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Diazinon toxicity: Effect on protein and nucleic acid metabolism in the liver of zebrafish, Brachydanio rerio (Cyprinidae)
The Science of the Total Environment, 76 (1988) 63-68 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
63
DIAZINON TOXICITY: EFFECT ON PROTEIN A N D NUCLEIC ACID METABOLISM IN THE LIVER OF ZEBRAFISH, B R A C H Y D A N I O R E R I O (CYPRINIDAE)
BADRE ALAM ANSARI and KAUSHAL KUMAR
Department of Zoology, University of Gorakhpur, Gorakhpur 273 009 (India) (Received January 3rd, 1988; accepted February 18th, 1988)
ABSTRACT Four-month old adult siblings of zebrafish were exposed to four concentrations of diazinon for up to 168 h. DNA, RNA, protein and total free amino acid content were monitored in the liver. The DNA, RNA and protein contents were significantly reduced, whereas the amino acid content was significantly enhanced. All these changes showed dose- as well as time-dependent response.
INTRODUCTION
Restrictions on the use of organochlorine pesticides imposed in several countries have promoted the use of organophosphate (OP) compounds. Diazinon (O,O-diethyl-O-(2-isopropyl-4-methyl-pyrinidyl-6) thionophosphate) is a promising OP compound and is recommended for the control of a variety of household insects, soil insects, and pests of fruit, vegetables, field crops, lawns and ornamentals [1]. Despite all the above-mentioned advantages, the possibility of water contamination by diazinon due to degradation, pH, etc. [2] cannot be overlooked, as it has already been detected in several rivers in the U.S.A. at concentrations slightly below 1.0#g 1-1 [3]. The major victims of this contamination are nontarget aquatic animals, especially fish. As the OP pesticides are known inhibitors of acetylcholinesterase (ACHE) [4, 5] and are also known to affect several systems in fish - - inducing skeletal deformities and lowering the gonadosomatic index [6], affecting basic cell constituents and the activities of phosphatases [7, 8] as well as the embryonic stages and the hatchlings [9], and causing pathological alterations in the liver and ovary [10, 11] - - we commenced a study on the effect of diazinon on the basic cell constituents and enzyme systems of zebrafish. In the first instance, we looked at its effect on AChE and phosphatases [12]. Then, since the liver is considered to be the main detoxifying organ [13, 14] and the toxicity of many compounds, including certain drugs, pesticides, hepatotoxins and chemical carcinogens, can be altered by induction or inhibition of certain hepatic microsomal enzymes [15], we thought it desirable to investigate the changes in the basic macromolecules of the liver of zebrafish. 0048-9697/88/$03.50
© 1988 Elsevier Science Publishers B.V.
