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Journal of Toxicology and Environmental Health: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
Chronic mercuric chloride intoxication in digestive system of Channa punctatus a
K. V. Sastry & P. K. Gupta
a
a
Department of Zoology , D.A.V. (P.G.) College , Muzaffarnagar, U.P., India Published online: 20 Oct 2009.
To cite this article: K. V. Sastry & P. K. Gupta (1978) Chronic mercuric chloride intoxication in digestive system of Channa punctatus , Journal of Toxicology and Environmental Health: Current Issues, 4:5-6, 777-783, DOI: 10.1080/15287397809529699 To link to this article: http://dx.doi.org/10.1080/15287397809529699
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
CHRONIC MERCURIC CHLORIDE INTOXICATION IN DIGESTIVE SYSTEM OF CHANNA PUNCTATUS K. V. Sastry, P. K. Gupta Department of Zoology, D.A.V. (P.G.) College, Muzaffarnagar, India
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The effect of exposure to a sublethal concentration (0.30 mg/l) of mercuric chloride on the activities of alkaline phosphatase, acid phosphatase, amylase, trypsin, and pepsin has been examined at intervals of 7, 15, and 30 d in the digestive system of a teleost fish, Channa punctatus. Inhibition of the activities of all these enzymes was noted after the first week of treatment. Treatment of the fish for 15 d resulted in marked increases in the activities of all the enzymes. A slight fall in enzyme activity was recorded after 30 d, but the overall activity was higher than in control fish.
INTRODUCTION Heavy metals produce toxic effects on the tissues and alter the physiological functioning of various systems in animals. Mercury and copper are among the most toxic heavy metals to fish. Toxicity of mercurial compounds has been reported in the nervous system (Chang and Hartmann, 1972a, 1972b, 1972c) and ¡n the kidney (Sahaphong and Trump, 1971; Ware et al., 1973). Hinton et al. (1973) observed liver damage, in the form of necrosis in portal areas and formation of connective tissue septa, resulting from methyl mercury poisoning. Chang et al. (1973) studied histochemical changes in rat kidney, liver, and brain after chronic mercuric chloride intoxication. Very little information is available on the histopathologic and biochemical alterations produced in the digestive system by exposure to this metal. In this paper we report alterations in enzyme activities that accompany chronic mercury intoxication in liver and different parts of the digestive tract of a teleost fish, Channa punctatus. METHODS AND MATERIALS Living fish were collected from local freshwater sources and maintained in laboratory aquariums. Specimens weighing 60 ± 5 g each were We thank Dr. V. P. Agrawal for criticism and help. Financial assistance from the University Grants Commission, New Delhi, is gratefully acknowledged. Requests for reprints should be sent to K. V. Sastry, Department of Zoology, D.A.V. (P.G.) College, Muzaffarnagar (U.P.), India. 777 Journal of Toxicology and Environmental Health, 4:777-783,1978 Copyright © 1978 by Hemisphere Publishing Corporation 0098-4108/78/040777-07$2.25
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selected and were acclimatized to laboratory conditions for 3 d before experimentation. Preliminary bioassays conducted in the laboratory under static conditions showed that 0.30 mg/l is a sublethal concentration. Fish were divided into 2 groups of 30 fish each. The first group was treated with the calculated dose of mercuric chloride up to 4 wk, while second group served as controls. From both groups, 10 fish each were sacrificed at intervals of 7, 15, and 30 d. The different parts of the alimentary canal and liver were quickly excised from fish 1 h after feeding, and the tissues were freed from the adjoining tissues and immediately frozen at —4°C. For the preparation of the enzyme extracts of the stomach, intestine, and pyloric ceca, these portions were slit open longitudinally and stretched on clean glass slides. The luminal contents were washed out with cold distilled water, the mucosa was scraped with a glass slide, and the scrapings were weighed to the nearest milligram. Homogenates of the mucosal linings of the different regions of the alimentary canal and liver were prepared in 0.25 M sucrose solution in a Potter-Elvehjem homogenizer. During homogenization, the homogenizer tube was kept in a container packed with crushed ice so that a temperature near 0°C was maintained. Homogenates were centrifuged for 20 min at 1000 Xg in a cold room, and the clear supernatant fluids were used as the source of enzymes. Enzyme extracts were kept frozen until required. The entire process of dissection of animals and collection of the mucosal lining consumed about 0.5 h. The activity of alkaline and acid phosphatase was determined by the method of Bodansky (1965). The substrate solution for alkaline phosphatase was prepared by dissolving 0.5 g sodium (3-glycerophosphate and 0.424 g sodium diethylbarbiturate in 100 ml double-distilled water. A 9-ml portion of the substrate solution was preincubated at 37°C for 15 min and 1 ml enzyme extract was added to it. The mixture was incubated at 37°C for exactly 1 h. At the end of the incubation period, enzyme activity was stopped with 2 ml 30% trichloroacetic acid (TCA). After filtering, 1 ml acidic ammonium molybdate solution and 0.4 ml aminonaphthosulfonic acid solution were added to 8 ml filtrate and the mixture was diluted to 10 ml. The intensity of color that developed after 5 min was read at 660 nm. For controls, 9 ml substrate solution was preincubated with 2 ml 30% TCA, and the enzyme extract was added after 15 min. A blank with 8 ml 5% TCA and standard were similarly prepared. For acid phosphatase a similar procedure was followed, except that the substrate solution prepared for alkaline phosphate was adjusted to pH 5 with 1 N acetic acid. Amylase activity was estimated by the method of Bemfield (1955). Merck's soluble starch (1 g) was dissolved in 100 ml 0.02 M phosphatase buffer, pH 6.9, containing 0.0067 M NaCI. A 1-ml portion of the substrate solution was incubated with 1 ml enzyme extract for 1 h at 37°C.
