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Hemolymph acid phosphatase activity pattern in copper-stressed bivalves
JOURNALOFINVERTEBRATEPATHOLOGY
55,118-125(1990)
Hemolymph
Acid Phosphatase Activity Pattern in Copper-Stressed Bivalves K. SURESH AND A. MOHANDAS'
School of Environmental
Studies, Cochin University of Science and Technology, Fine Arts Avenue, Cochin-682 016, Kerala, India Received April 10, 1989; accepted May 8, 1989
The activity pattern of the lysosomal marker enzyme, acid phosphatase, was studied for 120 hr in the hemolymph of two clam species, Sunetta scripta and Villorita cyprinoides var. cochinensis, exposed to three sublethal concentrations of copper. Fifty specimens of S. scripta were exposed to each of the concentrations of copper (1, 3, and 5 ppm). Fifty specimens of V. cyprinoides var. cochinensis were exposed to 0. IS, 0.30 and 0.45 ppm of copper. The enzyme activity was estimated every 24 hr. The results indicate that (1) the activity levels of hemolymph acid phosphatase in clams exposed to sublethal concentrations of copper vary from species to species and are also dependent on the concentration of metal ions used; (2) the metal ion can cause destabilization of the lysosomal membrane and the consequent release of the enzyme into the hemolymph or can trigger hypersynthesis of acid phosphatase, which is subsequently released into the hemolymph; (3) copper ions can inhibit the activity of the enzyme; and (4) depending upon the period of exposure and the concentration of the metal ion, enzyme synthesis can also be adversely affected. e ww Academic Press, Inc.
KEY WORDS:
Sunetta scripta; Villorita cyprinoides var. cochinensis; hemolymph; acid phos-
phatase; copper.
INTRODUCTION The capacity of bivalves to accumulate potentially toxic heavy metals in their tissues far in excess of environmental levels is well documented (Roberts, 1976; Phillips, 1977; Calabrese et al., 1984). This indicates that these organisms have evolved some form of control or tolerance at the cellular and subcellular levels. Heavy metals, however, have been found to exert inhibitory effects on several physiological processes (see Akberali and Trueman, 1985; Suresh and Mohandas, 1987), to inhibit the activity of certain enzymes (Mahler, 1961; Yoshino et al., 1966; Dixon and Webb, 1967), to activate (Iordachesw et al., 1978) or influence the rate of action by activation, inactivation, uncoupling reactions or mechanisms yet to be defined (Have, 1969; Spom et al., 1970; Vallee and Wacker, 1970), to affect the functions of several cellular constituents, including lysosomes (Stemlieb and Goldfischer, 1976; Moore, 1977, 1980; Pickwell and Steinert, 1984; Moore et al., 1984; ’ To whom correspondence should be addressed.
and others), and cellular immunity (Cheng and Sullivan, 1984). An organism’s response to any perturbation of its environment will appear first at the molecular level. Detection of appropriate indicator molecules, released or elicited by specific pollutants, could give a sensitive and early measure of a stress response (Livingstone, 1982; Pickwell and Steinert, 1984). Lysosomes have been found to be an ideal starting point for investigations of generalized cellular injury in marine mollusks because many cell types in these animals are particularly rich in lysosomes (Summer, 1969) and because these organelles are noted for their compartmentalization and accumulation of a wide variety of organic chemicals and metals. The lysosomal system has also been shown to be very sensitive to changes in the intra- and extracellular environment, and subsequently to be involved directly or indirectly in controlling many physiological and pathological processes (Chandy and Patel, 1985). Lysosomes are an effective detoxication system until the storage capacity of 118
0022-2011190 $1.50 Copyright 6 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
ACID
PHOSPHATASE
IN
COPPER-STRESSED
lysosomes is overloaded or they are damaged by the accumulated contaminants (Moore et al., 1985). When destabilization occurs (Moore and Lowe, 1985; Bayne et al., 1985), lysosomes burst, releasing lysosomal hydrolases into the cytoplasm and extracellular environment (Lauwerys and Buchet, 1972; Moore, 1976; Bayne et al., 1976; Baccino, 1978; Pickwell and Steinert, 1984). Quantitative measurement of the levels of marker lysosomal enzymes has been suggested as one of the best methods to use as a reliable indicator of the stress imposed by environmental pollutants (Cheng, 1983a). As very little information is available in this area, i.e., the activity pattern of lysosomal enzymes under heavy metal stress, it was thought worthwhile to investigate the activity pattern of the lysosomal marker enzyme, acid phosphatase, in the hemolymph of two clam species, Sunetta scripta and Villorita cyprinoides var. cochinensis, exposed to three sublethal concentrations of copper. The sublethal concentrations were determined from the results of previous bioassays. Blood parameters have been recognized as valuable tools in assessing the conditions of the organisms and their responses to physicochemical changes in the environment (Carr and Neff, 1984; Jyothirmayi and Rao, 1987), and, moreover, the circulating hemocytes are considered to be the primary source of hydrolytic enzymes in the serum of mollusks (Cheng and Rodrick, 1980). MATERIALS
AND METHODS