64 MATERIALS AND METHODS Z e b r a f i s h w e r e c o l l e c t e d , s t o c k e d a n d b r e d i n t h e l a b o r a t o r y i n 35 1 g l a s s a q u a r i a [16]. T h e a q u a r i a w e r e e q u i p p e d w i t h a t h e r m o s t a t (Temp. 25-27 ° C ) and were c o n t i n u o u s l y a e r a t e d t h r o u g h s t o n e diffusers c o n n e c t e d to a m e c h a n i c a l a i r c o m p r e s s o r . F o u r - m o n t h old a d u l t s i b l i n g s w e r e u s e d i n t h e p r e s e n t s t u d y to a v o i d i n t e r f e r e n c e of age. Six r e p l i c a t e s , e a c h o f 20 fish, w e r e e x p o s e d to f o u r d i f f e r e n t c o n c e n t r a t i o n s of d i a z i n o n , i.e. 0.64, 1.06, 1.48 a n d 1.91 m g 1-1 (30, 50, 70 a n d 90% o f t h e 96-h LCs0 v a l u e [12]). A c e t o n e w a s u s e d as c a r r i e r for t h e p e s t i c i d e . A f t e r c o m p l e t i o n of' t h e f o u r s e l e c t e d e x p o s u r e p e r i o d s (24, 72, 120 a n d 168 h) t h e r e q u i r e d n u m b e r o f fish w e r e s a c r i f i c e d , t o g e t h e r w i t h t h e c o r r e s p o n d i n g c o n t r o l fish f r o m t h e aquaria containing acetone only. Their liver was removed, weighed and p r o c e s s e d as follows. F o r n u c l e i c a c i d ( R N A a n d D N A ) e s t i m a t i o n s , t h e m e t h o d of S c h n e i d e r [17] TABLE 1 Effect of diazinon on RNA content (pg/mg wet tissue) in the liver of B r a c h y d a n i o rerio a Concentration of diazinon (mg 1 ') 0.0 (control) 0.64 1.06 1.48 1.91
Exposure period (h) 24 150.42 (100) 148.70 (99) 130.46 (87) 125.35 (83) 124.59 (83)
72 _+ 4.29 + 3.10 _+ 3.46 i 4.05 ± 3.39
149.51 (100) 137.18 (92) 124.53 (83) 110.30 (74) 99.61 (67)
120 + 4.39 ± 2.23 ± 3.34 ± 3.75 ± 4.12
153.86 (100) 123.90 (81) 111.20 (72) 96.96 (63) 94.36 (61)
168 + 4.65 +_ 2.76 ± 6.89 ± 5.63 t 4.35
153.74 (100) 103.70 (67) 93.05 (61) 73.94 (48) 65.60 (43)
+ 4.84 + 3.88 ± 2.61 ± 4.23 ± 3.63
Summary of the computation for ANOVA of the data of Table 1 Source of variation
df
Sum of squares
Mean squares
F value
P<
Between concentrations Between exposure periods Interaction Within boxes Total
4 3 12 100 119
48333.90 22851.46 8008.68 8414.06 87608.10
12083.47 7617.15 667.39 84.14
143.61 90.52 7.93
0.001 0.001 0.001
aRNA was extracted from the liver of the fish exposed to diazinon (diluted in acetone) for 24, 72, 120 and 168h at 0.64, 1.06, 1.48 and 1.91mgl 1. Values represent means + SE (N = 6). Figures in parentheses indicate the percentage change rounded off to nearest values with control value taken as 100%.
65 TABLE 2 Effect of diazinon on protein content ~g/mg wet tissue) in the liver of B r a c h y d a n i o Concentration of diazinon (mg l- 1)
Exposure period (h) 24
72
0.0 (control)
332.94 + 10.52
328.67 (100) 318.56 (97) 296.41 (90) 277.32 (84) 257.16 (78)
(100) O.64 1.06 1.48 1.91
337.10 (101) 313.80 (94) 290.39 (87) 275.85 (83)
+_ 8.20 _+ 3.40 _+ 5.45 + 3.65
120 _+ 8.10 _+ 6.44 _+ 3.79 +_ 7.12 _+ 6.32
336.18 (100) 289.65 (86) 270.55 (80) 251.35 (75) 238.75 (71)
rerio a
168 _+ 9.10 _+ 5.77 _+ 4.41 _+ 4.85 + 2.77
338.83 (100) 248.10 (73) 237.29 (70) 207.86 (61) 188.63 (56)
_+ 10.30 _+ 3.40 _+ 2.65 _+ 3.04 _+ 3.11
Summary of the computation for ANOVA of the data of Table 2 Source of variation
df
Sum of squares
Mean squares
F value
P<
Between concentrations Between exposure periods Interaction Within boxes Total
4 3 12 100 119
129291.70 72800.00 23781.40 18768.30 244641.40
32322.92 24266.66 1981.78 187.68
172.22 129.29 10.55
0.001 0.001 0.001
aProtein was extracted from the liver of the fish exposed to diazinon (diluted in acetone). Other details as in Table 1.
