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Interaction of insecticides with the Ca2+-pump activity of sarcoplasmic reticulum
PESTICIDE
BIOCHEMISTRY
AND
Interaction
PHYSIOLOGY
17, 185-190 (1982)
of Insecticides with the Ca2+-Pump Sarcoplasmic Reticulum
Activity
of
M. C. ANTUNES-MADEIRA AND VITOR M. C. MADEIRA Centro
de Biologia
Celular,
Universidade
de Coimbra,
Coimbra,
Portugal
Received September 24, 1981; accepted December 30, 1981 The organophosphorus insecticides, parathion and azinphos (10-5- 10m4M), significantly stimulate the Ca*+-pump activity of sarcoplasmic reticulum, while malathion has a limited effect. The rates of Ca2+ translocation and ATP hydrolysis are both stimulated and, apparently, the CaZ+/ATP ratio is improved. Parathion and azinphos maximally increase this ratio by 26 and 14%, respectively. The organochlorine compounds, DDT and aldrin, also stimulate the Caz+ pump, and lindane has a reduced effect. These effects are smaller than those observed for parathion and azinphos. The order of effectiveness is similar to the toxicity of the compounds to mammals and can be described as follows: parathion > azinphos > DDT = aldrin > malathion = lindane.
INTRODUCTION
Organophosphorus and carbamate insecticides are known to be powerful inhibitors of acetylcholinesterase (1 - 3). However, some of their effects, viz., chronic toxicity, remain unexplained. Organochlorine compounds of DDT type may exert their actions by poisoning the axonic membranes (4-6), and cyclodiene compounds may interact at the level of synapse (2, 4). In all cases, the precise biochemical mechanisms are not completely understood. However, since all the compounds are lipophilic, it can be predicted that they are incorporated in biomembranes where they may induce chemical and physical changes which alter their native properties. Therefore, the investigation of the effects in well-defined membrane mechanisms is of great interest to clarify the mode of action of insecticide compounds. The simplicity of sarcoplasmic reticulum membranes makes the intrinsic Ca2+-pump system an attractive and useful model for studying the interactions of physiologically active compounds. The activity of the Ca2+-pump system intrinsically associated with the membrane is modulated by membrane lipids (7-10). This modulation probably occurs through lipid-protein interac-
tions (11). The lipophilic insecticide compounds may be easily retained in lipid moieties of biomembranes. Therefore, these compounds may induce alterations which perturb the normal membrane mechanisms and, presumably, also the Ca2+pump system of sarcoplasmic reticulum. It is shown that the insecticide compounds specifically modulate the Ca”+pump activity of sarcoplasmic reticulum, in the sense that the Ca2+ transiocation and ATP hydrolysis are both stimulated. MATERIALS
AND
METHODS
Fragmented sarcoplasmic reticulum was obtained from white muscles of rabbits, as described elsewhere (12). The purity of preparations was routinely checked by electrophoretic estimation of the Caz+ATPase enzyme which amounted to about 80% of the total protein (13). The Ca2+ uptake activity was determined as previously described (14), with a Ca2* electrode of neutral carrier type generously supplied by Dr. W. Simon (Laboratorium fur Organische Chemie, Zurich). The reaction media contained 50 mit4 KCl, 5 mill MgC12, 60 PM CaCl,, 5 mM Tris-Cl, pH 6.9, and 0.12 mg of membrane protein/ml. The reactions monitored at 20°C were initi18.5 0048-3575/82/020185-06$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
186
ANTUNES-MADEIRA
AND MADEIRA I
I
I
1
I
1 -0
I 100
..C”
-4oa
/
s
-50
FIG. 1. Effect of parathion (A) and azinphos (B) on the Caz+-pump activity of sarcoplasmic reticulum. The activities were recorded with the aid of electrodes. The insecticides were added at concentrations @M) shown on the traces. The effect of X-537A addition (20 pM) is shown for one of the curves. Note the response of the Ca 2+ electrode to the addition of 20 nmol CaZ+. The reaction media contained 50 mM KCI, 5 mM M&l,, 5 mM Tris -Cl, pH 6.9.60 pM CaC&, 0.2 mM ATP, and 0.3 mg of protein in 2.5 ml total volume. For technical reasons, the Ca2+ uptake was Gtitiated by adding Mg-ATP and the ATP hydrolysis by adding CaCl,.
