JOURNAL OF APPLIED TOXICOLOGY, VOL. 10( l), 55-57 (1990)
Chlordecone Interaction of Calmodulin Binding with Phosphodiesterase P. J. S. Vig and D. Desaiaht Department of Neurology, University of Mississippi School of Medicine, Jackson, MS 39216, USA
B. D. Mehrotra Department of Chemistry, Tougaloo College, Tougaloo, MS 391 74, USA
Key words: organochlorine compounds; calmodulin; phosphodiesterase.
The effects of organochlorine (O.C.) compounds, such as aldrin, dieldrin, endrin, isodrin, chlordecone and mirex, on calmodulin (CaM) activity were investigated. Changes induced by 0 . C compounds on biological and physical properties of CaM were monitored in terms of phosphodiesterase stimulation and tyrosine fluorescence, respectively. None of the O.C. compounds altered tyrosine fluorescence of CaM in the presence of Ca2+.Except for chlordecone, none of the O.C. compounds inhibited CaM-activated phosphodiesterase (PDE). Chlordecone significantly decreased ( P < 0.05) CaM-activated PDE in a concentration-dependent manner without affecting the basal enzyme. Combination of chlordecone with W-7 (CaM antagonist) increased the inhibitory effect of W-7on CaM activity. These results suggest that O.C. compounds may not be changing the tyrosine fluorescence of CaM. Among the O.C. compounds tested, chlordecone is a specific inhibitor of CaM-activated PDE.
INTRODUCTION Calmodulin (CaM) has been recognized widely as a Ca' ' receptor protein mediating the Ca2+ function.'. Toxic compounds that can interact with CaM can alter a number of vital cellular processes.ss Exposure to organochlorine (O.C.) compounds results in a marked alteration in the body metabolism, including disturbances in the central nervous system, which may be correlated with the neurotoxic effects of these compounds."' Our earlier studies have shown that O.C. insecticides interact with CaM-dependent enzymes, especially membrane-bound Ca2+ ATPase and adenylate cyclase.'-'" However, whether these compounds directly interact with CaM is still not clearly understood. The present investigation was carried out to understand the possible interaction of O.C. compounds with CaM and its biological activity. EXPERIMENTAL Stock solutions of aldrin, dieldrin, endrin, isodrin, chlordecone and W-7 were prepared in ethanol and that of mirex in acetone, and appropriate dilutions were made to obtain the required concentrations as shown in the results section. CaM activity was measured according to the method of Wallace et al." The reaction mixture contained 40 mM Tris-HCI (pH 8.0), 3 mM MgSo,, 50 p,M CaCI,, 20 p,g CaM-deficient PDE, CaM (100 ng mlF') and varying concentrations of O.C. compounds. The basal enzyme was assayed in the absence of CaM. The reaction was initiated by the addition of [3H]cAMP (2 mM containing 200 000 cpm
t
Author to whom correspondence should be addressed.
026@437X/90/010055-03$05.00 0 1990 by John Wiley & Sons, Ltd.
per assay). After 10 min of incubation at 30"C, the reaction was terminated by placing the tubes in a water-bath (for 2 min) maintained at 95°C. After an additional 10 min of incubation with PDE-free snake venom (1 mg ml-l) at 30"C, [3HH]adenosinewas separated from untreated [3H]cAMP using a Dowex 1x8 anion exchanger (33% slurry, pH 5.0) and radioactivity in the supernatant was counted by a liquid scintillation counter (Searle, Isocap/300). Quench correction was done using an external standard. Protein concentrations were determined by the method of Lowry et al. Tyrosine fluorescence was measured13 by assigning an arbitrary value to Ca2+-CaM, and all other samples (containing O.C. compounds) were compared against this value. The excitation and emission wavelengths were 276 and 300 nm, with excitation and emission slits of 1 0 n m , respectively. Chemicals All O.C. compounds (purity > 98%) were obtained from Pesticide Repository, US Environmental Protection Agency, Research Triangle Park, NC. [2,8-3H]Adenosine 3'5'-cyclic phosphate (specific activity 33 Ci mmol-') was obtained from Amersham Corporation, Arlington Heights, IL. Other chemicals used were from Sigma Chemical Company, St. Louis, MO. Statistical analysis The data were analyzed by Students' t-test. Differences from controls were considered significant at P < 0.05. RESULTS None of the O.C. compounds altered the tyrosine fluorescence of CaM in the presence of Ca2+ (data not Received 10 May 1989 Accepted (revised) 7 Augur 1989
P. J. S. VIG ET A L .
1001
80
60
40
20 0.0
12.5
25.0
37.5
50.0
62.5
75.0
Concentration ( phd ) Figure 1. Effect of chlordecone, rnirex and W-7 on calrnodulin activity (nmol CAMP hydrolyzed rng- ' protein min-'1. Each value is a mean -tSE of three estimations. * Significantly different from control. P < 0.05.
shown). None of the O.C. compounds, except for chlordecone, inhibited CaM activity. An illustrative example of mirex and chlordecone is shown in Fig. 1. W-7, a calmodulin antagonist, significantly inhibited (I' < 0.05) CaM activity in a dose-dependent manner without affecting the basal enzyme (Fig. 1). However, when W-7 was used in combination with chlordecone (6.25 pM), an additive effect on CaM activity was observed (Fig. 2).