u s i n g orcinol a n d d i p h e n y l a m i n e r e a g e n t s respectively was followed; the v a l u e s are expressed in m i c r o g r a m s per m i l l i g r a m of wet liver. P r o t e i n was e s t i m a t e d b y t h e m e t h o d of L o w r y et al. [18] u s i n g f o l i n - p h e n o l r e a g e n t , w h i l e t h e t o t a l free a m i n o a c i d c o n t e n t w a s d e t e r m i n e d b y t h e m e t h o d of S p i e s [19] using ninhydrin reagent. These values are also expressed in micrograms per m i l l i g r a m o f w e t l i v e r . A N O V A w a s u s e d t o t e s t for s i g n i f i c a n c e . RESULTS F o l l o w i n g d i a z i n o n e x p o s u r e , t h e R N A a n d p r o t e i n c o n t e n t s s h o w e d signific a n t t i m e - as w e l l as d o s e - d e p e n d e n t i n h i b i t i o n ( T a b l e s 1 a n d 2). T h e D N A content, although showing some enhancement during initial exposures (Table 3), l a t e r f o l l o w e d t h e t y p i c a l t r e n d of i n h i b i t i o n s h o w n b y R N A a n d p r o t e i n . T h e t o t a l free a m i n o a c i d c o n t e n t s s h o w e d a s i g n i f i c a n t i n c r e a s e a t a l l conc e n t r a t i o n s a n d e x p o s u r e p e r i o d s ( T a b l e 4).
66 TABLE 3 Effect of diazinon on DNA content ~ g / m g wet tissue) in the liver of B r a c h y d a n i o rerio a Concentration of diazinon (mg l- l)
Exposure period (h)
0.00 (control)
55.99 (100) 82.66 (148) 75.50 (135) 66.91 (120) 57.23 (102)
0.64 1.06 1.48 1.91
24
72 + 2.21 ± 2.42 ± 3.13 _+ 3.00 ± 1.98
55.20 (100) 67.05 (121) 57.54 (104) 46.31 (84) 38.03 (69)
120 + 3.68 ± 1.96 ± 2.25 ± 2.54 ± 2.33
58.26 (100) 48.60 (83) 46.83 (80) 41.10 (71) 33.29 (57)
168 ± 2.28 ± 2.61 ± 3.91 +_ 2.22 ± 2.53
56.80 (100) 45.43 (80) 39.99 (70) 30.18 (53) 28.61 (50)
± 3.61 ± 1.88 ± 1.78 ± 1.75 + 2.73
Summary of the computation for ANOVA of the data of Table 3 Source of variation
df
Sum of squares
Mean squares
F value
P <
Between concentrations Between exposure periods Interaction Within boxes Total
4 3 12 100 119
7310.26 12756.08 4107.56 3406.75 27580.65
1827.57 4252.02 342.29 34.06
53.65 124.83 10.04
0.001 0.001 0.001
aDNA was extracted from the liver of the fish exposed to diazinon (diluted in acetone). Other details as in Table 1.
