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Biochemical alteration in freshwater fish Channa punctatus due to latices of Euphorbia royleana and Jatropha gossypifolia
Environmental Toxicology and Pharmacology 12 (2002) 129 /136 www.elsevier.com/locate/etap
Biochemical alteration in freshwater fish Channa punctatus due to latices of Euphorbia royleana and Jatropha gossypifolia Digvijay Singh a,b, Ajay Singh b,* a
National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow 226 002, UP, India b Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur 273 009, UP, India Received 20 June 2001; received in revised form 24 October 2001; accepted 3 April 2002
Abstract Exposure of fish over 96 h to 40 and 80% of LC50 (24 h) of aqueous latex extracts of Euphorbia royleana and Jatropha gossypifolia of family Euphorbiaceae significantly altered the level of total protein, total free amino acids, nucleic acids, pyruvate, lactate, glycogen and as well as protease activity also in muscle, liver and gonadal tissue of the fish Channa punctatus . The alterations in all the biochemical parameters were significantly dose-dependent. Withdrawal study also shows that there is a partial recovery in the levels of glycogen, pyruvate, lactate and nucleic acids, but nearly complete recovery in total protein, total free amino acids level and protease activity in all the three tissues of the fish after the 7th day of the withdrawal of treatment, which supports the view that the plant product is safer in use as pesticides for control of common weed fishes in culture ponds. # 2002 Published by Elsevier Science B.V. Keywords: Euphorbia royleana ; Jatropha gossypifolia ; Channa punctatus ; Metabolism
1. Introduction Fish has encountered several serious aquatic pollution problems since past couple of years. Unlimited use of chemical pesticides and fertilizer as a pond treatment has not only affected the aquatic ecosystem adversely, but also the immune system of the aquatic animals (Richard et al., 1991). In recent years, the use of medicinal plants as effective alternatives of synthetic pesticides and fertilizers has gained importance espe"cially to combat problem both in fish and aquatic environment (Dahiya et al., 2000), because they are highly toxic to the target pests (Marston and Hostettmann, 1985; Singh and Agarwal, 1988, 1992; Singh et al., 1996a, 1998a,b). Euphorbia royleana and Jatropha gossypifolia are common medicinal plants of India having high molluscicidal properties (Singh and Agarwal, 1984a,b, 1988, 1990). We are interested in knowing the mode of action and long-term effect of these plant products on non-
* Corresponding author E-mail address: [email protected] (A. Singh).
target animals, because these substances cannot be put to commercial use without a study of these aspects as well. In the present study, the effect of sub-lethal doses of latex of E. royleana and J. gossypifolia is examined on nitrogenous and carbohydrate metabolism of freshwater fish Channa punctatus . C. punctatus is an important fish of Indian capture fishery.
2. Materials and methods 2.1. Tested animals The fish C. punctatus (Bloch), commonly called snake headed fish (17.59/1.20 cm), was collected from Ramgarh lake of Gorakhpur district. The collected animals were stored in glass aquaria containing 100 l of dechlorinated tap water. Prior to the experiment, the fishes were allowed to acclimate to laboratory conditions for 7 days. Diseased, injured and dead fish (if any) were removed as soon as possible to prevent the decomposition of the body. Water was changed every
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D. Singh, A. Singh / Environmental Toxicology and Pharmacology 12 (2002) 129 /136
24 h. Average-sized adult animals were used for the experiment. 2.2. Preparation of test substances Plants of E. royleana and J. gossypifolia (Euphorbiaceae) were collected from botanical garden of DDU Gorakhpur University, Gorakhpur and identified by plant taxonomy laboratory of the department. The latex of both the plants was collected by cutting the stem apices, and the drawn latex was lyophilised at /40 8C and dried powder was used for the experiments.
2.6. Biochemical estimations After termination of treatment, muscle, liver and gonadal tissues were dissected and homogenates were prepared for the measurement of the following biochemical parameters. 2.6.1. Estimation of protein The protein content of the samples was determined by the method of Lowry et al. (1951). Standard curves were prepared with different concentrations of bovine serum albumin. Values have been expressed as mg protein/mg of tissue.
2.3. Treatment conditions The acclimatized fishes were treated with sub-lethal doses [40 and 80% of LC50 (24 h)] of the latices of both the plant E. royleana and J. gossypifolia for 96 h exposure periods according to the method of Singh and Agarwal (1988). Six aquaria were set up for each dose, and each aquarium contained 10 fishes in 6 l of dechlorinated tap water. LC50 (24 h) of E. royleana and J. gossypifolia were 21.0 mg/l and 1316 mg/l, respectively, against C. punctatus (Singh and Singh, 2000). 2.4. Observation During exposure to sub-lethal concentrations, observations were made to detect the physical symptoms of poisoning in the fish. Behaviour symptoms like the fishes starting to experience respiratory distress, gulping of air at the upper surface were observed in the beginning of the experiment. 2.5. Withdrawal experiment In order to see the effect of withdrawal of the pesticide treatment, the fishes were exposed for 96 h to 80% of LC50 (24 h), and the one half of the animal was sacrificed and the level of total protein, total free amino acid, DNA, RNA, glycogen pyruvate, lactate and activity of protease was measured. The other half was transferred to latex-free water, which was changed every 24 h for the next 6 days. Following this, the level of total protein, total free amino acid, DNA, RNA, glycogen, pyruvate, lactate and activity of protease was measured in muscle, liver and gonadal tissue of the fishes. Control animals were kept in dechlorinated tap water without any treatment. Each assay was replicated six times, and values (mean9/SE) have been expressed as mg/mg in the case of total protein, total free amino acid, DNA, RNA, lactate and pyruvate and mg/g in the case of glycogen. Student’s t-test was applied to locate significant (P B/ 0.05) differences between treated and control animals (Sokal and Rohalf, 1973).
2.6.2. Estimation of total free amino acids Quantitative estimation of total free amino acids in the tissue was made according to the method of Spices (1957). Standard curves using the same procedure were drawn with known amounts of glycine. Free amino acids have been expressed as mg/mg of tissue. 2.6.3. Estimation of nucleic acids Estimation of DNA and RNA was performed by the methods of Schneider (1957) using diphenylamine and orcinol reagents, respectively. Homogenates (1 mg/ml, w/v) were prepared in 5% Trichloroacetic acid (TCA) at 90 8C, centrifuged at 5000 g for 20 min, and supernatant was used for estimation. Both DNA and RNA have been expressed as mg/mg tissue. 2.6.4. Estimation of glycogen content The anthrone method of Van der Vies (1954) as modified by Mahendru and Agarwal (1982) was used for the estimation of glycogen level in the tissue. The optical density was compared with a set of glucose standard of varying concentrations. Result has been expressed as mg glycogen/g tissue. 2.6.5. Estimation of pyruvate Pyruvate level was measured according to Friedemann and Haugen (1943). Homogenate (50 mg/ml, w/v) was prepared in 10% TCA. Sodium pyruvate was taken as standard. Result has been expressed as mg pyruvate/ mg tissue. 2.6.6. Estimation of lactate Lactate was estimated according to Barker and Summerson (1941), modified by Huckabee (1961). Homogenate (50 mg/ml, w/v) was prepared in 10% cold TCA. Sodium lactate was taken as standard. Lactate level has been expressed as mg/mg of tissue. 2.6.7. Protease Protease activity was estimated by the method of Moore and Stein (1954). Homogenate (50 mg/ml, w/v) was prepared in cold distilled water. Optical density was
D. Singh, A. Singh / Environmental Toxicology and Pharmacology 12 (2002) 129 /136
measured at 570 nm. The enzyme activity was expressed in mmol of tyrosine equivalent/mg protein/h.