Specimens of S. scripta were collected from the sea off Cochin (salinity 30 t 2%0), southern India, and V. cyprinoides var. cochinensis from Cochin backwaters (salinity I5 2 4%0). They were immediately brought to the laboratory where specimens of S. scripta were maintained in recirculating seawater tanks with a salinity of 30%0 and V. cyprinoides var. cochinensis in recirculating seawater tanks with a salinity of 15%0. Particular care was taken to ensure that in none of the tanks were overcrowd-
BIVALVES
119
ing and oxygen supply limiting factors. The experiments were carried out at room temperature (29” + 1°C) and the clams were fed cultured algae, Synechocystis sp . Fifty specimens of S. scripta of the 38- to 40-mm size group were transferred to each of the three 20-liter tanks containing filtered sea water of 30%0 salinity and dosed with 1, 3, and 5 ppm of Cu2+, respectively. These three batches of clams constituted the experimental group. The 50 clams in a fourth 20-liter tank containing filtered sea water of 30%0 salinity served as controls. Fifty specimens of V. cyprinoides var. cochinensis of the 38- to 40-mm size group were transferred to each of the three 20-liter tanks containing filtered sea water of 15%0salinity and dosed with 0.15, 0.30, and 0.45 ppm of Cu2+, respectively. These three batches of clams constituted the experimental group. The 50 clams in a fourth 20-liter tank containing filtered sea water with a salinity of 15%0 served as controls. Copper sulfate (&SO4 * 5H,O, Glaxo Laboratories, Bombay, India) was used. The water in the tanks was changed daily, and the concentrations of the metal ions in the experimental tanks were maintained at their respective levels throughout the experimental period of 5 days. Every 24 hr for 5 days, a 0.5-ml sample of hemolymph was withdrawn from the posterior adductor muscle sinus of each of the clams of each species of the experimental batches by using a l-ml tuberculin syringe fitted with a 23-gauge hypodermic needle. This sample was expelled into a clean test tube. To estimate hemolymph acid phosphatase activity, a O.l-ml sample of hemolymph was taken from the test tube and pipetted into another test tube containing 1 ml of frozen 0.1 M citrate buffer (pH 3.6) and kept frozen until use. At the time of analysis, the bufferhemolymph mixture was kept in a water bath at 37°C and 0.1 ml of substrate (2 mg of p-nitrophenol phosphate sodium salt (Merck) in 0.1 ml) was added to start the reaction. After incubation for 1 hr at 37”C, the reaction was stopped by adding 2 ml of
120
SURESH
AND
MOHANDAS
0.25 N NaOH. The liberated p-nitrophenol was read at 410 nm in a Hitachi Model 20020 spectrophotometer. The enzyme activity is expressed as pmol of p-nitrophenol liberated/mg of proteink. Another sample of 0.1 ml of hemolymph was used to estimate protein, following the method of Lowry et al. (1951). Collection of hemolymph samples from clams comprising the control groups was carried out and enzyme activity measured in the same manner as for the experimental groups. The two-tailed t test (Zar, 1974)was employed to determine the statistically significant differences between the enzyme activity values of each of the three experimental batches and that of the controls in each clam species.
exposed to three sublethal concentrations of copper for 5 days are given in Tables 1 and 2, respectively. In S. scripta, hemolymph acid phosphatase activity values of clams dosed with 1ppm of copper when compared with those of the controls were significantly higher at 24 (P < 0.01) and 48 hr (P < 0.01). The hemolymph acid phosphatase activity values of clams dosed with 3 ppm of copper were significantly lower than the control value at 120 hr (P < 0.05). Clams dosed with 5 ppm of copper showed significantly higher values than the controls at 48 hr (P < 0.01) but significantly lower values at 96 (P < 0.05) and 120 hr (P < 0.02). In V. cyprinoides var. cochinensis, hemolymph acid phosphatase activity values of clams dosed with 0.15 ppm copper were significantly higher at 24 (P < 0.05), 48 (P < RESULTS 0.05), and 96 hr (P < 0.01) than the control Hemolymph acid phosphatase activity values for the respective days. The value values in S. scripta and V. cyprinoides var. for clams dosed with 0.30 ppm of copper cochinensis under normal conditions and was significantly lower than the control
HEMOLYMPH
ACID
PHOSPHATASE
Sunetta scripta
TABLE 1 ACTIVITY VALUES (PmoVmg PROTEIN/hT) EXPOSED TO SUBLETHAL CONCENTRATIONS
IN 38- TO 40-mm OF COPPER
SIZE GROUP
Hours Group
24
48
72
96
120
Control N Mean value *SD Range
10 0.2724 0.0645 0.1731-0.3586
10 0.2913 0.0546 0.2051-0.3659
0.2814 0.0607 0.1595-0.3516
10 0.2683 0.0775 0.1742-0.3686
10 0.2690 0.0703 0.1905-0.4563
1 ppm of Cu*+ N Mean value *SD Range
10 0.3679 0.0624 0.2941-0.4733
10 0.4361 0.1222 0.2333-0.6500
10 0.2675 0.1040 0.1375-0.4579
10 0.2057 0.0609 0.1300-0.3019
10 0.2212 0.0675 0.1446-0.3433
3 ppm of Cu*+ N Mean value *SD Range
10 0.3311 0.0886 0.2129-0.5034
10 0.3549 0.1317 0.1856-0.5615
10 0.2687 0.0675 0.2101-0.4423
10 0.2077 0.0528 0.1181-0.2827
10 0.2000 0.0475 0.1198-0.2973
5 ppm of Cu*+ N Mean value aSD Range
10 0.2208 0.0796 0.1267-0.3516
10 0.4154 0.0905 0.2417-0.5604
10 0.2652 0.0712 0.1667-0.3925
10 0.2026 0.0410 0.1663-0.3095
10 0.1973 0.0416 0.1382-0.2692
10
ACID
HEMOLYMPH
PHOSPHATASE
ACID
Villorita
PHOSPHATASE cyprinoides VAR.