DISCUSSION
Our results indicate that there were significant changes in the nucleic acid, protein and total free amino acid contents in the liver of diazinon-exposed zebrafish. The increased DNA content during the initial stages of exposure suggests that the tissue first attempted to repair the damage caused by diazinon. In reparative regeneration of tissue the surviving cells are stimulated to divide faster to compensate for the damage. During mitosis the DNA strand is first replicated, therefore the content in that particular cell is duplicated. The initial increase in DNA thus indicates that the majority of surviving cells are engaged in active mitosis. However, this does not last long due to continuous stress from the pesticide, and finally the DNA content falls significantly (ANOVA, P < 0.001). As the DNA functions as "primer" in the DNA and RNA polymerase reactions, the synthesis of both DNA and RNA would be expected to be inhibited and this was in fact observed in the present study. The reason for this may be increased degradation of the macromolecules or the interference of
67 TABLE 4 Effect of diazinon on total free amino acid content ~g/mg wet tissue) in the liver of B r a c h y d a n i o rerio a
Concentration of diazinon (mg 1-1)
Exposure period (h)
0.00 (control)
37.47 (100) 45.66 (122) 48.65 (130) 51.28 (137) 56.21
0.64 1.06 1.48 1.91
24
72 _+ 7.80 + 3.69 _+ 3.29 +_ 2.62 _+ 2.61
(150)
45.37 (100) 51.25 (113) 57.95 (128) 58.67 (129) 72.93
120 + 4.26 + 2.09 _+ 2.32 + 4.44 +_ 5.37
(161)
44.54 (100) 64.63 (145) 78.98 (177) 84.65 (190) 89.59
168 _+ 3.31 _+ 2.59 + 2.39 _+ 2.55 +_ 1.57
(201)
43.82 (100) 76.23 (174) 83.79 (191) 88.04 (201) 93.38
_+ 2.49 + 4.15 _+ 2.25 + 2.54 _+ 2.34
(213)
Summary of the computation for ANOVA of the data of Table 4 Source of variation
df
Sum of squares
Mean squares
F value
P<
Between concentrations Between exposure periods Interaction Within boxes Total
4 3 12 100 119
15813.34 15304.45 4502.94 4863.34 40484.07
3953.33 5101.48 375.24 48.63
81.29 104.90 7.71
0.001 0.001 0.001
aTotal free amino acid was extracted from the liver of the fish exposed to diazinon (diluted in acetone). Other details as in Table 1.
d i a z i n o n i n s o m e m e t a b o l i c p a t h w a y s n e c e s s a r y for R N A s y n t h e s i s . T h e p r o t e i n c o n t e n t w a s a l s o f o u n d to h a v e b e e n i n h i b i t e d i n t h e p r e s e n t i n v e s t i g a t i o n . A s R N A p l a y s a v i t a l r o l e i n p r o t e i n s y n t h e s i s , t h e d e c r e a s e d p r o t e i n c o n t e n t is t o some e x t e n t explained. At the same time, the a m i n o acid c o n t e n t s i n c r e a s e d s i g n i f i c a n t l y ; t h i s c o u l d h a v e b e e n c a u s e d by: (i) d e c r e a s e d u p t a k e of a m i n o a c i d s i n t o t h e p o l y p e p t i d e c h a i n , o r (ii) i n c r e a s e d p r o t e i n h y d r o l y s i s . T h e s e t w o factors, t o g e t h e r with decreased R N A synthesis, r e s u l t in the d i s r u p t i o n of protein synthesis observed in this study. T h u s , i t c a n b e c o n c l u d e d t h a t d i a z i n o n is n o t o n l y a n A C h E i n h i b i t o r [12] b u t i t a l s o d i s t u r b s t h e b a s i c cell c o n s t i t u e n t s o f fish. REFERENCES 1 2 3
G.L. Berg (Ed.), Farm Chemicals Handbook, Meister, Willoughby, Ohio, 1977. D.T. Allison and R.O. Hermanutz, U.S. EPA, National Environmental Research Center, Ecological Research Series, EPA-600/3-77-060, 1977. J.A. Shulze, D.B. Manigold and F.L. Andrews, Pestic. Monit. J., 7 (1973) 73.