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Enzyme activity was stopped by boiling the incubation mixtures in a water bath for 5 min. The reducing sugars liberated were quantitatively estimated by the method of Folin and Wu, as given by Oser (1965). The activity of the two proteases was determined by the method of Rick (1965), using hemoglobin as the substrate. Hemoglobin (2.0 g) was suspended in 50 ml double-distilled water, and 36 g urea and 8 ml 1 /V NaOH were added. After 60 min, 10 ml 1 N boric acid and 44 ml 5% CaCI2 were added. The mixture was adjusted to pH 7.5 for trypsin and diluted to 100 ml with double-distilled water. To 5 ml substrate, 1 ml enzyme extract was added, and the solution was incubated at 37°C. After 1 h, 10 ml 5% TCA was added and the tyrosine liberated was determined by the Folin phenol reagent at 578 nm. For the estimation of pepsin activity, 2 g hemoglobin was dissolved in 0.06 N HCI and the solution was made up to 100 ml with distilled water. The pH was 1.8. The rest of the procedure was similar to that for trypsin. For each enzyme, triplicate samples were analyzed and the incubations were repeated three times. The total protein content in the homogenates was determined in TCA precipitates by the method of Lowry et al. (1951), using bovine serum albumin as the standard. The test described by Fisher (1950) was employed for statistical analysis of the results. RESULTS AND DISCUSSION The results of the experiments conducted are presented in Tables 1-3. We observed an overall inhibition of enzyme activity during the first week of mercuric chloride intoxication, showing that the physiological functioning of the digestive system is affected. However, during the second and fourth weeks, the level of enzyme activity was restored or there was a slight elevation in activity. Alkaline phosphatase is a brush border enzyme involved in transphosphorylation reactions. The activity of enzyme has been correlated with the absorption of nutrients across the intestinal wall (Verzar and McDougall, 1936). The decrease in activity observed during the first week of treatment may result from the cellular damage and indicates that the transphosphorylation reaction and absorption processes in the intestine are inhibited. Hinton et al. (1973) and Kendall (1975) reported similar inhibition in hepatic alkaline phosphatase activity. However, Hindriks et al. (1973) showed that zinc and magnesium are necessary for alkaline phosphatase activity in mammalian intestine and placenta and that mercury can replace them. If a similar condition exists in fish, the slight increase in alkaline phosphatase activity observed in the second and fourth weeks of treatment may be caused by greater availability of mercury in the intestine. In the liver and stomach, although a marginal increase in activity was noted, it was statistically insignificant. Cellular damage is usually accompanied by an increase in acid phosphatase activity; the inhibition observed in the first week may be caused by direct binding of
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TABLE 1. Activities of Alkaline and Acid Phosphatases in Experimental and Control Fish" Experimental Enzymes
Tissue
Alkaline phosphatase
Liver Stomach Intestine Pyloric ceca Liver Stomach Intestine Pyloric ceca
o Acid phosphatase
Control 0.0192 0.0205 0.0184 0.0185 0.0216 0.0217 0.0187 0.0195
± ± i ± ± ± ± ±
0.0019 0.0024 0.0015 0.0010 0.0010 0.0016 0.0015 0.0010
7d 0.0102 ± 0.0165 t 0.0218 ± 0.0145 ± 0.0145 ± 0.0160 ± 0.0248 ± 0.0171 t
Difference 0.0001 0.0002 0.0003 0.0005 0.0030 0.0002 0.0004 0.0002
5.84 6 2.10 2.906 4.44 e &ASb 4.75 6 5.08o 2.92 6
15 d 0.0232 ± 0.0248 ± 0.0283 ± 0.0265 ± 0.0246 ± 0.0283 ± 0.0270 ± 0.0228 ±
0.0005 0.0006 0.0014 0.0002 0.0001 0.0002 0.0003 0.0002
Difference 2.50 2.26 5.93 6 9.75o 3.70 6 5.50 6 7.25Ö 4.02 ö
30 d 0.0220 0.0225 0.0246 0.0263 0.0220 0.0225 0.0246 0.0263
± ± ± ± i ± ± ±
"Activity is expressed as milligrams of inorganic phosphate liberated per milligram of protein per hour at 37°C. Values are means ± SE. This difference is statistically significant at the 95% confidence interval.