3. Results Table 1 indicates a significant (P B/0.05) dose-dependent decrease in the protein level in the muscle, liver and gonadal tissues of the fish C. punctatus exposed for 40 and 80% of 24 h LC50 of the latices of E. royleana. Exposure to 40 and 80% of 24 h LC50 of aqueous latex extracts resulted in a decrease of the protein level in the muscle to 80 and 67%, in the liver tissue to 72 and 64%, while in the gonads to 73 and 57% of the control value, respectively (Table 1). Exposure to 40 and 80% of 24 h LC50 of the latex extracts up to 96 h resulted in an increase of total free amino acid level in the muscle to 114 and 137%, in the liver to 123 and 162%, respectively, while in the gonads to 155 and 173% of the control, on exposure to 40 and 80% of 24 h LC50, respectively. The sub-lethal concentration resulted in significant decline in nucleic acid level (DNA and RNA) in the muscle, liver and gonadal tissue of the fish. In the case of DNA level, decreases in the muscle to 86 and 74%, in the liver to 85 and 75%, while in the gonadal tissue to 46 and 34% of
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control, respectively, were observed, and the same trend was observed in the RNA level, the decrease noted to 80 and 68% in the muscle, 85 and 71% in the liver and 32 and 24% in the gonads of the control, respectively (Table 1). Protease activity was significantly increased in all the tissues after the sub-lethal exposure. It was increased up to 124 and 138% in the muscle, 140 and 162% in the liver and 118 and 138% in the gonads after 96 h of exposure to 40 and 80%, respectively, of LC50 (24 h) of latex of E. royleana (Table 1). The sub-lethal exposure to aqueous latex extracts of E. royleana caused a significant reduction in the muscle, liver and gonadal glycogen content of the fish (Table 1). The 96 h exposure to 40 and 80% of 24 h LC50 of the latex caused a decrease in muscle glycogen content to 68 and 49%, in the liver to 64 and 47%, while in gonads to 65 and 51% of the control, respectively. The 96 h exposure to 40 and 80% of 24 h LC50 of the latex caused a significant decrease in the muscle pyruvate level to 51 and 33%, in liver to 61% to 38%, while in the gonads to 49 and 31% of control, respectively. The 96 h exposure to 40 and 80% of 24 h LC50 of the latex caused a increase in the muscle lactate level to 120 and 165%, in liver to 131 and 172%, while in the gonads to 118 and 162% of the control, respectively (Table 1).
Table 1 Change in total protein, total free amino acid, nucleic acids, glycogen, pyruvate, and lactate levels and activity of protease in different tissues of C. punctatus after 96 h of exposure to 40 and 80% of LC50 (24 h) of latices of E. royleana Parameter
Tissue
Control
40% of LC50
Protein (mg/mg)
Muscle Liver Gonad
154.892.01 (100) 132.191.37 (100) 1.2791.00 (100)
Amino acids (mg/mg)
Muscle Liver Gonad
27.390.73 (100) 19.390.73 (100) 17.690.78 (100)
DNA (mg/mg)
Muscle Liver Gonad
142.4490.75 (100) 140.0190.71 (100) 141.291.18 (100)
122.4995.34a (86) 119.094.34a (85) 64.591.35a (46)
104.093.45a (74) 105.094.56a (75) 48.290.71a (34)
RNA (mg/mg)
Muscle Liver Gonad
103.0090.28 (100) 100.090.29 (100) 106.590.92 (100)
82.4093.23a (80) 85.094.23a (85) 34.291.42a (32)
70.0493.23a (68) 71.093.54a (71) 25.891.53a (24)
Protease (tyrosine/mg protein/h)
Muscle Liver Gonad
0.64290.011 (100) 0.68990.2017 (100) 0.69890.0167 (100)
0.79690.0234a (124) 0.96590.0165a (140) 0.82490.0128a (118)
0.88690.0283a (138) 1.11790.0136a (162) 0.86690.0732a (138)
Glycogen (mg/g)
Muscle Liver Gonad
1.7390.01 (100) 1.9890.02 (100) 1.7390.01 (100)
1.1890.07a (68) 1.2890.11a (64) 1.1290.03a (65)
0.8690.01a (49) 0.9390.03a (47) 0.8890.02a (51)
Pyruvate (mg/mg)
Muscle Liver Gonad
2.41690.018 (100) 3.07690.018 (100) 2.13390.036 (100)
1.23290.016a (51) 1.87690.036a (61) 1.04590.017a (49)
0.79790.028a (33) 1.16890.008a (38) 0.66190.023a (31)
Lactate (mg/mg)
Muscle Liver Gonad
2.81690.018 (100) 2.23390.023 (100) 3.81690.083 (100)
3.37990.092a (120) 2.92590.023a (131) 4.50290.088a (118)
4.64690.064a (165) 3.84090.076a (172) 6.18190.092a (162)
124.191.89a (80) 95.090.80a (72) 93.891.09a (73) 31.391.19a (114) 23.890.71a (123) 27.390.73a (155)
80% of LC50 103.591.26a (67) 84.390.88a (64) 73.291.31a (57) 37.690.96a (137) 31.391.35a (162) 30.591.01a (173)
a Significant (P B 0.05), when Student’s ‘t ’-test was applied between treated and control groups. Values are mean9SE of six replicates. Values in parenthesis are % change with control taken as 100%.
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The latex of J. gossypifolia caused a significant reduction in the protein level in the muscle, liver and gonadal tissue. In case of muscle protein, treatment of 40 and 80% of 24 h LC50 of the latex extracts caused a reduction to 82 and 70%, in the liver to 77 and 67% and in the gonads to 76 and 64% of the controls, respectively. Treatment of sub-lethal concentrations of the latex extracts enhanced significantly free amino acid levels in the muscle, liver and gonadal tissues. The increase of total free amino acid in the muscle was to 110 and 132%, in the liver to 126 and 144% and in the gonadal tissue to 137 and 151% of control, respectively (Table 2). Sub-lethal concentration of the latex extract resulted in a significant decline in the DNA and RNA level in all the tissues of the fish (Table 2). In the case of DNA, 96 h of exposure to 40 and 80% of 24 h LC50 of the latex extract resulted in a decrease of DNA level in the muscle to 89 and 56%, in the liver to 81 and 42% and in the gonads to 50 and 43% of the control, respectively. For RNA, the decrease was noted to 69 and 29% in the muscle, in the liver to 72 and 38% and in the gonads to 39 and 31% of the control, respectively (Table 2). Protease activity was significantly increased in all the tissues after the sub-lethal exposure. It was increased up to 114 and 127% in the muscle, 132 and 152% in the liver
and 116 and 126% in the gonads after 96 h of exposure to 40 and 80%, respectively, of LC50 (24 h) of latex of J. gossypifolia (Table 2). The latex extract of J. gossypifolia resulted in a significant (P B/0.05) reduction in the muscle, liver and gonadal glycogen content of the fish (Table 2). After 96 h of exposure to 40 and 80% of 24 h LC50 of the latex extracts, there was a decrease in the muscle glycogen level to 73 and 64%, in the liver to 68 and 53% and in the gonads to 72 and 62% of the control, respectively. The 96 h exposure to 40 and 80% of 24 h LC50 of the latex caused a decrease in the muscle pyruvate level to 41 and 27%, in the liver to 51 and 32%, while in the gonads to 39 and 25% of the controls, respectively. The 96 h exposure to 40 and 80% of 24 h LC50 of the latex caused a increase in the muscle lactate level to 131 and 143%, in the liver to 141 and 150%, while in the gonads to 129 and 140% of the control, respectively (Table 2). Tables 3 and 4 show that 144 h after the termination of the treatment with latices of both the plants, there was nearly a complete recovery in the protein, amino acid levels and in the protease activity, while a highly significant recovery was observed in the nucleic acid, glycogen, pyruvate and lactate levels.