68 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
R.D. O'Brien, Insecticides, action and Metabolism, Academic Press, New York, 1967. B.A. Ansari and K. Kumar, Toxicol. Lett., 20 (1984) 283-287. K. Kumar and B.A. Ansari, Pestic. Sci., 15 (1984) 107-111. K. Kumar and B.A. Ansari, Ecotoxicol. Environ. Saf., 12 (1986) 199-205. B.A. Ansari and K. Kumar, Int. Rev. Gesamten. Hydrobiol., 72 (1987) 517-528. B.A. Ansari and K. Kumar, Acta Hydrochim. Hydrobiol., 14 (1986) 567-570. B.A. Ansari, R. Srivastava and K. Kumar, Bolm. Fisiol. Anita. Univ. Sao Paulo, 10 (1986) 95-101. B.A. Ansari and K. Kumar, Bolm. Fisiol. Anim. Univ. Sao Paulo, 11 (1987) 27-34. B.A. Ansari, M. Aslam and K. Kumar, Acta Hydrochim. Hydrobiol., 15(1987) 301-306. R.L. Metcalf, The chemistry and biology of pesticides, in R. White-Stevens (Ed.), Pesticides in the Environment, Dekker, New York, Vol. 1, 1971. K.V. Sastry and P.K. Gupta, Environ. Res., 16 (1978) 270-278. E. Hodgson and F.E. Guthrie (Eds), Introduction to Biochemical Toxicology, Elsevier-North Holland Inc., New York, 1980. B.A. Ansari and K. Kumar, J. Adv. Zool., 3 (1982) 88-89. W.C. Schneider, in S.P. Colowick and N.O. Kaplan (Eds), Methods in Enzymology, Academic Press, New York, Vol. 3, 1957. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951). J.R. Spies, in S.P. Colowick and N.O. Kaplan (Eds), Methods in Enzymology, Academic Press, New York, Vol. 3, 1957.
63
DIAZINON TOXICITY: EFFECT ON PROTEIN A N D NUCLEIC ACID METABOLISM IN THE LIVER OF ZEBRAFISH, B R A C H Y D A N I O R E R I O (CYPRINIDAE)
BADRE ALAM ANSARI and KAUSHAL KUMAR
Department of Zoology, University of Gorakhpur, Gorakhpur 273 009 (India) (Received January 3rd, 1988; accepted February 18th, 1988)
ABSTRACT Four-month old adult siblings of zebrafish were exposed to four concentrations of diazinon for up to 168 h. DNA, RNA, protein and total free amino acid content were monitored in the liver. The DNA, RNA and protein contents were significantly reduced, whereas the amino acid content was significantly enhanced. All these changes showed dose- as well as time-dependent response.
INTRODUCTION
Restrictions on the use of organochlorine pesticides imposed in several countries have promoted the use of organophosphate (OP) compounds. Diazinon (O,O-diethyl-O-(2-isopropyl-4-methyl-pyrinidyl-6) thionophosphate) is a promising OP compound and is recommended for the control of a variety of household insects, soil insects, and pests of fruit, vegetables, field crops, lawns and ornamentals [1]. Despite all the above-mentioned advantages, the possibility of water contamination by diazinon due to degradation, pH, etc. [2] cannot be overlooked, as it has already been detected in several rivers in the U.S.A. at concentrations slightly below 1.0#g 1-1 [3]. The major victims of this contamination are nontarget aquatic animals, especially fish. As the OP pesticides are known inhibitors of acetylcholinesterase (ACHE) [4, 5] and are also known to affect several systems in fish - - inducing skeletal deformities and lowering the gonadosomatic index [6], affecting basic cell constituents and the activities of phosphatases [7, 8] as well as the embryonic stages and the hatchlings [9], and causing pathological alterations in the liver and ovary [10, 11] - - we commenced a study on the effect of diazinon on the basic cell constituents and enzyme systems of zebrafish. In the first instance, we looked at its effect on AChE and phosphatases [12]. Then, since the liver is considered to be the main detoxifying organ [13, 14] and the toxicity of many compounds, including certain drugs, pesticides, hepatotoxins and chemical carcinogens, can be altered by induction or inhibition of certain hepatic microsomal enzymes [15], we thought it desirable to investigate the changes in the basic macromolecules of the liver of zebrafish. 0048-9697/88/$03.50
© 1988 Elsevier Science Publishers B.V.