0.0003 0.0002 0.0005 0.0003 0.0003 0.0002 0.0005 0.0003
Difference 1.75 1.05 4.84 6 9.28& 0.47 0.66 4.61 6 8.09 6
TABLE 2. Activity of Amylase in Experimental and Control Fish0 Experimental Tissue Liver Stomach Intestine Pyloric ceca
Control 0.1070 0.0713 0.0667 0.0698
± ± ± ±
7d
0.0030 0.0092 0.0044 0.0048
0.1020 0.0421 0.0767 0.0463
Difference
+ 0.0030 ± 0.0004 + 0.0013 ± 0.0009
15 d
1.56 3.89* 2.70 5.92*
0.1190 ± 0.0710 ± 0.0730 ± 0.0830 ±
Difference
0.0004 0.0028 0.0003 0.0004
5.0* 0.04 1.75 3.41*
30 d 0.1240 0.0979 0.0860 0.0939
±0.0004 ± 0.0020 ± 0.0011 ± 0.0012
Difference 7.91* 3.41* 5.21* 6.02*
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"Activity is expressed as milligrams of maltose liberated per milligram of protein per hour at 37°C. Values are means ± SE. This difference is statistically significant at the 95% confidence interval.
00
TABLE 3. Activities of Trypsin and Pepsin in Experimental and Control Fish Experimental Enzyme Trypsin Pepsin
Tissue
Control
7d
Difference
15 d
Difference
30 d
Difference
Intestine Pyloric ceca Stomach
0.0860 + 0.0092 0.0640 ± 0.0070 0.4230 ± 0.0346
0.1180 ± 0.0032 0.0710± 0.0026 0.0630 ± 0.0070
4.10* 1.66 12.50*
0.1120 ± 0.0060 0.0900 ± 0.0100 0.7880 + 0.0120
2.95* 2.62 12.16*
0.2250 ± 0.0100 0.1300 ± 0.0126 0.0469 ± 0.0130
12.63* 6.66* 12.53*
^Activity is expressed as milligrams of glycine liberated per milligram of protein per hour at 37°C. Values are means ± SE. This difference is statistically significant at the 95% confidence interval.
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mercury to the enzyme protein. In contrast after 15 d, the enzyme activity was markedly elevated. Increased acid phosphatase activity in the intestinal mucosa of animals treated with manganese was reported by Chandra and Imam (1973), and was attributed by Zonek et al. (1966) to increased pinocytosis. After 30 d of exposure there was a slight increase in enzyme activity, but this was insignificant in liver and stomach. Amylase, trypsin, and pepsin activities were inhibited in the first week and elevated after 15 and 30 d of exposure. In our earlier studies (Sastry and Gupta, 1977a, 1977b) similar alterations in digestive enzyme activities were observed in C. punctatus exposed to 1.8 mg/l of mercuric chloride and 3.8 mg/l of lead nitrate. Heavy metals such as mercury and lead are known to be inhibitors of enzyme activities. Sugai (1944) observed a similar inhibition of trypsin activity in vitro by nickel. This inhibition may result from direct binding of the heavy metal to the enzyme protein, leading to a decrease in activity, or to toxic effects in tissue resulting in structural damage to the cell organelles and decreased synthesis of enzyme protein. Although in vitro inhibition of enzymes by heavy metals is well known, the mechanism is not clear. The increased activity of enzymes, as pointed out by Christensen (1975), may result from enzyme induction. The initial inhibition of enzyme activity observed here may be caused by structural damage and binding of the mercury to the enzymes. The return to normal or elevation of the enzyme activity may be attributed to repair of the cells and enzyme induction.