ACTIVITY
cochinensis
IN COPPER-STRESSED
121
BIVALVES
TABLE 2 VALUES (kmol/mg PRoTEINihr) IN 38- TO 40-mm SIZE GROUP EXPOSED TO SUBLETHAL CONCENTRATIONS OF COPPER Hours
Group
24
48
72
96
120 --~
Control N Mean value *SD Range
10 0.5913 0.1712 0.3984-1.0152
10 0.4764 0.1515 0.2484-0.6909
10 0.5862 0.2009 0.3750-1.1007
10 0.5983 0.2268 0.3491-1.0187
10 0.6180 0.1920 0.3250-0.9206
0.15 ppm of Cu*+ N Mean value *SD Range
10 0.8191 0.2040 0.4854-l .2317
10 0.6428 0.1603 0.3585-0.9859
10 0.4353 0.1621 0.2295-0.6957
10 1.0634 0.3036 0.6273-1.6129
10 0.7067 0.2352 0.4200-1.0595
0.30 ppm of Cu*+ N Mean value +SD Range
10 0.6894 0.1721 0.4419-l .1233
10 0.5933 0.2222 0.2941-1.0147
10 0.7072 0.2929 0.3947-1.2195
10 0.6958 0.1926 0.4091-1.0319
10 0.3350 0.0972 0.2037-0.4507
0.45 ppm of Cd+ N Mean value tSD Range
10 0.7684 0.2224 0.369k1.1151
10 0.3153 0.0845 0.1744-0.4324
10 0.1600 0.0477 0.0866-0.2468
10 0.5510 0.2145 0.2568-0.8974
10 0.1558 0.0144 0.1362-0.1754
value at 120 hr (P < 0.01). The values for clams dosed with 0.45 ppm of copper were significantly lower than control values at 48 (P < 0.02), 72 (P < O.Ol), and 120 hr (P < 0.01). DISCUSSION In S. scripta and V. cyprinoides var. cochinensis exposed to copper, the observed differences in the time that the maximum and minimum activity levels of hemolymph acid phosphatase occurred are attributed to species differences and to the concentrations of the metal ion used. Apart from these, the onset of destabilization and reduction of latency of the enzyme might have also influenced the activity pattern of the enzyme. Many xenobiotics are known to induce alterations in the bounding membrane of the lysosome, leading to destabilization (see Moore and Lowe, 1985; Nott and Moore, 1987) and the subsequent release of
the lysosomal hydrolases into the cytosol (Moore, 1976; Baccino, 1978; and others). Roesijadi (1980) observed increased acid phosphatase activity in the gills of the clam Protothaca staminea at sublethal exposures to copper. Harrison and Berger (1982) found no significant reduction in lysosomal integrity in Mytilus edulis exposed to low copper concentrations but at the highest concentration significant reduction in lysosomal latency was observed. Pickwell and Steinert (1984) found that at low concentrations of copper, hemolymph lysozyme levels in M. edulis did not differ significantly from control values. Copper is believed to damage lysosomes by promoting lipid peroxidation (Baccino, 1978). The observed increase in hemolymph acid phosphatase activity levels in the early time periods in S. scriptu and V. cyprinoides var. cochinensis dosed with 1 and 0.15 ppm of copper, respectively, can be attributed to the release of the enzyme into the he-
122
SURESH
AND
molymph compartment from the lysosomes after the destabilization of the lysosomal membrane caused by copper ions. Since destabilization of the lysosomal membrane is believed to take place in the early time periods no significant quantum of enzyme was released into the hemolymph at later time periods, and hence no significant variation in the enzyme activity values at later time periods. Alternatively, metal ions might trigger the hypersynthesis of lysosomal hydrolase, acid phosphatase, which is released into the hemolymph. When the metal ion concentration is low, the hypersynthesis is not immediately inactivated by the metal ions and this is reflected as increased enzyme activity in the hemolymph in the early time periods. However, as the clams continue to be exposed to copper, hypersynthesis of the enzyme is inactivated and hence no significant difference in the values was observed at later time periods, i.e., at 72, 96, and 120 hr in S. scripta and at 120 hrs in V. cyprinoides var. cochinensis. Chandy and Pate1 (1985) indicated that at lower concentrations the metal ions that entered the system are engulfed into lysosomes and subsequently transformed into biologically inactive forms. Chandy and Pate1 (1985) added that the presence of metal ions causes increased availability of lysosomal acid hydrolases to metabolize and sequester the metal in some nontoxic form and eventually excrete it. Hypersynthesis and release of acid hydrolases from hemocytes into the hemolymph when mollusks are challenged with biotic and abiotic agents has already been indicated (see Cheng, 1983a, b; Cheng and Mohandas, 1985) with the involvement of recognition sites on the surface membranes of these cells. In S. scripta dosed with 3 ppm of copper and V. cyprinoides var. cochinensis dosed with 0.30 ppm of copper, the absence of a significant increase in enzyme activity in the early time periods is believed to occur for two reasons: (1) because of the fairly high metal ion concentration, the peak en-