Table 2 Change in total protein, total free amino acid, nucleic acids, glycogen, pyruvate and lactate levels and activity of protease in different tissues of C. punctatus after 96 h of exposure to 40 and 80% of LC50 (24 h) of latices of J. gossypifolia Parameter
Tissue
Control
Protein (mg/mg)
Muscle Liver Gonad
155.392.09 (100) 127.692.54 (100) 128.391.08 (100)
Amino acids (mg/mg)
Muscle Liver Gonad
27.690.78 (100) 19.690.61 (100) 18.690.70 (100)
DNA (mg/mg)
Muscle Liver Gonad
140.391.28 (100) 137.091.0 (100) 140.592.24 (100)
124.8693.45a (89) 110.9794.23a (81) 71.391.35a (50)
78.5693.22a (56) 57.5493.23a (42) 59.891.53a (43)
RNA (mg/mg)
Muscle Liver Gonad
105.091.45 (100) 104.291.37 (100) 106.891.31 (100)
72.4592.89a (69) 75.0293.43a (72) 41.891.58a (39)
30.4591.39a (29) 39.5993.23a (38) 32.891.40a (31)
Protease (tyrosine/mg protein/h)
Muscle Liver Gonad
0.66690.016 (100) 0.68390.0184 (100) 0.69890.018 (100)
0.75990.0765a (114) 0.90190.0049a (132) 0.81090.0983a (116)
0.84690.0873a (127) 1.03890.0068a (152) 0.88090.0345a (126)
Glycogen (mg/g)
Muscle Liver Gonad
1.7490.01 (100) 2.0290.04 (100) 1.7590.01 (100)
1.2790.01a (73) 1.3890.01a (68) 1.2690.03a (72)
1.1290.009a (64) 1.0790.013a (53) 1.0890.01a (62)
Pyruvate (mg/mg)
Muscle Liver Gonad
2.63390.036 (100) 2.91390.021 (100) 3.13390.023 (100)
1.07990.053a (41) 1.48590.063a (51) 1.22190.087a (39)
1.07190.013a (27) 0.93290.016a (32) 0.78390.025a (25)
Lactate (mg/mg)
Muscle Liver Gonad
2.87690.004 (100) 3.25590.002 (100) 4.11290.004 (100)
3.76790.056a (131) 4.58990.055a (141) 5.30490.048a (129)
4.11290.068a (143) 4.88290.025a (150) 5.75690.068a (140)
a
Details are as given in Table 1.
40% of LC50 128.091.02a (82) 99.591.19a (77) 98.391.54a (76) 30.591.19a (110) 24.891.37a (126) 25.591.35a (137)
80% of LC50 110.091.16a (70) 86.691.43a (67) 82.591.62a (64) 36.691.32a (132) 28.390.88a (144) 28.190.77a (151)
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Table 3 Changes in total protein, total free amino acids, nucleic acid (DNA and RNA), glycogen, pyruvate, lactate and protease in different tissues of the fish C. punctatus after exposure to 96 h against 80% of LC50 (24 h) of E. royleana and 7th day after withdrawal Parameter
Tissues
Control
80% of LC50
7th day after withdrawal a
103.591.26 (67) 88.390.52a (66) 72.590.95a (58)
151.890.78 (98) 128.491.16 (96) 117.591.39 (94)
Protein (mg/mg)
Muscle Liver Gonad
154.690.78 (100) 133.891.03 (100) 125.191.24 (100)
Amino acid (mg/mg)
Muscle Liver Gonad
26.391.35 (100) 21.091.29 (100) 19.090.80 (100)
DNA (mg/mg)
Muscle Liver Gonad
143.591.52 (100) 140.691.12 (100) 139.591.29 (100)
93.793.46a (65) 89.9893.46a (64) 59.991.35a (43)
121.991.31a (84) 116.791.46a (83) 99.091.37a (71)
RNA (mg/mg)
Muscle Liver Gonad
103.891.07 (100) 100.090.94 (100) 106.390.88 (100)
73.791.31a (74) 73.091.25a (73) 35.191.43a (33)
76.5090.92a (84) 86.090.78a (86) 86.1091.42a (81)
Glycogen (mg/g)
Muscle Liver Gonad
1.7890.02 (100) 2.0890.05 (100) 1.7990.07 (100)
0.8990.015a (50) 0.9690.004a (46) 0.8790.008a (49)
1.1790.048a (66) 1.2590.095a (60) 1.1390.12a (63)
Pyruvate (mg/mg)
Muscle Liver Gonad
2.5190.017 (100) 3.08690.018 (100) 2.21390.086 (100)
0.8790.006a (35) 1.2090.02a (39) 0.6690.007 (30)
1.6890.18a (67) 2.0090.02a (65) 1.4190.018 (64)
Lactate (mg/mg)
Muscle Liver Gonad
2.9190.015 (100) 2.4790.018 (100) 3.7990.02 (100)
4.8890.012a (168) 4.3290.16a (175) 6.2590.08a (165)
3.4990.12a (120) 3.0890.018a (125) 4.6990.15a (124)
Protease (tyrosine/mg protein/h)
Muscle Liver Gonad
0.679690.12 (100) 0.698590.015 (100) 0.708690.016 (100)
0.951490.08a (140) 1.15290.012a (165) 0.999190.017a (141)
a
33.991.31a (129) 32.590.96a (155) 30.291.35a (159)
27.390.85 (104) 22.890.96 (109) 20.990.79 (110)
0.713690.008 (104) 0.747390.008 (107) 0.765290.005 (108)
Details are as given in Table 1.
4. Discussion Singh and Singh (2000) reported earlier that the latices of E. royleana and J. gossypifolia are toxic to C. punctatus at higher concentration. It is also clear from the present study that the treatment with sub-lethal doses of 40 and 80% of LC50 (24 h) continued up to 96 h significantly altered the levels of total protein, total free amino acid, nucleic acids, glycogen, pyruvate and lactate, and activity of protease. The rate of alteration in all the cases was significantly (P B/0.05) dosedependent. The decrease in protein level observed in the present study may be due to their degradation and also to their possible utilization for metabolic purposes. Bradbury et al. (1987) pointed out that the decreased protein content might also be attributed to the destruction or necrosis of cells and consequent impairment in protein synthesis machinery. The quantity of protein is dependent on the rate of protein synthesis, or on the rate of its degradation. The quantity of protein may also be affected due to impaired incorporation of amino acids into polypeptide chains (Singh et al., 1996b). The synthesis of RNA plays an important role in protein synthesis. The inhibition of RNA synthesis at transcriptional level, thus, may affect
the protein level. In the present study, a significant decline in RNA level in exposed fish was observed. The decrease in RNA level may also have been a cause of protein depletion. On the other hand, increase in protease activity may be the cause of increased protein degradation. Enhanced protease activity and decreased protein level have resulted in marked elevation of free amino acid content in the fish tissues. The accumulation of free amino acids can also be attributed to the lesser use of amino acids (Seshagiri Rao et al., 1987) and their involvement in the maintenance of an acid /base balance (Moorthy et al., 1984). It has been suggested that the stress condition in general induces elevation in the transamination pathway (Natarajan, 1985). It is supported by the observation of Malla Reddy and Bashamohideen (1995), Begam et al. (1994). The enzyme aminotransaminase provides a link between carbohydrate and protein metabolism, as they inter-convert the metabolites such as a-ketogluterate, pyruvate and oxaloacetate on the one hand, and alanin, asparate and glutamate on the other. Extracts of both the plants also decreased the level of nucleic acids significantly in all the tested tissues of the fish. Several reports are available on the reduction in DNA and RNA level on exposure to different pesticides
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Table 4 Changes in total protein, total free amino acids, nucleic acid (DNA and RNA), glycogen, pyruvate, lactate and protease in different tissues of the fish C. punctatus after exposure to 96 h against 80% of LC50 (24 h) of J. gossypifolia and 7th day after withdrawal Parameter
Tissues
Control
80% of LC50
7th day after withdrawal a
108.5091.03 (70) 88.5491.96a (67) 77.6991.46a (63)
151.9091.38 (98) 128.1991.52 (97) 117.1691.91 (95)
Protein (mg/mg)
Muscle Liver Gonad
155.092.94 (100) 132.1691.03 (100) 123.3391.23 (100)
Amino acid (mg/mg)
Muscle Liver Gonad
26.591.38 (100) 19.6690.78 (100) 17.8391.66 (100)
34.9891.66a (132) 27.9190.78a (142) 27.2791.46a (153)
DNA (mg/mg)
Muscle Liver Gonad
144.3391.96 (100) 139.8391.91 (100) 138.8391.52 (100)
98.1490.98a (68) 90.8891.96a (65) 73.5791.03a (53)
125.5692.94a (87) 120.2591.46a (86) 98.5691.26a (71)
RNA (mg/mg)
Muscle Liver Gonad
103.091.29 (100) 99.591.46 (100) 104.8391.03 (100)
70..0491.52a (68) 65.6791.46a (66) 34.5991.03a (33)
89.6191.38a (87) 85.5791.05a (86) 84.9191.08a (81)
Glycogen (mg/g)
Muscle Liver Gonad
1.7490.02 (100) 2.0390.019 (100) 1.7090.03 (100)
1.1490.08a (66) 1.0990.17a (54) 1.0290.08a (60)
1.3090.06a (75) 1.3390.15a (66) 1.1090.02a (65)
Pyruvate (mg/mg)
Muscle Liver Gonad
2.4590.02 (100) 3.0590.03 (100) 2.2090.08 (100)
0.8590.02a (35) 0.8590.04a (28) 0.6690.05a (30)
1.5990.15a (65) 2.0490.36a (67) 1.3890.26a (63)
Lactate (mg/mg)
Muscle Liver Gonad
2.8790.04 (100) 2.3990.06 (100) 3.6190.14 (100)
4.1691.05a (145) 3.7090.76a (155) 5.1990.98a (144)
3.3090.98a (115) 2.8290.79a (118) 4.3390.78a (120)
Protease (tyrosine/mg protein/h)
Muscle Liver Gonad
0.66590.008 (100) 0.68090.003 (100) 0.71290.004 (100)
0.85790.008a (129) 1.06090.005a (156) 0.92590.012a (130)
0.68490.004 (103) 0.72090.017 (106) 0.76190.005 (107)
a
28.6291.03 (108) 21.2391.26 (108) 19.6191.05 (110)
Details are as given in Table 1.