REFERENCES Bernfield, P. 1955. Amylases α and β. Methods Enzymol. 1:149. Bodansky, A. 1965. Determination of serum phosphatase activity. In Hawk's Physiological Chemistry, ed. B. L. Oser, p. 1118. New York: McGraw-Hill. Chandra, S. V. and Imam, Z. 1973. Manganese induced histochemical and histological alterations in gastrointestinal mucosa of guinea pig. Acta Pharmacol. Toxicol. (Kbh.) 33:449-458. Chang, L. W. and Hartmann, H. A. 1972a. Ultrastructure studies of the nervous system after mercury intoxication. I. Pathological changes in the nerve cell bodies. Acta Neutropathol. (Berl.) 20:122-138. Chang, L. W. and Hartmann, H. A. 1972b. Ultrastructural studies of the nervous system after mercury intoxication. II. Pathological changes in the nerve fibres. Acta Neuropathol. (Berl.) 20:316-334. Chang, L. W. and Hartmann, H. A. 1972c. Electron microscopic histochemical studies on the localization and distribution of mercury in the nervous system after mercury intoxication. Exp. Neurol. 35:122-137. Chang, L. W., Ware, R. A., and Desnoyers, P. A. 1973. A histochemical study on some enzyme change in the kidney, liver and brain after chronic mercury intoxication in rat. Food Cosmet. Toxicol. 11:283-286. Christensen, G. M. 1975. Biochemical effects of methyl mercuric chloride, cadmium chloride and lead nitrate on embryos and elevins of the brook trout, Salvelinus fontinalis. Toxicol. Appl. Pharmacol. 32:191-197. Fisher, R. A. 1950. Statistical Methods for Research Workers, 11th ed. London: Oliver & Boyd. Hindriks, F. R., Groen, A., and Kroon, A. M. 1973. On the relation of heavy metals to the activity
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and heat stability of alkaline phosphatase from human placenta. Biochim. Biophys. Acta 315:94-102. Hinton, D. E., Kendall, M. W., and Silver, B. B. 1973. Use of histologic and histochemical assessment in the prognosis of the effect of aquatic pollutants. In Biological Methods for the Assessment of Water Quality, eds. J. Cairns, Jr. and K. L. Dickson, pp. 194-208. Philadelphia: American Society for Testing and Materials. Kendall, M. W. 1975. Acute effects of methyl mercury toxicity in channel cat fish (Ictalurus punctatus) kidney. Bull. Environ. Contam. Toxicol. 13:570. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265. Oser, B. L., ed. 1965. Hawk's Physiological Chemistry, p. 1052. New York: McGraw-Hill. Rick, W. 1965. Trypsin, pepsin pepsinogen, uropepsinogen. In Methods of Enzymatic Analysis, ed. H. U. Bergmeyer, p. 807. New York: Academic Press. Sahaphong, G. S. and Trump, B. F. 1971. Studies of cellular injury in isolated kidney tubules of the flounder. Am. J. Pathol. 63:277-297. Sastry, K. V. and Gupta, P. K. 1977a. Effect of mercuric chloride on the digestive system of Channa punctatus. Bull. Environ. Contam. Toxicol., in press. Sastry, K. V. and Gupta, P. K. 1977b. Alterations in the activity of some digestive enzymes of Channa punctatus exposed to lead nitrate. Bull. Environ. Contam. Toxicol. 19:549-556. Sugai, K. 1944. Studies on proteases. VI. The effect of the addition of various salts on the tryptic activity. J. Biochem. (Tokyo) 36:91-100. Verzar, F. and McDougall, E. F. 1936. Absorption from the Intestine. London: Longmans Green. Ware, R. A., Chang, L. W., and Burkholder, P. M. 1973. Ultrastructural pathology of the kidney following organic and inorganic mercury intoxications. Fed. Proc. 32:823. Zonek, J., Oekowski, Z., and Jonderko, G. 1966. Cyto-chemical studies on the behavior of thiamine pyrophosphatase, NADH2-tetrazolium reductase and acid phosphatase in the cerebellum of rabbit chronically poisoned with manganese. Int. Arch. Gewerbepathol. Gewerbehyg. 22:1-9. Received August 28, 7977 Accepted December 15, 1977
Journal of Toxicology and Environmental Health: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
Chronic mercuric chloride intoxication in digestive system of Channa punctatus a
K. V. Sastry & P. K. Gupta
a
a
Department of Zoology , D.A.V. (P.G.) College , Muzaffarnagar, U.P., India Published online: 20 Oct 2009.