MOHANDAS
zyme activity as a result of lysosomal destabilization took place much earlier, i.e., before 24 hr, or (2) the hypersynthesis of the enzyme was also inactivated by the metal ions from the very first day, and hence no significant difference in activity levels was observed in the early time periods. However, as the clams continue to be under metal stress, the concentration of copper used affected enzyme synthesis, which is also reflected as a significant fall in enzyme activity by 120 hr. These two explanations also hold for the enzyme activity pattern seen in clams exposed to the highest concentrations. But the pattern of activity is different in that at the highest concentrations, the adverse effect of metal ions on enzyme synthesis started much earlier. With the result, the significant fall in hemolymph enzyme activity in S. scripta dosed with 5 ppm of copper and V. cyprinoides var. cochinensis dosed with 0.45 ppm of copper started by 96 and 48 hr, respectively . The results of the present study also indicated that at certain time periods the experimental specimens showed inconsistent enzyme activity values when compared with the controls, i.e., higher values at 48 hr, but no significant difference at 72 hr in S. scripta dosed with 5 ppm of copper and no significant difference in value at 72 hr and at 96 hr in V. cyprinoides var. cochinensis, dosed with 0.15 and 0.45 ppm of copper respectively. In this context it may be noted that lysosomes, even in single cells, are quite variable in their enzymatic constitution (Dean, 1977), and the heterogeneity in size and shape reflects the divergent functional activities of lysosomes in different cell types (Schellens et al., 1977). Moreover, the cytoplasmic granules in the granulocytes of several species of mollusks have been considered to be true lysosomes (see Mohandas et al., 1985). The finding of Yoshino and Cheng (1976) that not all of the cytoplasmic granules within the granulocytes of Mercenaria mercenaria include acid phosphatase activity prompted them to
ACID PHOSPHATASE
IN COPPER-STRESSED
suggest that these vesicles represent a chemically heterogeneous population or that there is a nonsynchronized chemical cycle that occurs within the granules (lysosomes). Thus, the observed inconsistency in enzyme activity values may be attributed to the nonsynchronized chemical cycle occurring in the lysosomes. It may also be noted that Granath and Yoshino (1983a) observed the existence of discrete lysosomal enzyme subpopulations within and between the hemocyte populations of two strains of Biomphalaria glabrata. Granath and Yoshino (1983b) subsequently suggested induced alterations in hemocyte enzyme activities that may include the selective switching of enzymes produced by individual hemocytes or the stimulation of enzyme production in previously nonenzymatically active cells. Based on the results obtained in the present study, it is concluded that (1) the activity levels of hemolymph acid phosphatase in clams exposed to sublethal concentrations of copper vary from species to species and are also dependent on the concentration of metal ions used; (2) copper ions can cause destabilization of the lysosomal membrane and the consequent release of the lysosomal hydrolase, acid phosphatase, into the hemolymph, or copper ions can trigger hypersynthesis of the lysosomal enzyme, which is subsequently released into the hemolymph; (3) copper ions can inhibit acid phosphatase activity; and (4) depending upon the period of exposure and the concentration of the metal ion, enzyme synthesis can be adversely affected. ACKNOWLEDGMENTS A personal fellowship awarded to the University Grants Commission, the facilities provided by the Cochin ence and Technology are gratefully
the first author by New Delhi, and University of Sciacknowledged.