(Tarig et al., 1977; Nordenskjold et al., 1979). Data attained in present investigation made it clear that these plant extracts are potential inhibitor of DNA synthesis, which resulted in the reduction in the RNA level. Mahendru (1981) suggested that anti-acetylcholinesterase compounds attack many enzymes responsible for normal metabolic pathway. Thus it is possible that latices of both plants might have inhibited the enzymes necessary for DNA synthesis. Reduction in glycogen level is thought to be the result of greater stress the organs experienced during the process of detoxification of active moieties and their metabolites. Several reports are available on the effect of muscular exercise on liver glycogen energy reserves in fish, which get depleted (Black et al., 1960, 1962; Chaudhary and Nath, 1985; Nath and Kumar, 1987; McLeary and Brown, 1975). Liver glycogen levels are depleted during acute hypoxia or physical disturbances in the fish (Heath and Fritechard, 1965). Anticholinesterase agents are known to inhibit acetylcholinesterase in nerve and other tissues, thereby causing an increase in the acetylcholine content after intoxication (Kaundinya and Ramamurthy, 1978). Nelson et al. (1976) reported that an increase in the Ach content has been shown to enhance the secretion of catecholamines in cod, Gardus morhua, which may bring
about glucogenolysis and hyperglycemia through the reserved level of cyclic AMP (Terrior and Perria, 1975). In this study, glycogenolysis seems to be the result of increased secretion of catecholamines due to the stress of plant extracts treatment, because the latices of euphorbious plants have potent anti-AChE activity (Singh and Agarwal, 1984a, 1987, 1990). Umminger (1977) reported that carbohydrate represents the principal and immediate energy source for animals exposed to stress conditions and that carbohydrate reserves deplete to meet energy demand. Decrement in pyruvate level is due to higher energy demand during exposure. In consonance with the increase in lactate content, there is a decrease in the pyruvate level, and this trend has been observed in all the tissues. The decrease in pyruvate level suggests the possibility of a shift towards anaerobic dependence due to a remarkable drop in aerobic segment. The decrease in pyruvate could be due to its conversion to lactate, or due to its mobilization to form amino acids, lipids, triglycerides and glycogen synthesis in addition to its role as a detoxification factor. The increase in lactate also suggests a shift towards anaerobiosis as a consequence of hypoxia leading to respiratory distress (Domsche et al., 1971). The development of such internal hypoxic conditions may be ultimately responsible for the shift to the less efficient anaerobic
D. Singh, A. Singh / Environmental Toxicology and Pharmacology 12 (2002) 129 /136
metabolism, evidenced by the change in lactate content observed during this study. It is evident that after 144 h of transfer into latex-free water, the effects were reversible in their action, and the level of protein, free amino acid and protease activity recovered completely, while nucleic acids (DNA and RNA), glycogen, and pyruvate lactate levels increased but still significantly different from the control. As glycogen is a storage material, metabolism of this biochemical pool will be lower, and therefore, the slower recovery can be explained. We therefore believe that these plant extracts may eventually be of great value for the control of aquatic target organisms as well as predatory and weed fishes.
References Barker, S.B., Summerson, W.H. 1941. Hawk’s Physiological Chemistry. Tata McGraw-Hill, Bombay, p. 1965. Begam, G., Vijayraghavan, S., Sharma, P.N., Husain, S. 1994. Study of dimethoate bioaccumulation in liver and muscle tissues of Clarius batrachus and its elimination following cessation of exposure. Pestic. Sci. 40, 201 /205. Black, E.C., Robertson, A.C., Hanslip, A.R., Chiu, W. 1960. Alteration in glycogen glucose and lactate in rainbow and kamloops trout, Salmo gairdneri following muscular activity. J. Fish Res. Bd. Can. 17, 487 /500. Black, E.C., Connor, A.R., Lam, K., Chiu, W. 1962. Changes in glycogen pyruvate and lactate in rainbow trout Salmo gairdneri during and following muscular activity. J. Fish Res. Bd. Can. 19, 409 /436. Bradbury, S.P., Symonic, D.M., Coats, J.R., Atchison, G.J. 1987. Toxicology of fenvalerete and its constituents isomers to the fathead minnow (Pimephales promelos ) and blue gill (Leponis macrochinus ). Bull. Environ. Contam. Toxicol. 38, 727 /735. Chaudhary, H.S., Nath, K. 1985. Effect of nickel intoxification on liver glycogen of a freshwater teleost Colisa fasciatus . Acta Hydrochem. Hydrobiol. 13, 245 /248. Dahiya, S., Kaur, A., Jain, N. 2000. Studies on difluoridation of water with Eichornia plant. J. Environ. Biol. 21 (4), 337 /340. Domsche, S., Domsche, W., Classen, M. 1971. Zum mechanisms deri Leber Zeuschadigung durch Alkaylphosphate. Natururissenchaflan 58, 575. Friedemann, T.E., Haugen, G.F. 1943. Pyruvic acid. I. Collection of blood for the determination of pyruvic acid and lactic acid. J. Biol. Chem. 144, 67 /77. Heath, A.G., Fritechard, A.W. 1965. Effect of severe hypoxia on carbohydrate energy. Stores and metabolism in two species of freshwater fish. Physiol. Zool. 38, 325 /334. Huckabee, W.E. 1961. In: Oser, B.L. (Ed.), Hawk’s Physiological Chemistry, 14th ed.. Tata McGraw-Hill, New Delhi, p. 1103. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randell, R.J. 1951. Protein measurement with foline phenol reagent. J. Biol. Chem. 193, 265 /275. Kaundinya, R.P., Ramamurthy, R. 1978. Effect of sumithion (Fentothion ) on some selected enzyme system in the fish, Tilapia mossambica (Peters). Indian J. Exp. Biol. 16, 809 /811. Mahendru, V.K. 1981. Studies on pharmacology of molluscicides on the gastropod Lymnaea acuminata . Ph.D. thesis, Department of Zoology, Gorakhpur University, Gorakhpur India.