64 MATERIALS AND METHODS Z e b r a f i s h w e r e c o l l e c t e d , s t o c k e d a n d b r e d i n t h e l a b o r a t o r y i n 35 1 g l a s s a q u a r i a [16]. T h e a q u a r i a w e r e e q u i p p e d w i t h a t h e r m o s t a t (Temp. 25-27 ° C ) and were c o n t i n u o u s l y a e r a t e d t h r o u g h s t o n e diffusers c o n n e c t e d to a m e c h a n i c a l a i r c o m p r e s s o r . F o u r - m o n t h old a d u l t s i b l i n g s w e r e u s e d i n t h e p r e s e n t s t u d y to a v o i d i n t e r f e r e n c e of age. Six r e p l i c a t e s , e a c h o f 20 fish, w e r e e x p o s e d to f o u r d i f f e r e n t c o n c e n t r a t i o n s of d i a z i n o n , i.e. 0.64, 1.06, 1.48 a n d 1.91 m g 1-1 (30, 50, 70 a n d 90% o f t h e 96-h LCs0 v a l u e [12]). A c e t o n e w a s u s e d as c a r r i e r for t h e p e s t i c i d e . A f t e r c o m p l e t i o n of' t h e f o u r s e l e c t e d e x p o s u r e p e r i o d s (24, 72, 120 a n d 168 h) t h e r e q u i r e d n u m b e r o f fish w e r e s a c r i f i c e d , t o g e t h e r w i t h t h e c o r r e s p o n d i n g c o n t r o l fish f r o m t h e aquaria containing acetone only. Their liver was removed, weighed and p r o c e s s e d as follows. F o r n u c l e i c a c i d ( R N A a n d D N A ) e s t i m a t i o n s , t h e m e t h o d of S c h n e i d e r [17] TABLE 1 Effect of diazinon on RNA content (pg/mg wet tissue) in the liver of B r a c h y d a n i o rerio a Concentration of diazinon (mg 1 ') 0.0 (control) 0.64 1.06 1.48 1.91
Exposure period (h) 24 150.42 (100) 148.70 (99) 130.46 (87) 125.35 (83) 124.59 (83)
72 _+ 4.29 + 3.10 _+ 3.46 i 4.05 ± 3.39
149.51 (100) 137.18 (92) 124.53 (83) 110.30 (74) 99.61 (67)
120 + 4.39 ± 2.23 ± 3.34 ± 3.75 ± 4.12
153.86 (100) 123.90 (81) 111.20 (72) 96.96 (63) 94.36 (61)
168 + 4.65 +_ 2.76 ± 6.89 ± 5.63 t 4.35
153.74 (100) 103.70 (67) 93.05 (61) 73.94 (48) 65.60 (43)
+ 4.84 + 3.88 ± 2.61 ± 4.23 ± 3.63
Summary of the computation for ANOVA of the data of Table 1 Source of variation
df
Sum of squares
Mean squares
F value
P<
Between concentrations Between exposure periods Interaction Within boxes Total
4 3 12 100 119
48333.90 22851.46 8008.68 8414.06 87608.10
12083.47 7617.15 667.39 84.14
143.61 90.52 7.93
0.001 0.001 0.001
aRNA was extracted from the liver of the fish exposed to diazinon (diluted in acetone) for 24, 72, 120 and 168h at 0.64, 1.06, 1.48 and 1.91mgl 1. Values represent means + SE (N = 6). Figures in parentheses indicate the percentage change rounded off to nearest values with control value taken as 100%.