To cite this article: K. V. Sastry & P. K. Gupta (1978) Chronic mercuric chloride intoxication in digestive system of Channa punctatus , Journal of Toxicology and Environmental Health: Current Issues, 4:5-6, 777-783, DOI: 10.1080/15287397809529699 To link to this article: http://dx.doi.org/10.1080/15287397809529699
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
CHRONIC MERCURIC CHLORIDE INTOXICATION IN DIGESTIVE SYSTEM OF CHANNA PUNCTATUS K. V. Sastry, P. K. Gupta Department of Zoology, D.A.V. (P.G.) College, Muzaffarnagar, India
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The effect of exposure to a sublethal concentration (0.30 mg/l) of mercuric chloride on the activities of alkaline phosphatase, acid phosphatase, amylase, trypsin, and pepsin has been examined at intervals of 7, 15, and 30 d in the digestive system of a teleost fish, Channa punctatus. Inhibition of the activities of all these enzymes was noted after the first week of treatment. Treatment of the fish for 15 d resulted in marked increases in the activities of all the enzymes. A slight fall in enzyme activity was recorded after 30 d, but the overall activity was higher than in control fish.
INTRODUCTION Heavy metals produce toxic effects on the tissues and alter the physiological functioning of various systems in animals. Mercury and copper are among the most toxic heavy metals to fish. Toxicity of mercurial compounds has been reported in the nervous system (Chang and Hartmann, 1972a, 1972b, 1972c) and ¡n the kidney (Sahaphong and Trump, 1971; Ware et al., 1973). Hinton et al. (1973) observed liver damage, in the form of necrosis in portal areas and formation of connective tissue septa, resulting from methyl mercury poisoning. Chang et al. (1973) studied histochemical changes in rat kidney, liver, and brain after chronic mercuric chloride intoxication. Very little information is available on the histopathologic and biochemical alterations produced in the digestive system by exposure to this metal. In this paper we report alterations in enzyme activities that accompany chronic mercury intoxication in liver and different parts of the digestive tract of a teleost fish, Channa punctatus. METHODS AND MATERIALS Living fish were collected from local freshwater sources and maintained in laboratory aquariums. Specimens weighing 60 ± 5 g each were We thank Dr. V. P. Agrawal for criticism and help. Financial assistance from the University Grants Commission, New Delhi, is gratefully acknowledged. Requests for reprints should be sent to K. V. Sastry, Department of Zoology, D.A.V. (P.G.) College, Muzaffarnagar (U.P.), India. 777 Journal of Toxicology and Environmental Health, 4:777-783,1978 Copyright © 1978 by Hemisphere Publishing Corporation 0098-4108/78/040777-07$2.25
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selected and were acclimatized to laboratory conditions for 3 d before experimentation. Preliminary bioassays conducted in the laboratory under static conditions showed that 0.30 mg/l is a sublethal concentration. Fish were divided into 2 groups of 30 fish each. The first group was treated with the calculated dose of mercuric chloride up to 4 wk, while second group served as controls. From both groups, 10 fish each were sacrificed at intervals of 7, 15, and 30 d. The different parts of the alimentary canal and liver were quickly excised from fish 1 h after feeding, and the tissues were freed from the adjoining tissues and immediately frozen at —4°C. For the preparation of the enzyme extracts of the stomach, intestine, and pyloric ceca, these portions were slit open longitudinally and stretched on clean glass slides. The luminal contents were washed out with cold distilled water, the mucosa was scraped with a glass slide, and the scrapings were weighed to the nearest milligram. Homogenates of the mucosal linings of the different regions of the alimentary canal and liver were prepared in 0.25 M sucrose solution in a Potter-Elvehjem homogenizer. During homogenization, the homogenizer tube was kept in a container packed with crushed ice so that a temperature near 0°C was maintained. Homogenates were centrifuged for 20 min at 1000 Xg in a cold room, and the clear supernatant fluids were used as the source of enzymes. Enzyme extracts were kept frozen until required. The entire process of dissection of animals and collection of the mucosal lining consumed about 0.5 h. The activity of alkaline and acid phosphatase was determined by the method of Bodansky (1965). The substrate solution for alkaline phosphatase was prepared by dissolving 0.5 g sodium (3-glycerophosphate and 0.424 g sodium diethylbarbiturate in 100 ml double-distilled water. A 9-ml portion of the substrate solution was preincubated at 37°C for 15 min and 1 ml enzyme extract was added to it. The mixture was incubated at 37°C for exactly 1 h. At the end of the incubation period, enzyme activity was stopped with 2 ml 30% trichloroacetic acid (TCA). After filtering, 1 ml acidic ammonium molybdate solution and 0.4 ml aminonaphthosulfonic acid solution were added to 8 ml filtrate and the mixture was diluted to 10 ml. The intensity of color that developed after 5 min was read at 660 nm. For controls, 9 ml substrate solution was preincubated with 2 ml 30% TCA, and the enzyme extract was added after 15 min. A blank with 8 ml 5% TCA and standard were similarly prepared. For acid phosphatase a similar procedure was followed, except that the substrate solution prepared for alkaline phosphate was adjusted to pH 5 with 1 N acetic acid. Amylase activity was estimated by the method of Bemfield (1955). Merck's soluble starch (1 g) was dissolved in 100 ml 0.02 M phosphatase buffer, pH 6.9, containing 0.0067 M NaCI. A 1-ml portion of the substrate solution was incubated with 1 ml enzyme extract for 1 h at 37°C.