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Mahendru, V.K., Agarwal, R.A. 1982. Changes in metabolism in various organs of the snail Lymnaea acuminata following exposure to trichlorfon. Acta Pharmacol. Toxicol. 48, 377 /381. Malla Reddy, P., Bashamohideen, Md. 1995. Alteration in protein metabolism in selected tissues of fish Cyprinus caprio during sublethal concentration of cypermethrin. Environ. Contam. Toxicol. 36, 183 /190. Marston, A., Hostettmann, K. 1985. Plant molluscicides. Phytochemistry 24, 639 /652. McLeary, D.J., Brown, D.A. 1975. Effect of acute exposure to bleached kraft pulpmill effluent on carbohydrate metabolism of juvenile coho Salmon onchorhynchus (Bistuch) during rest and exercise. J. Fish Res. Bd. Can. 32, 753 /760. Moore, S., Stein, W.H. 1954. In: Colowick, Kaplan (Eds.), Methods in Enzymology, vol. II. Academic, New York. Moorthy, K.S., Kashi Reddy, B., Swamy, K.S., Chethy, C.S. 1984. Changes in respiration and ionic content in the tissues of freshwater mussel exposed to methyl-parathion toxicity. Toxicol. Lett. 21, 287 /291. Natarajan, G.M. 1985. Inhibition of branchial enzymes in snake head fish (Channa striatus ) by oxy demetom-methyl. Pest Biochem. Physiol. 23, 41 /46. Nath, K., Kumar, K. 1987. Toxic impact of hexavalent chromium on the blood pyruvate of a teleost Colisa fasciatus . Acta Hydrochem. Hydrobiol. 5, 531 /534. Nelson, S., Abrahamsson, R., Grove, D.J. 1976. Sympathetic nervous control of adrenalin release from the head kidney of cod Gardus morhua . Comp. Biochem. Physiol., C. 55, 123 /127. Nordenskjold, M., Soderhall, J., Moldeus, P. 1979. Studies on DNA strand break induced in human fibroblast by chemical mutagens and carcinogens. Mutat. Res. 63, 393 /400. Richard, R.H., Inglis, V., Frerichs, G.N., Milloar, S.D., 1991. Working paper from the conference: problems of chemoptherapy; from the theory of reality, Paris, 12 March. Schneider, W.C. 1957. Determination of nucleic acids in tissue by pantose analysis. In: Calowick, S.P., Kaplon, N.O. (Eds.), Enzymology. Academic, New York, p. 680. Seshagiri Rao, K., Srinivas Moorthy, Kashi Reddy, B., Swamy, K.S., Chethy, C.S. 1987. Effect of benthiocarb on protein metabolism of teleost, Sarotherodon mossambica . Indian J. Environ. Health. 29, 440 /450. Singh, A., Agarwal, R.A. 1988. Possibility of using latex of euphorbiales for snail control. Sci. Total. Environ. 77, 231 /236. Singh, A., Agarwal, R.A. 1990. Molluscicidal and anti-cholinesterase activity of euphorbiales. Biol. Agric. Hortic. 7, 81 /91. Singh, A., Agarwal, R.A. 1992. Toxicity of the latex of euphorbiales. Effect on acid and alkaline phosphatase of the snail Lymnaea acuminata . Biol. Agric. Hortic. 8, 211 /219. Singh, A., Singh, D.K., Mishra, T.N., Agarwal, R.A. 1996a. Molluscicides of plant origin. Biol. Agric. Hortic. 13, 205 /252. Singh, D.K., Agarwal, R.A. 1984a. Correlation of the anti-cholinesterase and molluscicidal activity of the latex of Euphorbia royleana Bioss. on Lymnaea acuminata . J. Nat. Prod. 47, 702 /705. Singh, D.K., Agarwal, R.A. 1984b. Alteration of biogenic amine level in the snail Lymnaea acuminata by the latex of Euphorbia royleana . Toxicol. Lett. 21, 309 /314. Singh, D.K., Agarwal, R.A. 1987. Effect of the synthetic pyrethroids peremethrin on the snail Lymnaea acuminata . Sci. Total Environ. 67, 263 /267. Singh, D., Singh, A. 2000. The acute toxicity of plant origin pesticides to the fresh water fish Channa punctatus . Acta Hydrochim. Hydrobiol. 28, 92 /94. Singh, K., Singh, A., Singh, D.K. 1998a. The use of piperonyl butoxide and MGK-264 to improve the efficacy of plant derived molluscicides. Pest Sci. 54, 145 /149. Singh, K., Singh, A., Singh, D.K. 1996b. Molluscicidal activity of neem (Azadirachta indica ). J. Ethanopharmacol. 52, 35 /40.
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Singh, K., Singh, A., Singh, D.K. 1998b. Synergistic effect of MGK264 and piperonyl butoxide on the toxicity of plant molluscicides. Chemosphere 36 (15), 3055 /3060. Sokal, R.R., Rohalf, R.J. 1973. Introduction to Biostatistic. Freeman, San Francisco, CA, p. 368. Spices, J.R. 1957. Colorimetric procedures for amino acids. In: Calowick, S.P., Kaplon, N.O. (Eds.), Methods of Enzymology. Academic, New York, p. 468. Tarig, S.S., Haqui, M., Adhami, U.M. 1977. Effect of thioptera on RNA and total protein synthesis content in the testis of albino rats. Indian J. Exp. Biol. 15, 804 /805.
Terrior, M., Perria, J. 1975. Cyclic 3,5 adenosin-monophoshate levels in plasma of rainbow trot Salmo gairdeneri following adrenalin administration and consentrained exercise. Experementia 31, 196 / 199. Umminger, B.L. 1977. Relation of whole blood sugar concentration in vertebrate to standard metabolic rate. Comp. Biochem. Physiol. 55, 457 /460. Van der Vies, J. 1954. Two methods for determination of glycogen in liver. Biochem. J. 57, 410 /416.
Biochemical alteration in freshwater fish Channa punctatus due to latices of Euphorbia royleana and Jatropha gossypifolia Digvijay Singh a,b, Ajay Singh b,* a
National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow 226 002, UP, India b Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur 273 009, UP, India Received 20 June 2001; received in revised form 24 October 2001; accepted 3 April 2002
Abstract Exposure of fish over 96 h to 40 and 80% of LC50 (24 h) of aqueous latex extracts of Euphorbia royleana and Jatropha gossypifolia of family Euphorbiaceae significantly altered the level of total protein, total free amino acids, nucleic acids, pyruvate, lactate, glycogen and as well as protease activity also in muscle, liver and gonadal tissue of the fish Channa punctatus . The alterations in all the biochemical parameters were significantly dose-dependent. Withdrawal study also shows that there is a partial recovery in the levels of glycogen, pyruvate, lactate and nucleic acids, but nearly complete recovery in total protein, total free amino acids level and protease activity in all the three tissues of the fish after the 7th day of the withdrawal of treatment, which supports the view that the plant product is safer in use as pesticides for control of common weed fishes in culture ponds. # 2002 Published by Elsevier Science B.V. Keywords: Euphorbia royleana ; Jatropha gossypifolia ; Channa punctatus ; Metabolism
1. Introduction Fish has encountered several serious aquatic pollution problems since past couple of years. Unlimited use of chemical pesticides and fertilizer as a pond treatment has not only affected the aquatic ecosystem adversely, but also the immune system of the aquatic animals (Richard et al., 1991). In recent years, the use of medicinal plants as effective alternatives of synthetic pesticides and fertilizers has gained importance espe"cially to combat problem both in fish and aquatic environment (Dahiya et al., 2000), because they are highly toxic to the target pests (Marston and Hostettmann, 1985; Singh and Agarwal, 1988, 1992; Singh et al., 1996a, 1998a,b). Euphorbia royleana and Jatropha gossypifolia are common medicinal plants of India having high molluscicidal properties (Singh and Agarwal, 1984a,b, 1988, 1990). We are interested in knowing the mode of action and long-term effect of these plant products on non-
* Corresponding author E-mail address: [email protected] (A. Singh).
target animals, because these substances cannot be put to commercial use without a study of these aspects as well. In the present study, the effect of sub-lethal doses of latex of E. royleana and J. gossypifolia is examined on nitrogenous and carbohydrate metabolism of freshwater fish Channa punctatus . C. punctatus is an important fish of Indian capture fishery.
2. Materials and methods 2.1. Tested animals The fish C. punctatus (Bloch), commonly called snake headed fish (17.59/1.20 cm), was collected from Ramgarh lake of Gorakhpur district. The collected animals were stored in glass aquaria containing 100 l of dechlorinated tap water. Prior to the experiment, the fishes were allowed to acclimate to laboratory conditions for 7 days. Diseased, injured and dead fish (if any) were removed as soon as possible to prevent the decomposition of the body. Water was changed every
1382-6689/02/$ - see front matter # 2002 Published by Elsevier Science B.V. PII: S 1 3 8 2 - 6 6 8 9 ( 0 2 ) 0 0 0 1 6 - 9
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24 h. Average-sized adult animals were used for the experiment. 2.2. Preparation of test substances Plants of E. royleana and J. gossypifolia (Euphorbiaceae) were collected from botanical garden of DDU Gorakhpur University, Gorakhpur and identified by plant taxonomy laboratory of the department. The latex of both the plants was collected by cutting the stem apices, and the drawn latex was lyophilised at /40 8C and dried powder was used for the experiments.
2.6. Biochemical estimations After termination of treatment, muscle, liver and gonadal tissues were dissected and homogenates were prepared for the measurement of the following biochemical parameters. 2.6.1. Estimation of protein The protein content of the samples was determined by the method of Lowry et al. (1951). Standard curves were prepared with different concentrations of bovine serum albumin. Values have been expressed as mg protein/mg of tissue.