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55,118-125(1990)
Hemolymph
Acid Phosphatase Activity Pattern in Copper-Stressed Bivalves K. SURESH AND A. MOHANDAS'
School of Environmental
Studies, Cochin University of Science and Technology, Fine Arts Avenue, Cochin-682 016, Kerala, India Received April 10, 1989; accepted May 8, 1989
The activity pattern of the lysosomal marker enzyme, acid phosphatase, was studied for 120 hr in the hemolymph of two clam species, Sunetta scripta and Villorita cyprinoides var. cochinensis, exposed to three sublethal concentrations of copper. Fifty specimens of S. scripta were exposed to each of the concentrations of copper (1, 3, and 5 ppm). Fifty specimens of V. cyprinoides var. cochinensis were exposed to 0. IS, 0.30 and 0.45 ppm of copper. The enzyme activity was estimated every 24 hr. The results indicate that (1) the activity levels of hemolymph acid phosphatase in clams exposed to sublethal concentrations of copper vary from species to species and are also dependent on the concentration of metal ions used; (2) the metal ion can cause destabilization of the lysosomal membrane and the consequent release of the enzyme into the hemolymph or can trigger hypersynthesis of acid phosphatase, which is subsequently released into the hemolymph; (3) copper ions can inhibit the activity of the enzyme; and (4) depending upon the period of exposure and the concentration of the metal ion, enzyme synthesis can also be adversely affected. e ww Academic Press, Inc.
KEY WORDS:
Sunetta scripta; Villorita cyprinoides var. cochinensis; hemolymph; acid phos-
phatase; copper.
INTRODUCTION The capacity of bivalves to accumulate potentially toxic heavy metals in their tissues far in excess of environmental levels is well documented (Roberts, 1976; Phillips, 1977; Calabrese et al., 1984). This indicates that these organisms have evolved some form of control or tolerance at the cellular and subcellular levels. Heavy metals, however, have been found to exert inhibitory effects on several physiological processes (see Akberali and Trueman, 1985; Suresh and Mohandas, 1987), to inhibit the activity of certain enzymes (Mahler, 1961; Yoshino et al., 1966; Dixon and Webb, 1967), to activate (Iordachesw et al., 1978) or influence the rate of action by activation, inactivation, uncoupling reactions or mechanisms yet to be defined (Have, 1969; Spom et al., 1970; Vallee and Wacker, 1970), to affect the functions of several cellular constituents, including lysosomes (Stemlieb and Goldfischer, 1976; Moore, 1977, 1980; Pickwell and Steinert, 1984; Moore et al., 1984; ’ To whom correspondence should be addressed.
and others), and cellular immunity (Cheng and Sullivan, 1984). An organism’s response to any perturbation of its environment will appear first at the molecular level. Detection of appropriate indicator molecules, released or elicited by specific pollutants, could give a sensitive and early measure of a stress response (Livingstone, 1982; Pickwell and Steinert, 1984). Lysosomes have been found to be an ideal starting point for investigations of generalized cellular injury in marine mollusks because many cell types in these animals are particularly rich in lysosomes (Summer, 1969) and because these organelles are noted for their compartmentalization and accumulation of a wide variety of organic chemicals and metals. The lysosomal system has also been shown to be very sensitive to changes in the intra- and extracellular environment, and subsequently to be involved directly or indirectly in controlling many physiological and pathological processes (Chandy and Patel, 1985). Lysosomes are an effective detoxication system until the storage capacity of 118
0022-2011190 $1.50 Copyright 6 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
ACID
PHOSPHATASE
IN
COPPER-STRESSED
lysosomes is overloaded or they are damaged by the accumulated contaminants (Moore et al., 1985). When destabilization occurs (Moore and Lowe, 1985; Bayne et al., 1985), lysosomes burst, releasing lysosomal hydrolases into the cytoplasm and extracellular environment (Lauwerys and Buchet, 1972; Moore, 1976; Bayne et al., 1976; Baccino, 1978; Pickwell and Steinert, 1984). Quantitative measurement of the levels of marker lysosomal enzymes has been suggested as one of the best methods to use as a reliable indicator of the stress imposed by environmental pollutants (Cheng, 1983a). As very little information is available in this area, i.e., the activity pattern of lysosomal enzymes under heavy metal stress, it was thought worthwhile to investigate the activity pattern of the lysosomal marker enzyme, acid phosphatase, in the hemolymph of two clam species, Sunetta scripta and Villorita cyprinoides var. cochinensis, exposed to three sublethal concentrations of copper. The sublethal concentrations were determined from the results of previous bioassays. Blood parameters have been recognized as valuable tools in assessing the conditions of the organisms and their responses to physicochemical changes in the environment (Carr and Neff, 1984; Jyothirmayi and Rao, 1987), and, moreover, the circulating hemocytes are considered to be the primary source of hydrolytic enzymes in the serum of mollusks (Cheng and Rodrick, 1980). MATERIALS
AND METHODS