65 TABLE 2 Effect of diazinon on protein content ~g/mg wet tissue) in the liver of B r a c h y d a n i o Concentration of diazinon (mg l- 1)
Exposure period (h) 24
72
0.0 (control)
332.94 + 10.52
328.67 (100) 318.56 (97) 296.41 (90) 277.32 (84) 257.16 (78)
(100) O.64 1.06 1.48 1.91
337.10 (101) 313.80 (94) 290.39 (87) 275.85 (83)
+_ 8.20 _+ 3.40 _+ 5.45 + 3.65
120 _+ 8.10 _+ 6.44 _+ 3.79 +_ 7.12 _+ 6.32
336.18 (100) 289.65 (86) 270.55 (80) 251.35 (75) 238.75 (71)
rerio a
168 _+ 9.10 _+ 5.77 _+ 4.41 _+ 4.85 + 2.77
338.83 (100) 248.10 (73) 237.29 (70) 207.86 (61) 188.63 (56)
_+ 10.30 _+ 3.40 _+ 2.65 _+ 3.04 _+ 3.11
Summary of the computation for ANOVA of the data of Table 2 Source of variation
df
Sum of squares
Mean squares
F value
P<
Between concentrations Between exposure periods Interaction Within boxes Total
4 3 12 100 119
129291.70 72800.00 23781.40 18768.30 244641.40
32322.92 24266.66 1981.78 187.68
172.22 129.29 10.55
0.001 0.001 0.001
aProtein was extracted from the liver of the fish exposed to diazinon (diluted in acetone). Other details as in Table 1.
u s i n g orcinol a n d d i p h e n y l a m i n e r e a g e n t s respectively was followed; the v a l u e s are expressed in m i c r o g r a m s per m i l l i g r a m of wet liver. P r o t e i n was e s t i m a t e d b y t h e m e t h o d of L o w r y et al. [18] u s i n g f o l i n - p h e n o l r e a g e n t , w h i l e t h e t o t a l free a m i n o a c i d c o n t e n t w a s d e t e r m i n e d b y t h e m e t h o d of S p i e s [19] using ninhydrin reagent. These values are also expressed in micrograms per m i l l i g r a m o f w e t l i v e r . A N O V A w a s u s e d t o t e s t for s i g n i f i c a n c e . RESULTS F o l l o w i n g d i a z i n o n e x p o s u r e , t h e R N A a n d p r o t e i n c o n t e n t s s h o w e d signific a n t t i m e - as w e l l as d o s e - d e p e n d e n t i n h i b i t i o n ( T a b l e s 1 a n d 2). T h e D N A content, although showing some enhancement during initial exposures (Table 3), l a t e r f o l l o w e d t h e t y p i c a l t r e n d of i n h i b i t i o n s h o w n b y R N A a n d p r o t e i n . T h e t o t a l free a m i n o a c i d c o n t e n t s s h o w e d a s i g n i f i c a n t i n c r e a s e a t a l l conc e n t r a t i o n s a n d e x p o s u r e p e r i o d s ( T a b l e 4).
66 TABLE 3 Effect of diazinon on DNA content ~ g / m g wet tissue) in the liver of B r a c h y d a n i o rerio a Concentration of diazinon (mg l- l)
Exposure period (h)
0.00 (control)
55.99 (100) 82.66 (148) 75.50 (135) 66.91 (120) 57.23 (102)
0.64 1.06 1.48 1.91
24
72 + 2.21 ± 2.42 ± 3.13 _+ 3.00 ± 1.98
55.20 (100) 67.05 (121) 57.54 (104) 46.31 (84) 38.03 (69)
120 + 3.68 ± 1.96 ± 2.25 ± 2.54 ± 2.33
58.26 (100) 48.60 (83) 46.83 (80) 41.10 (71) 33.29 (57)
168 ± 2.28 ± 2.61 ± 3.91 +_ 2.22 ± 2.53
56.80 (100) 45.43 (80) 39.99 (70) 30.18 (53) 28.61 (50)
± 3.61 ± 1.88 ± 1.78 ± 1.75 + 2.73
Summary of the computation for ANOVA of the data of Table 3 Source of variation
df
Sum of squares
Mean squares
F value
P <
Between concentrations Between exposure periods Interaction Within boxes Total
4 3 12 100 119
7310.26 12756.08 4107.56 3406.75 27580.65
1827.57 4252.02 342.29 34.06
53.65 124.83 10.04
0.001 0.001 0.001
aDNA was extracted from the liver of the fish exposed to diazinon (diluted in acetone). Other details as in Table 1.