2.3. Treatment conditions The acclimatized fishes were treated with sub-lethal doses [40 and 80% of LC50 (24 h)] of the latices of both the plant E. royleana and J. gossypifolia for 96 h exposure periods according to the method of Singh and Agarwal (1988). Six aquaria were set up for each dose, and each aquarium contained 10 fishes in 6 l of dechlorinated tap water. LC50 (24 h) of E. royleana and J. gossypifolia were 21.0 mg/l and 1316 mg/l, respectively, against C. punctatus (Singh and Singh, 2000). 2.4. Observation During exposure to sub-lethal concentrations, observations were made to detect the physical symptoms of poisoning in the fish. Behaviour symptoms like the fishes starting to experience respiratory distress, gulping of air at the upper surface were observed in the beginning of the experiment. 2.5. Withdrawal experiment In order to see the effect of withdrawal of the pesticide treatment, the fishes were exposed for 96 h to 80% of LC50 (24 h), and the one half of the animal was sacrificed and the level of total protein, total free amino acid, DNA, RNA, glycogen pyruvate, lactate and activity of protease was measured. The other half was transferred to latex-free water, which was changed every 24 h for the next 6 days. Following this, the level of total protein, total free amino acid, DNA, RNA, glycogen, pyruvate, lactate and activity of protease was measured in muscle, liver and gonadal tissue of the fishes. Control animals were kept in dechlorinated tap water without any treatment. Each assay was replicated six times, and values (mean9/SE) have been expressed as mg/mg in the case of total protein, total free amino acid, DNA, RNA, lactate and pyruvate and mg/g in the case of glycogen. Student’s t-test was applied to locate significant (P B/ 0.05) differences between treated and control animals (Sokal and Rohalf, 1973).
2.6.2. Estimation of total free amino acids Quantitative estimation of total free amino acids in the tissue was made according to the method of Spices (1957). Standard curves using the same procedure were drawn with known amounts of glycine. Free amino acids have been expressed as mg/mg of tissue. 2.6.3. Estimation of nucleic acids Estimation of DNA and RNA was performed by the methods of Schneider (1957) using diphenylamine and orcinol reagents, respectively. Homogenates (1 mg/ml, w/v) were prepared in 5% Trichloroacetic acid (TCA) at 90 8C, centrifuged at 5000 g for 20 min, and supernatant was used for estimation. Both DNA and RNA have been expressed as mg/mg tissue. 2.6.4. Estimation of glycogen content The anthrone method of Van der Vies (1954) as modified by Mahendru and Agarwal (1982) was used for the estimation of glycogen level in the tissue. The optical density was compared with a set of glucose standard of varying concentrations. Result has been expressed as mg glycogen/g tissue. 2.6.5. Estimation of pyruvate Pyruvate level was measured according to Friedemann and Haugen (1943). Homogenate (50 mg/ml, w/v) was prepared in 10% TCA. Sodium pyruvate was taken as standard. Result has been expressed as mg pyruvate/ mg tissue. 2.6.6. Estimation of lactate Lactate was estimated according to Barker and Summerson (1941), modified by Huckabee (1961). Homogenate (50 mg/ml, w/v) was prepared in 10% cold TCA. Sodium lactate was taken as standard. Lactate level has been expressed as mg/mg of tissue. 2.6.7. Protease Protease activity was estimated by the method of Moore and Stein (1954). Homogenate (50 mg/ml, w/v) was prepared in cold distilled water. Optical density was
D. Singh, A. Singh / Environmental Toxicology and Pharmacology 12 (2002) 129 /136
measured at 570 nm. The enzyme activity was expressed in mmol of tyrosine equivalent/mg protein/h.
3. Results Table 1 indicates a significant (P B/0.05) dose-dependent decrease in the protein level in the muscle, liver and gonadal tissues of the fish C. punctatus exposed for 40 and 80% of 24 h LC50 of the latices of E. royleana. Exposure to 40 and 80% of 24 h LC50 of aqueous latex extracts resulted in a decrease of the protein level in the muscle to 80 and 67%, in the liver tissue to 72 and 64%, while in the gonads to 73 and 57% of the control value, respectively (Table 1). Exposure to 40 and 80% of 24 h LC50 of the latex extracts up to 96 h resulted in an increase of total free amino acid level in the muscle to 114 and 137%, in the liver to 123 and 162%, respectively, while in the gonads to 155 and 173% of the control, on exposure to 40 and 80% of 24 h LC50, respectively. The sub-lethal concentration resulted in significant decline in nucleic acid level (DNA and RNA) in the muscle, liver and gonadal tissue of the fish. In the case of DNA level, decreases in the muscle to 86 and 74%, in the liver to 85 and 75%, while in the gonadal tissue to 46 and 34% of
131
control, respectively, were observed, and the same trend was observed in the RNA level, the decrease noted to 80 and 68% in the muscle, 85 and 71% in the liver and 32 and 24% in the gonads of the control, respectively (Table 1). Protease activity was significantly increased in all the tissues after the sub-lethal exposure. It was increased up to 124 and 138% in the muscle, 140 and 162% in the liver and 118 and 138% in the gonads after 96 h of exposure to 40 and 80%, respectively, of LC50 (24 h) of latex of E. royleana (Table 1). The sub-lethal exposure to aqueous latex extracts of E. royleana caused a significant reduction in the muscle, liver and gonadal glycogen content of the fish (Table 1). The 96 h exposure to 40 and 80% of 24 h LC50 of the latex caused a decrease in muscle glycogen content to 68 and 49%, in the liver to 64 and 47%, while in gonads to 65 and 51% of the control, respectively. The 96 h exposure to 40 and 80% of 24 h LC50 of the latex caused a significant decrease in the muscle pyruvate level to 51 and 33%, in liver to 61% to 38%, while in the gonads to 49 and 31% of control, respectively. The 96 h exposure to 40 and 80% of 24 h LC50 of the latex caused a increase in the muscle lactate level to 120 and 165%, in liver to 131 and 172%, while in the gonads to 118 and 162% of the control, respectively (Table 1).
Table 1 Change in total protein, total free amino acid, nucleic acids, glycogen, pyruvate, and lactate levels and activity of protease in different tissues of C. punctatus after 96 h of exposure to 40 and 80% of LC50 (24 h) of latices of E. royleana Parameter
Tissue
Control
40% of LC50
Protein (mg/mg)
Muscle Liver Gonad
154.892.01 (100) 132.191.37 (100) 1.2791.00 (100)
Amino acids (mg/mg)
Muscle Liver Gonad
27.390.73 (100) 19.390.73 (100) 17.690.78 (100)
DNA (mg/mg)
Muscle Liver Gonad
142.4490.75 (100) 140.0190.71 (100) 141.291.18 (100)
122.4995.34a (86) 119.094.34a (85) 64.591.35a (46)
104.093.45a (74) 105.094.56a (75) 48.290.71a (34)
RNA (mg/mg)
Muscle Liver Gonad
103.0090.28 (100) 100.090.29 (100) 106.590.92 (100)
82.4093.23a (80) 85.094.23a (85) 34.291.42a (32)
70.0493.23a (68) 71.093.54a (71) 25.891.53a (24)
Protease (tyrosine/mg protein/h)
Muscle Liver Gonad
0.64290.011 (100) 0.68990.2017 (100) 0.69890.0167 (100)
0.79690.0234a (124) 0.96590.0165a (140) 0.82490.0128a (118)
0.88690.0283a (138) 1.11790.0136a (162) 0.86690.0732a (138)
Glycogen (mg/g)
Muscle Liver Gonad
1.7390.01 (100) 1.9890.02 (100) 1.7390.01 (100)
1.1890.07a (68) 1.2890.11a (64) 1.1290.03a (65)
0.8690.01a (49) 0.9390.03a (47) 0.8890.02a (51)
Pyruvate (mg/mg)
Muscle Liver Gonad
2.41690.018 (100) 3.07690.018 (100) 2.13390.036 (100)
1.23290.016a (51) 1.87690.036a (61) 1.04590.017a (49)
0.79790.028a (33) 1.16890.008a (38) 0.66190.023a (31)
Lactate (mg/mg)
Muscle Liver Gonad
2.81690.018 (100) 2.23390.023 (100) 3.81690.083 (100)
3.37990.092a (120) 2.92590.023a (131) 4.50290.088a (118)
4.64690.064a (165) 3.84090.076a (172) 6.18190.092a (162)
124.191.89a (80) 95.090.80a (72) 93.891.09a (73) 31.391.19a (114) 23.890.71a (123) 27.390.73a (155)
80% of LC50 103.591.26a (67) 84.390.88a (64) 73.291.31a (57) 37.690.96a (137) 31.391.35a (162) 30.591.01a (173)
a Significant (P B 0.05), when Student’s ‘t ’-test was applied between treated and control groups. Values are mean9SE of six replicates. Values in parenthesis are % change with control taken as 100%.