DISCUSSION
Although reduced levels of CaM in rat brain after chlordecone treatment have been reported,' the exact mechanism of its neurotoxicity is still not clear. Our earlier reports showed that all of these O.C. compounds, except for mirex, inhibited CaM-regulated Ca2+ ATPases in brain and heart of rats with varying potencies."' The present data show that none of these O.C. compounds with the exception of chlordecone have any effect on CaM. This may be due to the lipophilic nature of these compounds, which may cause a disintegration and/or disorientation of membrane components resulting in non-availability of particulate CaM to activate membrane-bound CaM-dependent enzymes; in the case of PDE, these compounds may not interact with the CaM binding sites of the enzyme. Furthermore, these compounds may not interact with CaM so as to bring about a conformational change,
fQH CHLORDECONE m w-7 CHLORDECONE+ W-7
*
+
Figure 2. Additive effect of W-7 (25 pM) and chlordecone (6.25 pM) on calrnodulin activity. Each value is a mean *SE of three estimations. * Chlordecone vs. chlordecone + W-7 ( P < 0.05). t W-7 vs. chlordecone + W-7 ( P < 0.05).
other than that of Ca2', as is evident from tyrosine fluorescence studies. Mirex (a structural analog of chlordecone), being non-neurotoxic, did not inhibit CaM activity. However, chlordecone seems to interact with the CaM binding sites on PDE and/or with the sites that are distinct from the W-7 binding sites of CaM, as evidenced by the additive effect of these two compounds on PDE. Further data are necessary to identify chlordecone binding sites on CaM/PDE. Acknowledgement These studies were supportcd by Grant R R 081 10 from NIHIMBRS.
CHLORDECONE INHIBITION OF CALMODULIN ACTIVITY
1. W. Y. Cheung. Calmodulin plays a pivotal role in cellular regulation. Science 207, 1!327 (1980). 2. C. B. Klee, T. H. Crouch and P. G. Richman, Calmodulin. Ann. Rev. Biochem. 49, 489-518 (1980). 3. W. Y. Cheung, Calmodulin: its potential role in cell proliferation and heavy metal toxicity. Fed. Proc. 43, 2995-2999 (1984). 4. J. L. Cox and S. J. Harrison, Jr., Correlation of metal toxicity with in vitro calmodulin inhibition. Biochem. Biophys. Res. Commun. 115, 106-111 (1983). 5. K. S. Moorthy, I. K. Ahammed Sahib, J. E. Uzodinma, C. H. Trottman and D. Desaiah, Effect of mercury, cadmium and lead on calmodulin regulated calcium, pump activity in rat brain and heart. Trends Life Sci. 1, 37-48 (1986). 6. D. Desaiah, Biochemical mechanisms of chlordecone neurotoxicity: a review. Neurotoxicology 3, 103-1 10 (1982). 7. D. Desaiah, C. S. Chetty and K. S. Prasada Rao, Chlordecone inhibition of calmodulin activated calcium ATPase in rat brain synaptosomes. J. Toxicol. Environ. Health 16, 189-195 (1985). 8. B. D. Mehrotra, S. Ravichandra Reddy and D. Desaiah,
9.
10.
11. 12. 13.
57
Effect of subchronic dieldrin treatment on calmodulinregulated Ca2+ pump activity in rat brain. J. Toxicol. Environ. Health 25, 461-469 (1988). B. D. Mehrotra, K. S. Moorthy, S. Ravichandra Reddy and D. Desaiah, Effects of cylcodiene compounds on calcium pump activity in rat brain and heart. Toxicology 54, 17-29 ( 1989). P. S. Kodavanti, B. D. Mehrotra, C. S.Chetty and D. Desaiah, Effect of selected insecticides on rat brain synaptosomal adenylate cyclase and phosphodiesterase. J. Toxicol. Environ. Health 25, 207-215 (1988). R. W. Wallace, E. A. Tallant and W. Y. Cheung, Assay of calmodulin by Caz+-dependent phosphodiesterase. Methods Enzymol. 102, 39-47 (1983). 0. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurement with Fohn phenol reagent. J. Biol. Chem. 193, 265-275 (1951). S. H. Chao, Y. Suzuki, J. R. Zysk and W. Y. Cheung, Activation of calmodulin by various metal cations as a function of ionic radius. Mol. fharmacol. 26, 7 5 8 2 (1984).