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Enzyme activity was stopped by boiling the incubation mixtures in a water bath for 5 min. The reducing sugars liberated were quantitatively estimated by the method of Folin and Wu, as given by Oser (1965). The activity of the two proteases was determined by the method of Rick (1965), using hemoglobin as the substrate. Hemoglobin (2.0 g) was suspended in 50 ml double-distilled water, and 36 g urea and 8 ml 1 /V NaOH were added. After 60 min, 10 ml 1 N boric acid and 44 ml 5% CaCI2 were added. The mixture was adjusted to pH 7.5 for trypsin and diluted to 100 ml with double-distilled water. To 5 ml substrate, 1 ml enzyme extract was added, and the solution was incubated at 37°C. After 1 h, 10 ml 5% TCA was added and the tyrosine liberated was determined by the Folin phenol reagent at 578 nm. For the estimation of pepsin activity, 2 g hemoglobin was dissolved in 0.06 N HCI and the solution was made up to 100 ml with distilled water. The pH was 1.8. The rest of the procedure was similar to that for trypsin. For each enzyme, triplicate samples were analyzed and the incubations were repeated three times. The total protein content in the homogenates was determined in TCA precipitates by the method of Lowry et al. (1951), using bovine serum albumin as the standard. The test described by Fisher (1950) was employed for statistical analysis of the results. RESULTS AND DISCUSSION The results of the experiments conducted are presented in Tables 1-3. We observed an overall inhibition of enzyme activity during the first week of mercuric chloride intoxication, showing that the physiological functioning of the digestive system is affected. However, during the second and fourth weeks, the level of enzyme activity was restored or there was a slight elevation in activity. Alkaline phosphatase is a brush border enzyme involved in transphosphorylation reactions. The activity of enzyme has been correlated with the absorption of nutrients across the intestinal wall (Verzar and McDougall, 1936). The decrease in activity observed during the first week of treatment may result from the cellular damage and indicates that the transphosphorylation reaction and absorption processes in the intestine are inhibited. Hinton et al. (1973) and Kendall (1975) reported similar inhibition in hepatic alkaline phosphatase activity. However, Hindriks et al. (1973) showed that zinc and magnesium are necessary for alkaline phosphatase activity in mammalian intestine and placenta and that mercury can replace them. If a similar condition exists in fish, the slight increase in alkaline phosphatase activity observed in the second and fourth weeks of treatment may be caused by greater availability of mercury in the intestine. In the liver and stomach, although a marginal increase in activity was noted, it was statistically insignificant. Cellular damage is usually accompanied by an increase in acid phosphatase activity; the inhibition observed in the first week may be caused by direct binding of
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TABLE 1. Activities of Alkaline and Acid Phosphatases in Experimental and Control Fish" Experimental Enzymes
Tissue
Alkaline phosphatase
Liver Stomach Intestine Pyloric ceca Liver Stomach Intestine Pyloric ceca
o Acid phosphatase
Control 0.0192 0.0205 0.0184 0.0185 0.0216 0.0217 0.0187 0.0195
± ± i ± ± ± ± ±
0.0019 0.0024 0.0015 0.0010 0.0010 0.0016 0.0015 0.0010
7d 0.0102 ± 0.0165 t 0.0218 ± 0.0145 ± 0.0145 ± 0.0160 ± 0.0248 ± 0.0171 t
Difference 0.0001 0.0002 0.0003 0.0005 0.0030 0.0002 0.0004 0.0002
5.84 6 2.10 2.906 4.44 e &ASb 4.75 6 5.08o 2.92 6
15 d 0.0232 ± 0.0248 ± 0.0283 ± 0.0265 ± 0.0246 ± 0.0283 ± 0.0270 ± 0.0228 ±
0.0005 0.0006 0.0014 0.0002 0.0001 0.0002 0.0003 0.0002
Difference 2.50 2.26 5.93 6 9.75o 3.70 6 5.50 6 7.25Ö 4.02 ö
30 d 0.0220 0.0225 0.0246 0.0263 0.0220 0.0225 0.0246 0.0263
± ± ± ± i ± ± ±
"Activity is expressed as milligrams of inorganic phosphate liberated per milligram of protein per hour at 37°C. Values are means ± SE. This difference is statistically significant at the 95% confidence interval.