DISCUSSION
Our results indicate that there were significant changes in the nucleic acid, protein and total free amino acid contents in the liver of diazinon-exposed zebrafish. The increased DNA content during the initial stages of exposure suggests that the tissue first attempted to repair the damage caused by diazinon. In reparative regeneration of tissue the surviving cells are stimulated to divide faster to compensate for the damage. During mitosis the DNA strand is first replicated, therefore the content in that particular cell is duplicated. The initial increase in DNA thus indicates that the majority of surviving cells are engaged in active mitosis. However, this does not last long due to continuous stress from the pesticide, and finally the DNA content falls significantly (ANOVA, P < 0.001). As the DNA functions as "primer" in the DNA and RNA polymerase reactions, the synthesis of both DNA and RNA would be expected to be inhibited and this was in fact observed in the present study. The reason for this may be increased degradation of the macromolecules or the interference of
67 TABLE 4 Effect of diazinon on total free amino acid content ~g/mg wet tissue) in the liver of B r a c h y d a n i o rerio a
Concentration of diazinon (mg 1-1)
Exposure period (h)
0.00 (control)
37.47 (100) 45.66 (122) 48.65 (130) 51.28 (137) 56.21
0.64 1.06 1.48 1.91
24
72 _+ 7.80 + 3.69 _+ 3.29 +_ 2.62 _+ 2.61
(150)
45.37 (100) 51.25 (113) 57.95 (128) 58.67 (129) 72.93
120 + 4.26 + 2.09 _+ 2.32 + 4.44 +_ 5.37
(161)
44.54 (100) 64.63 (145) 78.98 (177) 84.65 (190) 89.59
168 _+ 3.31 _+ 2.59 + 2.39 _+ 2.55 +_ 1.57
(201)
43.82 (100) 76.23 (174) 83.79 (191) 88.04 (201) 93.38
_+ 2.49 + 4.15 _+ 2.25 + 2.54 _+ 2.34
(213)
Summary of the computation for ANOVA of the data of Table 4 Source of variation
df
Sum of squares
Mean squares
F value
P<
Between concentrations Between exposure periods Interaction Within boxes Total
4 3 12 100 119
15813.34 15304.45 4502.94 4863.34 40484.07
3953.33 5101.48 375.24 48.63
81.29 104.90 7.71
0.001 0.001 0.001
aTotal free amino acid was extracted from the liver of the fish exposed to diazinon (diluted in acetone). Other details as in Table 1.
d i a z i n o n i n s o m e m e t a b o l i c p a t h w a y s n e c e s s a r y for R N A s y n t h e s i s . T h e p r o t e i n c o n t e n t w a s a l s o f o u n d to h a v e b e e n i n h i b i t e d i n t h e p r e s e n t i n v e s t i g a t i o n . A s R N A p l a y s a v i t a l r o l e i n p r o t e i n s y n t h e s i s , t h e d e c r e a s e d p r o t e i n c o n t e n t is t o some e x t e n t explained. At the same time, the a m i n o acid c o n t e n t s i n c r e a s e d s i g n i f i c a n t l y ; t h i s c o u l d h a v e b e e n c a u s e d by: (i) d e c r e a s e d u p t a k e of a m i n o a c i d s i n t o t h e p o l y p e p t i d e c h a i n , o r (ii) i n c r e a s e d p r o t e i n h y d r o l y s i s . T h e s e t w o factors, t o g e t h e r with decreased R N A synthesis, r e s u l t in the d i s r u p t i o n of protein synthesis observed in this study. T h u s , i t c a n b e c o n c l u d e d t h a t d i a z i n o n is n o t o n l y a n A C h E i n h i b i t o r [12] b u t i t a l s o d i s t u r b s t h e b a s i c cell c o n s t i t u e n t s o f fish. REFERENCES 1 2 3
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