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The latex of J. gossypifolia caused a significant reduction in the protein level in the muscle, liver and gonadal tissue. In case of muscle protein, treatment of 40 and 80% of 24 h LC50 of the latex extracts caused a reduction to 82 and 70%, in the liver to 77 and 67% and in the gonads to 76 and 64% of the controls, respectively. Treatment of sub-lethal concentrations of the latex extracts enhanced significantly free amino acid levels in the muscle, liver and gonadal tissues. The increase of total free amino acid in the muscle was to 110 and 132%, in the liver to 126 and 144% and in the gonadal tissue to 137 and 151% of control, respectively (Table 2). Sub-lethal concentration of the latex extract resulted in a significant decline in the DNA and RNA level in all the tissues of the fish (Table 2). In the case of DNA, 96 h of exposure to 40 and 80% of 24 h LC50 of the latex extract resulted in a decrease of DNA level in the muscle to 89 and 56%, in the liver to 81 and 42% and in the gonads to 50 and 43% of the control, respectively. For RNA, the decrease was noted to 69 and 29% in the muscle, in the liver to 72 and 38% and in the gonads to 39 and 31% of the control, respectively (Table 2). Protease activity was significantly increased in all the tissues after the sub-lethal exposure. It was increased up to 114 and 127% in the muscle, 132 and 152% in the liver
and 116 and 126% in the gonads after 96 h of exposure to 40 and 80%, respectively, of LC50 (24 h) of latex of J. gossypifolia (Table 2). The latex extract of J. gossypifolia resulted in a significant (P B/0.05) reduction in the muscle, liver and gonadal glycogen content of the fish (Table 2). After 96 h of exposure to 40 and 80% of 24 h LC50 of the latex extracts, there was a decrease in the muscle glycogen level to 73 and 64%, in the liver to 68 and 53% and in the gonads to 72 and 62% of the control, respectively. The 96 h exposure to 40 and 80% of 24 h LC50 of the latex caused a decrease in the muscle pyruvate level to 41 and 27%, in the liver to 51 and 32%, while in the gonads to 39 and 25% of the controls, respectively. The 96 h exposure to 40 and 80% of 24 h LC50 of the latex caused a increase in the muscle lactate level to 131 and 143%, in the liver to 141 and 150%, while in the gonads to 129 and 140% of the control, respectively (Table 2). Tables 3 and 4 show that 144 h after the termination of the treatment with latices of both the plants, there was nearly a complete recovery in the protein, amino acid levels and in the protease activity, while a highly significant recovery was observed in the nucleic acid, glycogen, pyruvate and lactate levels.
Table 2 Change in total protein, total free amino acid, nucleic acids, glycogen, pyruvate and lactate levels and activity of protease in different tissues of C. punctatus after 96 h of exposure to 40 and 80% of LC50 (24 h) of latices of J. gossypifolia Parameter
Tissue
Control
Protein (mg/mg)
Muscle Liver Gonad
155.392.09 (100) 127.692.54 (100) 128.391.08 (100)
Amino acids (mg/mg)
Muscle Liver Gonad
27.690.78 (100) 19.690.61 (100) 18.690.70 (100)
DNA (mg/mg)
Muscle Liver Gonad
140.391.28 (100) 137.091.0 (100) 140.592.24 (100)
124.8693.45a (89) 110.9794.23a (81) 71.391.35a (50)
78.5693.22a (56) 57.5493.23a (42) 59.891.53a (43)
RNA (mg/mg)
Muscle Liver Gonad
105.091.45 (100) 104.291.37 (100) 106.891.31 (100)
72.4592.89a (69) 75.0293.43a (72) 41.891.58a (39)
30.4591.39a (29) 39.5993.23a (38) 32.891.40a (31)
Protease (tyrosine/mg protein/h)
Muscle Liver Gonad
0.66690.016 (100) 0.68390.0184 (100) 0.69890.018 (100)
0.75990.0765a (114) 0.90190.0049a (132) 0.81090.0983a (116)
0.84690.0873a (127) 1.03890.0068a (152) 0.88090.0345a (126)
Glycogen (mg/g)
Muscle Liver Gonad
1.7490.01 (100) 2.0290.04 (100) 1.7590.01 (100)
1.2790.01a (73) 1.3890.01a (68) 1.2690.03a (72)
1.1290.009a (64) 1.0790.013a (53) 1.0890.01a (62)
Pyruvate (mg/mg)
Muscle Liver Gonad
2.63390.036 (100) 2.91390.021 (100) 3.13390.023 (100)
1.07990.053a (41) 1.48590.063a (51) 1.22190.087a (39)
1.07190.013a (27) 0.93290.016a (32) 0.78390.025a (25)
Lactate (mg/mg)
Muscle Liver Gonad
2.87690.004 (100) 3.25590.002 (100) 4.11290.004 (100)
3.76790.056a (131) 4.58990.055a (141) 5.30490.048a (129)
4.11290.068a (143) 4.88290.025a (150) 5.75690.068a (140)
a
Details are as given in Table 1.
40% of LC50 128.091.02a (82) 99.591.19a (77) 98.391.54a (76) 30.591.19a (110) 24.891.37a (126) 25.591.35a (137)
80% of LC50 110.091.16a (70) 86.691.43a (67) 82.591.62a (64) 36.691.32a (132) 28.390.88a (144) 28.190.77a (151)
D. Singh, A. Singh / Environmental Toxicology and Pharmacology 12 (2002) 129 /136
133
Table 3 Changes in total protein, total free amino acids, nucleic acid (DNA and RNA), glycogen, pyruvate, lactate and protease in different tissues of the fish C. punctatus after exposure to 96 h against 80% of LC50 (24 h) of E. royleana and 7th day after withdrawal Parameter
Tissues
Control
80% of LC50
7th day after withdrawal a
103.591.26 (67) 88.390.52a (66) 72.590.95a (58)
151.890.78 (98) 128.491.16 (96) 117.591.39 (94)
Protein (mg/mg)
Muscle Liver Gonad
154.690.78 (100) 133.891.03 (100) 125.191.24 (100)
Amino acid (mg/mg)
Muscle Liver Gonad
26.391.35 (100) 21.091.29 (100) 19.090.80 (100)
DNA (mg/mg)
Muscle Liver Gonad
143.591.52 (100) 140.691.12 (100) 139.591.29 (100)
93.793.46a (65) 89.9893.46a (64) 59.991.35a (43)
121.991.31a (84) 116.791.46a (83) 99.091.37a (71)
RNA (mg/mg)
Muscle Liver Gonad
103.891.07 (100) 100.090.94 (100) 106.390.88 (100)
73.791.31a (74) 73.091.25a (73) 35.191.43a (33)
76.5090.92a (84) 86.090.78a (86) 86.1091.42a (81)
Glycogen (mg/g)
Muscle Liver Gonad
1.7890.02 (100) 2.0890.05 (100) 1.7990.07 (100)
0.8990.015a (50) 0.9690.004a (46) 0.8790.008a (49)
1.1790.048a (66) 1.2590.095a (60) 1.1390.12a (63)
Pyruvate (mg/mg)
Muscle Liver Gonad
2.5190.017 (100) 3.08690.018 (100) 2.21390.086 (100)
0.8790.006a (35) 1.2090.02a (39) 0.6690.007 (30)
1.6890.18a (67) 2.0090.02a (65) 1.4190.018 (64)
Lactate (mg/mg)
Muscle Liver Gonad
2.9190.015 (100) 2.4790.018 (100) 3.7990.02 (100)
4.8890.012a (168) 4.3290.16a (175) 6.2590.08a (165)
3.4990.12a (120) 3.0890.018a (125) 4.6990.15a (124)
Protease (tyrosine/mg protein/h)
Muscle Liver Gonad
0.679690.12 (100) 0.698590.015 (100) 0.708690.016 (100)
0.951490.08a (140) 1.15290.012a (165) 0.999190.017a (141)
a
33.991.31a (129) 32.590.96a (155) 30.291.35a (159)
27.390.85 (104) 22.890.96 (109) 20.990.79 (110)
0.713690.008 (104) 0.747390.008 (107) 0.765290.005 (108)
Details are as given in Table 1.