0.0003 0.0002 0.0005 0.0003 0.0003 0.0002 0.0005 0.0003
Difference 1.75 1.05 4.84 6 9.28& 0.47 0.66 4.61 6 8.09 6
TABLE 2. Activity of Amylase in Experimental and Control Fish0 Experimental Tissue Liver Stomach Intestine Pyloric ceca
Control 0.1070 0.0713 0.0667 0.0698
± ± ± ±
7d
0.0030 0.0092 0.0044 0.0048
0.1020 0.0421 0.0767 0.0463
Difference
+ 0.0030 ± 0.0004 + 0.0013 ± 0.0009
15 d
1.56 3.89* 2.70 5.92*
0.1190 ± 0.0710 ± 0.0730 ± 0.0830 ±
Difference
0.0004 0.0028 0.0003 0.0004
5.0* 0.04 1.75 3.41*
30 d 0.1240 0.0979 0.0860 0.0939
±0.0004 ± 0.0020 ± 0.0011 ± 0.0012
Difference 7.91* 3.41* 5.21* 6.02*
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"Activity is expressed as milligrams of maltose liberated per milligram of protein per hour at 37°C. Values are means ± SE. This difference is statistically significant at the 95% confidence interval.
00
TABLE 3. Activities of Trypsin and Pepsin in Experimental and Control Fish Experimental Enzyme Trypsin Pepsin
Tissue
Control
7d
Difference
15 d
Difference
30 d
Difference
Intestine Pyloric ceca Stomach
0.0860 + 0.0092 0.0640 ± 0.0070 0.4230 ± 0.0346
0.1180 ± 0.0032 0.0710± 0.0026 0.0630 ± 0.0070
4.10* 1.66 12.50*
0.1120 ± 0.0060 0.0900 ± 0.0100 0.7880 + 0.0120
2.95* 2.62 12.16*
0.2250 ± 0.0100 0.1300 ± 0.0126 0.0469 ± 0.0130
12.63* 6.66* 12.53*
^Activity is expressed as milligrams of glycine liberated per milligram of protein per hour at 37°C. Values are means ± SE. This difference is statistically significant at the 95% confidence interval.
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782
K. V. SASTRY AND P. K. GUPTA
mercury to the enzyme protein. In contrast after 15 d, the enzyme activity was markedly elevated. Increased acid phosphatase activity in the intestinal mucosa of animals treated with manganese was reported by Chandra and Imam (1973), and was attributed by Zonek et al. (1966) to increased pinocytosis. After 30 d of exposure there was a slight increase in enzyme activity, but this was insignificant in liver and stomach. Amylase, trypsin, and pepsin activities were inhibited in the first week and elevated after 15 and 30 d of exposure. In our earlier studies (Sastry and Gupta, 1977a, 1977b) similar alterations in digestive enzyme activities were observed in C. punctatus exposed to 1.8 mg/l of mercuric chloride and 3.8 mg/l of lead nitrate. Heavy metals such as mercury and lead are known to be inhibitors of enzyme activities. Sugai (1944) observed a similar inhibition of trypsin activity in vitro by nickel. This inhibition may result from direct binding of the heavy metal to the enzyme protein, leading to a decrease in activity, or to toxic effects in tissue resulting in structural damage to the cell organelles and decreased synthesis of enzyme protein. Although in vitro inhibition of enzymes by heavy metals is well known, the mechanism is not clear. The increased activity of enzymes, as pointed out by Christensen (1975), may result from enzyme induction. The initial inhibition of enzyme activity observed here may be caused by structural damage and binding of the mercury to the enzymes. The return to normal or elevation of the enzyme activity may be attributed to repair of the cells and enzyme induction.