Chlordecone Interaction of Calmodulin Binding with Phosphodiesterase P. J. S. Vig and D. Desaiaht Department of Neurology, University of Mississippi School of Medicine, Jackson, MS 39216, USA
B. D. Mehrotra Department of Chemistry, Tougaloo College, Tougaloo, MS 391 74, USA
Key words: organochlorine compounds; calmodulin; phosphodiesterase.
The effects of organochlorine (O.C.) compounds, such as aldrin, dieldrin, endrin, isodrin, chlordecone and mirex, on calmodulin (CaM) activity were investigated. Changes induced by 0 . C compounds on biological and physical properties of CaM were monitored in terms of phosphodiesterase stimulation and tyrosine fluorescence, respectively. None of the O.C. compounds altered tyrosine fluorescence of CaM in the presence of Ca2+.Except for chlordecone, none of the O.C. compounds inhibited CaM-activated phosphodiesterase (PDE). Chlordecone significantly decreased ( P < 0.05) CaM-activated PDE in a concentration-dependent manner without affecting the basal enzyme. Combination of chlordecone with W-7 (CaM antagonist) increased the inhibitory effect of W-7on CaM activity. These results suggest that O.C. compounds may not be changing the tyrosine fluorescence of CaM. Among the O.C. compounds tested, chlordecone is a specific inhibitor of CaM-activated PDE.
INTRODUCTION Calmodulin (CaM) has been recognized widely as a Ca' ' receptor protein mediating the Ca2+ function.'. Toxic compounds that can interact with CaM can alter a number of vital cellular processes.ss Exposure to organochlorine (O.C.) compounds results in a marked alteration in the body metabolism, including disturbances in the central nervous system, which may be correlated with the neurotoxic effects of these compounds."' Our earlier studies have shown that O.C. insecticides interact with CaM-dependent enzymes, especially membrane-bound Ca2+ ATPase and adenylate cyclase.'-'" However, whether these compounds directly interact with CaM is still not clearly understood. The present investigation was carried out to understand the possible interaction of O.C. compounds with CaM and its biological activity. EXPERIMENTAL Stock solutions of aldrin, dieldrin, endrin, isodrin, chlordecone and W-7 were prepared in ethanol and that of mirex in acetone, and appropriate dilutions were made to obtain the required concentrations as shown in the results section. CaM activity was measured according to the method of Wallace et al." The reaction mixture contained 40 mM Tris-HCI (pH 8.0), 3 mM MgSo,, 50 p,M CaCI,, 20 p,g CaM-deficient PDE, CaM (100 ng mlF') and varying concentrations of O.C. compounds. The basal enzyme was assayed in the absence of CaM. The reaction was initiated by the addition of [3H]cAMP (2 mM containing 200 000 cpm
t
Author to whom correspondence should be addressed.
026@437X/90/010055-03$05.00 0 1990 by John Wiley & Sons, Ltd.
per assay). After 10 min of incubation at 30"C, the reaction was terminated by placing the tubes in a water-bath (for 2 min) maintained at 95°C. After an additional 10 min of incubation with PDE-free snake venom (1 mg ml-l) at 30"C, [3HH]adenosinewas separated from untreated [3H]cAMP using a Dowex 1x8 anion exchanger (33% slurry, pH 5.0) and radioactivity in the supernatant was counted by a liquid scintillation counter (Searle, Isocap/300). Quench correction was done using an external standard. Protein concentrations were determined by the method of Lowry et al. Tyrosine fluorescence was measured13 by assigning an arbitrary value to Ca2+-CaM, and all other samples (containing O.C. compounds) were compared against this value. The excitation and emission wavelengths were 276 and 300 nm, with excitation and emission slits of 1 0 n m , respectively. Chemicals All O.C. compounds (purity > 98%) were obtained from Pesticide Repository, US Environmental Protection Agency, Research Triangle Park, NC. [2,8-3H]Adenosine 3'5'-cyclic phosphate (specific activity 33 Ci mmol-') was obtained from Amersham Corporation, Arlington Heights, IL. Other chemicals used were from Sigma Chemical Company, St. Louis, MO. Statistical analysis The data were analyzed by Students' t-test. Differences from controls were considered significant at P < 0.05. RESULTS None of the O.C. compounds altered the tyrosine fluorescence of CaM in the presence of Ca2+ (data not Received 10 May 1989 Accepted (revised) 7 Augur 1989
P. J. S. VIG ET A L .
1001
80
60
40
20 0.0
12.5
25.0
37.5
50.0
62.5
75.0
Concentration ( phd ) Figure 1. Effect of chlordecone, rnirex and W-7 on calrnodulin activity (nmol CAMP hydrolyzed rng- ' protein min-'1. Each value is a mean -tSE of three estimations. * Significantly different from control. P < 0.05.
shown). None of the O.C. compounds, except for chlordecone, inhibited CaM activity. An illustrative example of mirex and chlordecone is shown in Fig. 1. W-7, a calmodulin antagonist, significantly inhibited (I' < 0.05) CaM activity in a dose-dependent manner without affecting the basal enzyme (Fig. 1). However, when W-7 was used in combination with chlordecone (6.25 pM), an additive effect on CaM activity was observed (Fig. 2).