4. Discussion Singh and Singh (2000) reported earlier that the latices of E. royleana and J. gossypifolia are toxic to C. punctatus at higher concentration. It is also clear from the present study that the treatment with sub-lethal doses of 40 and 80% of LC50 (24 h) continued up to 96 h significantly altered the levels of total protein, total free amino acid, nucleic acids, glycogen, pyruvate and lactate, and activity of protease. The rate of alteration in all the cases was significantly (P B/0.05) dosedependent. The decrease in protein level observed in the present study may be due to their degradation and also to their possible utilization for metabolic purposes. Bradbury et al. (1987) pointed out that the decreased protein content might also be attributed to the destruction or necrosis of cells and consequent impairment in protein synthesis machinery. The quantity of protein is dependent on the rate of protein synthesis, or on the rate of its degradation. The quantity of protein may also be affected due to impaired incorporation of amino acids into polypeptide chains (Singh et al., 1996b). The synthesis of RNA plays an important role in protein synthesis. The inhibition of RNA synthesis at transcriptional level, thus, may affect
the protein level. In the present study, a significant decline in RNA level in exposed fish was observed. The decrease in RNA level may also have been a cause of protein depletion. On the other hand, increase in protease activity may be the cause of increased protein degradation. Enhanced protease activity and decreased protein level have resulted in marked elevation of free amino acid content in the fish tissues. The accumulation of free amino acids can also be attributed to the lesser use of amino acids (Seshagiri Rao et al., 1987) and their involvement in the maintenance of an acid /base balance (Moorthy et al., 1984). It has been suggested that the stress condition in general induces elevation in the transamination pathway (Natarajan, 1985). It is supported by the observation of Malla Reddy and Bashamohideen (1995), Begam et al. (1994). The enzyme aminotransaminase provides a link between carbohydrate and protein metabolism, as they inter-convert the metabolites such as a-ketogluterate, pyruvate and oxaloacetate on the one hand, and alanin, asparate and glutamate on the other. Extracts of both the plants also decreased the level of nucleic acids significantly in all the tested tissues of the fish. Several reports are available on the reduction in DNA and RNA level on exposure to different pesticides
D. Singh, A. Singh / Environmental Toxicology and Pharmacology 12 (2002) 129 /136
134
Table 4 Changes in total protein, total free amino acids, nucleic acid (DNA and RNA), glycogen, pyruvate, lactate and protease in different tissues of the fish C. punctatus after exposure to 96 h against 80% of LC50 (24 h) of J. gossypifolia and 7th day after withdrawal Parameter
Tissues
Control
80% of LC50
7th day after withdrawal a
108.5091.03 (70) 88.5491.96a (67) 77.6991.46a (63)
151.9091.38 (98) 128.1991.52 (97) 117.1691.91 (95)
Protein (mg/mg)
Muscle Liver Gonad
155.092.94 (100) 132.1691.03 (100) 123.3391.23 (100)
Amino acid (mg/mg)
Muscle Liver Gonad
26.591.38 (100) 19.6690.78 (100) 17.8391.66 (100)
34.9891.66a (132) 27.9190.78a (142) 27.2791.46a (153)
DNA (mg/mg)
Muscle Liver Gonad
144.3391.96 (100) 139.8391.91 (100) 138.8391.52 (100)
98.1490.98a (68) 90.8891.96a (65) 73.5791.03a (53)
125.5692.94a (87) 120.2591.46a (86) 98.5691.26a (71)
RNA (mg/mg)
Muscle Liver Gonad
103.091.29 (100) 99.591.46 (100) 104.8391.03 (100)
70..0491.52a (68) 65.6791.46a (66) 34.5991.03a (33)
89.6191.38a (87) 85.5791.05a (86) 84.9191.08a (81)
Glycogen (mg/g)
Muscle Liver Gonad
1.7490.02 (100) 2.0390.019 (100) 1.7090.03 (100)
1.1490.08a (66) 1.0990.17a (54) 1.0290.08a (60)
1.3090.06a (75) 1.3390.15a (66) 1.1090.02a (65)
Pyruvate (mg/mg)
Muscle Liver Gonad
2.4590.02 (100) 3.0590.03 (100) 2.2090.08 (100)
0.8590.02a (35) 0.8590.04a (28) 0.6690.05a (30)
1.5990.15a (65) 2.0490.36a (67) 1.3890.26a (63)
Lactate (mg/mg)
Muscle Liver Gonad
2.8790.04 (100) 2.3990.06 (100) 3.6190.14 (100)
4.1691.05a (145) 3.7090.76a (155) 5.1990.98a (144)
3.3090.98a (115) 2.8290.79a (118) 4.3390.78a (120)
Protease (tyrosine/mg protein/h)
Muscle Liver Gonad
0.66590.008 (100) 0.68090.003 (100) 0.71290.004 (100)
0.85790.008a (129) 1.06090.005a (156) 0.92590.012a (130)
0.68490.004 (103) 0.72090.017 (106) 0.76190.005 (107)
a
28.6291.03 (108) 21.2391.26 (108) 19.6191.05 (110)
Details are as given in Table 1.
(Tarig et al., 1977; Nordenskjold et al., 1979). Data attained in present investigation made it clear that these plant extracts are potential inhibitor of DNA synthesis, which resulted in the reduction in the RNA level. Mahendru (1981) suggested that anti-acetylcholinesterase compounds attack many enzymes responsible for normal metabolic pathway. Thus it is possible that latices of both plants might have inhibited the enzymes necessary for DNA synthesis. Reduction in glycogen level is thought to be the result of greater stress the organs experienced during the process of detoxification of active moieties and their metabolites. Several reports are available on the effect of muscular exercise on liver glycogen energy reserves in fish, which get depleted (Black et al., 1960, 1962; Chaudhary and Nath, 1985; Nath and Kumar, 1987; McLeary and Brown, 1975). Liver glycogen levels are depleted during acute hypoxia or physical disturbances in the fish (Heath and Fritechard, 1965). Anticholinesterase agents are known to inhibit acetylcholinesterase in nerve and other tissues, thereby causing an increase in the acetylcholine content after intoxication (Kaundinya and Ramamurthy, 1978). Nelson et al. (1976) reported that an increase in the Ach content has been shown to enhance the secretion of catecholamines in cod, Gardus morhua, which may bring
about glucogenolysis and hyperglycemia through the reserved level of cyclic AMP (Terrior and Perria, 1975). In this study, glycogenolysis seems to be the result of increased secretion of catecholamines due to the stress of plant extracts treatment, because the latices of euphorbious plants have potent anti-AChE activity (Singh and Agarwal, 1984a, 1987, 1990). Umminger (1977) reported that carbohydrate represents the principal and immediate energy source for animals exposed to stress conditions and that carbohydrate reserves deplete to meet energy demand. Decrement in pyruvate level is due to higher energy demand during exposure. In consonance with the increase in lactate content, there is a decrease in the pyruvate level, and this trend has been observed in all the tissues. The decrease in pyruvate level suggests the possibility of a shift towards anaerobic dependence due to a remarkable drop in aerobic segment. The decrease in pyruvate could be due to its conversion to lactate, or due to its mobilization to form amino acids, lipids, triglycerides and glycogen synthesis in addition to its role as a detoxification factor. The increase in lactate also suggests a shift towards anaerobiosis as a consequence of hypoxia leading to respiratory distress (Domsche et al., 1971). The development of such internal hypoxic conditions may be ultimately responsible for the shift to the less efficient anaerobic
D. Singh, A. Singh / Environmental Toxicology and Pharmacology 12 (2002) 129 /136
metabolism, evidenced by the change in lactate content observed during this study. It is evident that after 144 h of transfer into latex-free water, the effects were reversible in their action, and the level of protein, free amino acid and protease activity recovered completely, while nucleic acids (DNA and RNA), glycogen, and pyruvate lactate levels increased but still significantly different from the control. As glycogen is a storage material, metabolism of this biochemical pool will be lower, and therefore, the slower recovery can be explained. We therefore believe that these plant extracts may eventually be of great value for the control of aquatic target organisms as well as predatory and weed fishes.
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