REFERENCES Bernfield, P. 1955. Amylases α and β. Methods Enzymol. 1:149. Bodansky, A. 1965. Determination of serum phosphatase activity. In Hawk's Physiological Chemistry, ed. B. L. Oser, p. 1118. New York: McGraw-Hill. Chandra, S. V. and Imam, Z. 1973. Manganese induced histochemical and histological alterations in gastrointestinal mucosa of guinea pig. Acta Pharmacol. Toxicol. (Kbh.) 33:449-458. Chang, L. W. and Hartmann, H. A. 1972a. Ultrastructure studies of the nervous system after mercury intoxication. I. Pathological changes in the nerve cell bodies. Acta Neutropathol. (Berl.) 20:122-138. Chang, L. W. and Hartmann, H. A. 1972b. Ultrastructural studies of the nervous system after mercury intoxication. II. Pathological changes in the nerve fibres. Acta Neuropathol. (Berl.) 20:316-334. Chang, L. W. and Hartmann, H. A. 1972c. Electron microscopic histochemical studies on the localization and distribution of mercury in the nervous system after mercury intoxication. Exp. Neurol. 35:122-137. Chang, L. W., Ware, R. A., and Desnoyers, P. A. 1973. A histochemical study on some enzyme change in the kidney, liver and brain after chronic mercury intoxication in rat. Food Cosmet. Toxicol. 11:283-286. Christensen, G. M. 1975. Biochemical effects of methyl mercuric chloride, cadmium chloride and lead nitrate on embryos and elevins of the brook trout, Salvelinus fontinalis. Toxicol. Appl. Pharmacol. 32:191-197. Fisher, R. A. 1950. Statistical Methods for Research Workers, 11th ed. London: Oliver & Boyd. Hindriks, F. R., Groen, A., and Kroon, A. M. 1973. On the relation of heavy metals to the activity
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MERCURIC CHLORIDE IN CHANNA PUNCTATUS
783
and heat stability of alkaline phosphatase from human placenta. Biochim. Biophys. Acta 315:94-102. Hinton, D. E., Kendall, M. W., and Silver, B. B. 1973. Use of histologic and histochemical assessment in the prognosis of the effect of aquatic pollutants. In Biological Methods for the Assessment of Water Quality, eds. J. Cairns, Jr. and K. L. Dickson, pp. 194-208. Philadelphia: American Society for Testing and Materials. Kendall, M. W. 1975. Acute effects of methyl mercury toxicity in channel cat fish (Ictalurus punctatus) kidney. Bull. Environ. Contam. Toxicol. 13:570. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265. Oser, B. L., ed. 1965. Hawk's Physiological Chemistry, p. 1052. New York: McGraw-Hill. Rick, W. 1965. Trypsin, pepsin pepsinogen, uropepsinogen. In Methods of Enzymatic Analysis, ed. H. U. Bergmeyer, p. 807. New York: Academic Press. Sahaphong, G. S. and Trump, B. F. 1971. Studies of cellular injury in isolated kidney tubules of the flounder. Am. J. Pathol. 63:277-297. Sastry, K. V. and Gupta, P. K. 1977a. Effect of mercuric chloride on the digestive system of Channa punctatus. Bull. Environ. Contam. Toxicol., in press. Sastry, K. V. and Gupta, P. K. 1977b. Alterations in the activity of some digestive enzymes of Channa punctatus exposed to lead nitrate. Bull. Environ. Contam. Toxicol. 19:549-556. Sugai, K. 1944. Studies on proteases. VI. The effect of the addition of various salts on the tryptic activity. J. Biochem. (Tokyo) 36:91-100. Verzar, F. and McDougall, E. F. 1936. Absorption from the Intestine. London: Longmans Green. Ware, R. A., Chang, L. W., and Burkholder, P. M. 1973. Ultrastructural pathology of the kidney following organic and inorganic mercury intoxications. Fed. Proc. 32:823. Zonek, J., Oekowski, Z., and Jonderko, G. 1966. Cyto-chemical studies on the behavior of thiamine pyrophosphatase, NADH2-tetrazolium reductase and acid phosphatase in the cerebellum of rabbit chronically poisoned with manganese. Int. Arch. Gewerbepathol. Gewerbehyg. 22:1-9. Received August 28, 7977 Accepted December 15, 1977