DISCUSSION
Although reduced levels of CaM in rat brain after chlordecone treatment have been reported,' the exact mechanism of its neurotoxicity is still not clear. Our earlier reports showed that all of these O.C. compounds, except for mirex, inhibited CaM-regulated Ca2+ ATPases in brain and heart of rats with varying potencies."' The present data show that none of these O.C. compounds with the exception of chlordecone have any effect on CaM. This may be due to the lipophilic nature of these compounds, which may cause a disintegration and/or disorientation of membrane components resulting in non-availability of particulate CaM to activate membrane-bound CaM-dependent enzymes; in the case of PDE, these compounds may not interact with the CaM binding sites of the enzyme. Furthermore, these compounds may not interact with CaM so as to bring about a conformational change,
fQH CHLORDECONE m w-7 CHLORDECONE+ W-7
*
+
Figure 2. Additive effect of W-7 (25 pM) and chlordecone (6.25 pM) on calrnodulin activity. Each value is a mean *SE of three estimations. * Chlordecone vs. chlordecone + W-7 ( P < 0.05). t W-7 vs. chlordecone + W-7 ( P < 0.05).
other than that of Ca2', as is evident from tyrosine fluorescence studies. Mirex (a structural analog of chlordecone), being non-neurotoxic, did not inhibit CaM activity. However, chlordecone seems to interact with the CaM binding sites on PDE and/or with the sites that are distinct from the W-7 binding sites of CaM, as evidenced by the additive effect of these two compounds on PDE. Further data are necessary to identify chlordecone binding sites on CaM/PDE. Acknowledgement These studies were supportcd by Grant R R 081 10 from NIHIMBRS.
CHLORDECONE INHIBITION OF CALMODULIN ACTIVITY
1. W. Y. Cheung. Calmodulin plays a pivotal role in cellular regulation. Science 207, 1!327 (1980). 2. C. B. Klee, T. H. Crouch and P. G. Richman, Calmodulin. Ann. Rev. Biochem. 49, 489-518 (1980). 3. W. Y. Cheung, Calmodulin: its potential role in cell proliferation and heavy metal toxicity. Fed. Proc. 43, 2995-2999 (1984). 4. J. L. Cox and S. J. Harrison, Jr., Correlation of metal toxicity with in vitro calmodulin inhibition. Biochem. Biophys. Res. Commun. 115, 106-111 (1983). 5. K. S. Moorthy, I. K. Ahammed Sahib, J. E. Uzodinma, C. H. Trottman and D. Desaiah, Effect of mercury, cadmium and lead on calmodulin regulated calcium, pump activity in rat brain and heart. Trends Life Sci. 1, 37-48 (1986). 6. D. Desaiah, Biochemical mechanisms of chlordecone neurotoxicity: a review. Neurotoxicology 3, 103-1 10 (1982). 7. D. Desaiah, C. S. Chetty and K. S. Prasada Rao, Chlordecone inhibition of calmodulin activated calcium ATPase in rat brain synaptosomes. J. Toxicol. Environ. Health 16, 189-195 (1985). 8. B. D. Mehrotra, S. Ravichandra Reddy and D. Desaiah,
9.
10.
11. 12. 13.
57
Effect of subchronic dieldrin treatment on calmodulinregulated Ca2+ pump activity in rat brain. J. Toxicol. Environ. Health 25, 461-469 (1988). B. D. Mehrotra, K. S. Moorthy, S. Ravichandra Reddy and D. Desaiah, Effects of cylcodiene compounds on calcium pump activity in rat brain and heart. Toxicology 54, 17-29 ( 1989). P. S. Kodavanti, B. D. Mehrotra, C. S.Chetty and D. Desaiah, Effect of selected insecticides on rat brain synaptosomal adenylate cyclase and phosphodiesterase. J. Toxicol. Environ. Health 25, 207-215 (1988). R. W. Wallace, E. A. Tallant and W. Y. Cheung, Assay of calmodulin by Caz+-dependent phosphodiesterase. Methods Enzymol. 102, 39-47 (1983). 0. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurement with Fohn phenol reagent. J. Biol. Chem. 193, 265-275 (1951). S. H. Chao, Y. Suzuki, J. R. Zysk and W. Y. Cheung, Activation of calmodulin by various metal cations as a function of ionic radius. Mol. fharmacol. 26, 7 5 8 2 (1984).
