o l 5:771-781 Bouck GR (1972) E f f e c t s o f d i u r n a l h y p o x i a on e l e c t r o p h o r e t i c p r o t e i n f r a c t i o n and o t h e r h e a l t h p a r a m e t e r s o f r o c k b a s s ( A m b l o p l i t e s rupestris). Trans Amer F i s h Soc 3 : 4 8 8 - 4 9 3 Clarke TT ( 1 9 6 4 ) Simplified " D i s c " (polyacrylamide gel) electrophoresis. Ann NY Acad Sci 124:428-436 Dalela RC, Rani S, Kumar V, Verma SR (1981) Effect of in vivo haematological alterations in a freshwater teleost, Mystus vittatus following subacute exposure to pesticide and their combinations. J Environ Biol 2(2):76-86 Damji K, Richters A (1989) Reduction in T lymphocyte subpopulations following acute exposure to 4 ppm nitrogen dioxide. Environ Res 49 (2):217-224 Dutta HM, Lall SB, Haghighi AZ (1983) Methyl mercury induced changes in the serum proteins of Bluegills Lepomis macrochirus (Teleostei). Ohio J Sci 83(3):119-122 E1-DomiatyNA (1987) Stress response of juvenile Clarias larva elicited by copper. Comp Biochem Physiol 88C:259-262 Gill TS, Tewari H, Pant JC (1991) Effect of water-copper and lead on the peripheral blood in rosy bard, Barbus (Puntius~ conchonius Hamilton. Bull Environ Contam Toxicol 46:606-612 Harvey RB, Kubena LF, Huff WE, Elissalda MH, Phillips TD (1991) Hematologic and immunologic toxicity of deoxynivalenol (DON) contaminated diets to growing chickens. Bull Environ Contam Toxicol 46:410-416 Menzel BW (1970) An electrophoretic analysis of the blood proteins of the subgenus Luxilus (Notropis cyprinidae). Ph.D. Thesis, Cornell University, Ithaca, New York Mishra J, Srivastava AK (1983) Malathion induced haematological and biochemical changes in the Indian catfish Heteropensutes f o s s i l i s . Environ Res 30:399-411 Mukhopadhyay PK, Dehadrai PV (1980) Biochemical changes in the airbreathing catfish Clarias batrachus exposed to malathion. Environ Pollut Set Ecol Biol 22:149-158 Ney JJ, Smith Jr LL (1976) Serum protein variability in geographically defined bluegill Lepomis macrochirus populations. Trans AmFish Soc 2:281-289 O'Brien RD (1960) Toxic phosphorus esters. Academic Press New York. Post G (1966) Serum protein fractions and antibody production in rainbow trout (Salmo gairdneri). J Fish Res Bd Can 23(12) 1957-1963 Qayyum MA, Gayazuddin M (1978) Early diagnosis of water pollution (heavy metal intoxication) by zone electrophoresis. Ind J Zool 6: 99-107 Rai R (1987) Responses of serum protein in a freshwater fish to 96
experimental mercury poisoning. J. Environ. Biol. 8(2):225-228 Richmonds C (1989) E f f e c t s o f m a l a t h i o n on some p h y s i o l o g i c a l , histological and b e h a v i o r a l a s p e c t s o f b l u e g i l l s u n f i s h Lepomis m a c r o c h i r u s . P h l } D i s s e r t a t i o n , Kent S t a t e U n i v e r s i t y , Kent, Ohio pp 156 Richmonds C, Dutta rIM (1989) Histopathological changes induced by malathion in the gills of bluegill Lepomis macrochirus. Bull Environ Contam Toxicol 43:123-130 Summerfelt RC (1964) Blood proteins, their pattern, variations, and functions in the golden shiner, Notemigonus crysoleucas, cryprinidae. PhD Thesis. Univ of Southern Illinois, Carbondale, Illinois Tuschiya K (1979) Lead in Friberg L, Nordberg GF, Vouk VB (eds) Handbook on the toxicology of metals. Elsevier/North Holland Biomedical Press, Amsterdam Received August 1, 1991; accepted February 8, 1992.
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