Specimens of S. scripta were collected from the sea off Cochin (salinity 30 t 2%0), southern India, and V. cyprinoides var. cochinensis from Cochin backwaters (salinity I5 2 4%0). They were immediately brought to the laboratory where specimens of S. scripta were maintained in recirculating seawater tanks with a salinity of 30%0 and V. cyprinoides var. cochinensis in recirculating seawater tanks with a salinity of 15%0. Particular care was taken to ensure that in none of the tanks were overcrowd-
BIVALVES
119
ing and oxygen supply limiting factors. The experiments were carried out at room temperature (29” + 1°C) and the clams were fed cultured algae, Synechocystis sp . Fifty specimens of S. scripta of the 38- to 40-mm size group were transferred to each of the three 20-liter tanks containing filtered sea water of 30%0 salinity and dosed with 1, 3, and 5 ppm of Cu2+, respectively. These three batches of clams constituted the experimental group. The 50 clams in a fourth 20-liter tank containing filtered sea water of 30%0 salinity served as controls. Fifty specimens of V. cyprinoides var. cochinensis of the 38- to 40-mm size group were transferred to each of the three 20-liter tanks containing filtered sea water of 15%0salinity and dosed with 0.15, 0.30, and 0.45 ppm of Cu2+, respectively. These three batches of clams constituted the experimental group. The 50 clams in a fourth 20-liter tank containing filtered sea water with a salinity of 15%0 served as controls. Copper sulfate (&SO4 * 5H,O, Glaxo Laboratories, Bombay, India) was used. The water in the tanks was changed daily, and the concentrations of the metal ions in the experimental tanks were maintained at their respective levels throughout the experimental period of 5 days. Every 24 hr for 5 days, a 0.5-ml sample of hemolymph was withdrawn from the posterior adductor muscle sinus of each of the clams of each species of the experimental batches by using a l-ml tuberculin syringe fitted with a 23-gauge hypodermic needle. This sample was expelled into a clean test tube. To estimate hemolymph acid phosphatase activity, a O.l-ml sample of hemolymph was taken from the test tube and pipetted into another test tube containing 1 ml of frozen 0.1 M citrate buffer (pH 3.6) and kept frozen until use. At the time of analysis, the bufferhemolymph mixture was kept in a water bath at 37°C and 0.1 ml of substrate (2 mg of p-nitrophenol phosphate sodium salt (Merck) in 0.1 ml) was added to start the reaction. After incubation for 1 hr at 37”C, the reaction was stopped by adding 2 ml of
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AND
MOHANDAS
0.25 N NaOH. The liberated p-nitrophenol was read at 410 nm in a Hitachi Model 20020 spectrophotometer. The enzyme activity is expressed as pmol of p-nitrophenol liberated/mg of proteink. Another sample of 0.1 ml of hemolymph was used to estimate protein, following the method of Lowry et al. (1951). Collection of hemolymph samples from clams comprising the control groups was carried out and enzyme activity measured in the same manner as for the experimental groups. The two-tailed t test (Zar, 1974)was employed to determine the statistically significant differences between the enzyme activity values of each of the three experimental batches and that of the controls in each clam species.
exposed to three sublethal concentrations of copper for 5 days are given in Tables 1 and 2, respectively. In S. scripta, hemolymph acid phosphatase activity values of clams dosed with 1ppm of copper when compared with those of the controls were significantly higher at 24 (P < 0.01) and 48 hr (P < 0.01). The hemolymph acid phosphatase activity values of clams dosed with 3 ppm of copper were significantly lower than the control value at 120 hr (P < 0.05). Clams dosed with 5 ppm of copper showed significantly higher values than the controls at 48 hr (P < 0.01) but significantly lower values at 96 (P < 0.05) and 120 hr (P < 0.02). In V. cyprinoides var. cochinensis, hemolymph acid phosphatase activity values of clams dosed with 0.15 ppm copper were significantly higher at 24 (P < 0.05), 48 (P < RESULTS 0.05), and 96 hr (P < 0.01) than the control Hemolymph acid phosphatase activity values for the respective days. The value values in S. scripta and V. cyprinoides var. for clams dosed with 0.30 ppm of copper cochinensis under normal conditions and was significantly lower than the control
HEMOLYMPH
ACID
PHOSPHATASE
Sunetta scripta
TABLE 1 ACTIVITY VALUES (PmoVmg PROTEIN/hT) EXPOSED TO SUBLETHAL CONCENTRATIONS
IN 38- TO 40-mm OF COPPER
SIZE GROUP
Hours Group
24
48
72
96
120
Control N Mean value *SD Range
10 0.2724 0.0645 0.1731-0.3586
10 0.2913 0.0546 0.2051-0.3659
0.2814 0.0607 0.1595-0.3516
10 0.2683 0.0775 0.1742-0.3686
10 0.2690 0.0703 0.1905-0.4563
1 ppm of Cu*+ N Mean value *SD Range
10 0.3679 0.0624 0.2941-0.4733
10 0.4361 0.1222 0.2333-0.6500
10 0.2675 0.1040 0.1375-0.4579
10 0.2057 0.0609 0.1300-0.3019
10 0.2212 0.0675 0.1446-0.3433
3 ppm of Cu*+ N Mean value *SD Range
10 0.3311 0.0886 0.2129-0.5034
10 0.3549 0.1317 0.1856-0.5615
10 0.2687 0.0675 0.2101-0.4423
10 0.2077 0.0528 0.1181-0.2827
10 0.2000 0.0475 0.1198-0.2973
5 ppm of Cu*+ N Mean value aSD Range
10 0.2208 0.0796 0.1267-0.3516
10 0.4154 0.0905 0.2417-0.5604
10 0.2652 0.0712 0.1667-0.3925
10 0.2026 0.0410 0.1663-0.3095
10 0.1973 0.0416 0.1382-0.2692
10
ACID
HEMOLYMPH
PHOSPHATASE
ACID
Villorita
PHOSPHATASE cyprinoides VAR.
ACTIVITY
cochinensis
IN COPPER-STRESSED
121
BIVALVES
TABLE 2 VALUES (kmol/mg PRoTEINihr) IN 38- TO 40-mm SIZE GROUP EXPOSED TO SUBLETHAL CONCENTRATIONS OF COPPER Hours
Group
24
48
72
96
120 --~
Control N Mean value *SD Range
10 0.5913 0.1712 0.3984-1.0152
10 0.4764 0.1515 0.2484-0.6909
10 0.5862 0.2009 0.3750-1.1007
10 0.5983 0.2268 0.3491-1.0187
10 0.6180 0.1920 0.3250-0.9206
0.15 ppm of Cu*+ N Mean value *SD Range
10 0.8191 0.2040 0.4854-l .2317
10 0.6428 0.1603 0.3585-0.9859
10 0.4353 0.1621 0.2295-0.6957
10 1.0634 0.3036 0.6273-1.6129
10 0.7067 0.2352 0.4200-1.0595
0.30 ppm of Cu*+ N Mean value +SD Range
10 0.6894 0.1721 0.4419-l .1233
10 0.5933 0.2222 0.2941-1.0147
10 0.7072 0.2929 0.3947-1.2195
10 0.6958 0.1926 0.4091-1.0319
10 0.3350 0.0972 0.2037-0.4507
0.45 ppm of Cd+ N Mean value tSD Range
10 0.7684 0.2224 0.369k1.1151
10 0.3153 0.0845 0.1744-0.4324
10 0.1600 0.0477 0.0866-0.2468
10 0.5510 0.2145 0.2568-0.8974
10 0.1558 0.0144 0.1362-0.1754
value at 120 hr (P < 0.01). The values for clams dosed with 0.45 ppm of copper were significantly lower than control values at 48 (P < 0.02), 72 (P < O.Ol), and 120 hr (P < 0.01). DISCUSSION In S. scripta and V. cyprinoides var. cochinensis exposed to copper, the observed differences in the time that the maximum and minimum activity levels of hemolymph acid phosphatase occurred are attributed to species differences and to the concentrations of the metal ion used. Apart from these, the onset of destabilization and reduction of latency of the enzyme might have also influenced the activity pattern of the enzyme. Many xenobiotics are known to induce alterations in the bounding membrane of the lysosome, leading to destabilization (see Moore and Lowe, 1985; Nott and Moore, 1987) and the subsequent release of
the lysosomal hydrolases into the cytosol (Moore, 1976; Baccino, 1978; and others). Roesijadi (1980) observed increased acid phosphatase activity in the gills of the clam Protothaca staminea at sublethal exposures to copper. Harrison and Berger (1982) found no significant reduction in lysosomal integrity in Mytilus edulis exposed to low copper concentrations but at the highest concentration significant reduction in lysosomal latency was observed. Pickwell and Steinert (1984) found that at low concentrations of copper, hemolymph lysozyme levels in M. edulis did not differ significantly from control values. Copper is believed to damage lysosomes by promoting lipid peroxidation (Baccino, 1978). The observed increase in hemolymph acid phosphatase activity levels in the early time periods in S. scriptu and V. cyprinoides var. cochinensis dosed with 1 and 0.15 ppm of copper, respectively, can be attributed to the release of the enzyme into the he-
122
SURESH
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molymph compartment from the lysosomes after the destabilization of the lysosomal membrane caused by copper ions. Since destabilization of the lysosomal membrane is believed to take place in the early time periods no significant quantum of enzyme was released into the hemolymph at later time periods, and hence no significant variation in the enzyme activity values at later time periods. Alternatively, metal ions might trigger the hypersynthesis of lysosomal hydrolase, acid phosphatase, which is released into the hemolymph. When the metal ion concentration is low, the hypersynthesis is not immediately inactivated by the metal ions and this is reflected as increased enzyme activity in the hemolymph in the early time periods. However, as the clams continue to be exposed to copper, hypersynthesis of the enzyme is inactivated and hence no significant difference in the values was observed at later time periods, i.e., at 72, 96, and 120 hr in S. scripta and at 120 hrs in V. cyprinoides var. cochinensis. Chandy and Pate1 (1985) indicated that at lower concentrations the metal ions that entered the system are engulfed into lysosomes and subsequently transformed into biologically inactive forms. Chandy and Pate1 (1985) added that the presence of metal ions causes increased availability of lysosomal acid hydrolases to metabolize and sequester the metal in some nontoxic form and eventually excrete it. Hypersynthesis and release of acid hydrolases from hemocytes into the hemolymph when mollusks are challenged with biotic and abiotic agents has already been indicated (see Cheng, 1983a, b; Cheng and Mohandas, 1985) with the involvement of recognition sites on the surface membranes of these cells. In S. scripta dosed with 3 ppm of copper and V. cyprinoides var. cochinensis dosed with 0.30 ppm of copper, the absence of a significant increase in enzyme activity in the early time periods is believed to occur for two reasons: (1) because of the fairly high metal ion concentration, the peak en-
MOHANDAS
zyme activity as a result of lysosomal destabilization took place much earlier, i.e., before 24 hr, or (2) the hypersynthesis of the enzyme was also inactivated by the metal ions from the very first day, and hence no significant difference in activity levels was observed in the early time periods. However, as the clams continue to be under metal stress, the concentration of copper used affected enzyme synthesis, which is also reflected as a significant fall in enzyme activity by 120 hr. These two explanations also hold for the enzyme activity pattern seen in clams exposed to the highest concentrations. But the pattern of activity is different in that at the highest concentrations, the adverse effect of metal ions on enzyme synthesis started much earlier. With the result, the significant fall in hemolymph enzyme activity in S. scripta dosed with 5 ppm of copper and V. cyprinoides var. cochinensis dosed with 0.45 ppm of copper started by 96 and 48 hr, respectively . The results of the present study also indicated that at certain time periods the experimental specimens showed inconsistent enzyme activity values when compared with the controls, i.e., higher values at 48 hr, but no significant difference at 72 hr in S. scripta dosed with 5 ppm of copper and no significant difference in value at 72 hr and at 96 hr in V. cyprinoides var. cochinensis, dosed with 0.15 and 0.45 ppm of copper respectively. In this context it may be noted that lysosomes, even in single cells, are quite variable in their enzymatic constitution (Dean, 1977), and the heterogeneity in size and shape reflects the divergent functional activities of lysosomes in different cell types (Schellens et al., 1977). Moreover, the cytoplasmic granules in the granulocytes of several species of mollusks have been considered to be true lysosomes (see Mohandas et al., 1985). The finding of Yoshino and Cheng (1976) that not all of the cytoplasmic granules within the granulocytes of Mercenaria mercenaria include acid phosphatase activity prompted them to
ACID PHOSPHATASE
IN COPPER-STRESSED
suggest that these vesicles represent a chemically heterogeneous population or that there is a nonsynchronized chemical cycle that occurs within the granules (lysosomes). Thus, the observed inconsistency in enzyme activity values may be attributed to the nonsynchronized chemical cycle occurring in the lysosomes. It may also be noted that Granath and Yoshino (1983a) observed the existence of discrete lysosomal enzyme subpopulations within and between the hemocyte populations of two strains of Biomphalaria glabrata. Granath and Yoshino (1983b) subsequently suggested induced alterations in hemocyte enzyme activities that may include the selective switching of enzymes produced by individual hemocytes or the stimulation of enzyme production in previously nonenzymatically active cells. Based on the results obtained in the present study, it is concluded that (1) the activity levels of hemolymph acid phosphatase in clams exposed to sublethal concentrations of copper vary from species to species and are also dependent on the concentration of metal ions used; (2) copper ions can cause destabilization of the lysosomal membrane and the consequent release of the lysosomal hydrolase, acid phosphatase, into the hemolymph, or copper ions can trigger hypersynthesis of the lysosomal enzyme, which is subsequently released into the hemolymph; (3) copper ions can inhibit acid phosphatase activity; and (4) depending upon the period of exposure and the concentration of the metal ion, enzyme synthesis can be adversely affected. ACKNOWLEDGMENTS A personal fellowship awarded to the University Grants Commission, the facilities provided by the Cochin ence and Technology are gratefully
the first author by New Delhi, and University of Sciacknowledged.
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