ated by addition of 0.2 mA4 Mg-ATP. The Ca2+-dependent ATP hydrolysis was recorded as previously described (15) by following the production of protons from ATP splitting. These data were normalized by comparison with liberation of inorganic phosphate detected by the method of Taussky and Shorr (16). The kinetics of Ca2+ translocation and ATP hydrolysis were normalized to the same scale and the molar ratio of Ca2+ taken up per ATP hydrolyzed (Ca2+/ATP) was calculated 15 set after initiation of the reactions. The insecticides under test were delivered from ethanolic solutions to the membrane suspensions 1 min before starting the reactions. The small volumes of added ethanol were without effect on the measured activities. The fraction volume of membranes was
estimated taking 800 A for vesicular radius, 55 A for membrane thickness, and 5 ~1 of vesicular volume/mg of protein. RESULTS
AND DISCUSSION
The organophosphorus insecticides, parathion and azinphos,’ at concentrations as low as 10e5 M (Fig. l), significantly increase the activities of Ca2+ translocation and ATP hydrolysis promoted by the Ca2+ pump of sarcoplasmic reticulum, while malathion is much less effective (Fig. 2). As 1 Abbreviations used: azinphos, o,o-diethyl-S-[4oxo-1,2,3-benzotriazin-3-(4H)-ylmethyllphosphorothioate; parathion, o,o-diethyl-o-p-nitrophenyl phosphorothioate; malathion, o,o-dimethyl phosphorodithioate of diethyl mercaptosuccinate; aldrin, 1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro-1,4-endo, S,S-exo-dimethanonaphthalene; p,p’DDT, l,l,l-trichloro-2,2-bis(p-chlorophenyl)ethane; lindane, 1,2,3,4,5,6-hexachlorocyclohexane.
EFFECTS
OF
INSECTICIDES
ON
SARCOPLASMIC
TIME (S) 2. Comparative effects of parathion (P), azinphos (A), and malathion (M) on the Ca2+-pump activity of sarcoplasmic reticulum. The concentrations of added insecticides were 10M4 M. The experimental conditions were identical to those of Fig. 1. FIG.
shown in Fig. 1, experimental artifacts can be ruled out, since the recordings of Ca2+ translocation can be completely reversed upon addition of the ionophore, X-537A. Furthermore, it is shown in Fig. 1 that the response of the Ca2+ electrode is fast enough to record the kinetics of Ca2+ translocation at 20°C. In addition to the pump activities, also the Ca2+/ATP ratio is increased by the organophosphorus compounds (Table 1). This ratio at the 15th set after initiation of
Ca’+lATP
Ca2+
187
PUMP
the reactions, is increased by 26 and 10% when parathion and azinphos (10e4 M) are added, respectively, and 20 and 14%, respectively, at 4 X 10e5 M. It appears that the effects of parathion and azinphos are rather specific since the added concentrations produce mild accumulations in the membrane. Thus, membrane concentrations up to 14 and 7 mM are expected for parathion and azinphos, respectively, as it can be predicted from the partition coefficients (17) and the fractions taken up by the organic phase (18), assuming a fraction volume of membranes of 1.2 x 10p4. These concentrations of insecticides produce molar ratios, lipid to insecticide, up to 33 and 62 for parathion and azinphos, respectively, when the nominal concentrations in the medium are 1O-4 M. The organochlorine compounds, DDT and aldrin, also activate the Caz+ pump (Fig. 3) and lindane has a limited effect (Fig. 4). The effects of these compounds are comparatively smaller than those described for parathion and azinphos, regarding the activities of the pump and also the Ca”+/ ATP ratio (Table 2). The relative effects of the various compounds can be more accurately compared by taking into account the membrane concentrations estimated from the partition coefficients. For nominal concentrations of 10m4M of chlorinated compounds, the estimated membrane concentrations are 476. 474, and 95 mA4 for aldrin, DDT, and lindane, respectively. These concentrations
TABLE 1 Ratio at the 15th set after lnitiation ofCa’+-Pump Organophosphorus
Concentration (PM) 0 10 20 40 60 80 100
RETICULUM
Activity,
as Aficted
by
Insecticides
Azinphos
Parathion
Malathion
Ca=+/ATP
% increase
Ca2+/ATP
% increase
1.60 1.68 1.78 1.92 1.90 1.93 2.02
5.0 11.2 20.0 18.8 20.6 26.3
1.60 1.73 1.82 1.81 1.81 1.76
8.1 13.8 13.1 13.1 10.0
Ca’+IATP 1.65 1.70 1.70 1.73
-
% increase 3.0 3.0 4.8
188
ANTUNES-MADEIRA
, I 0
I 10
I 20 TIME
AND
1
30
(S)
FIG. 3. Effect of aldrin (A) and DDT experimental conditions were identical
1 0
MADEIRA
I 10
I 20
1
3
TIME (S)
(B) on the Caz+-pump activity of sarcoplasmic to those described for Fig. I.
produce molar ratios, lipid to insecticide, of 0.95, 0.97, and 4.77, respectively. These fraction values are much higher than those estimated for the organophosphorus compounds. Therefore, the relative effects of chlorinated compounds are considerably lower than those of parathion and azinphos. It should be noted that the values given above are probably rough estimates since the partition coefftcients in hexane, rather
FIG. 4. Comparative effects of organochlorine insecticides on the Ca2+-pump activity of sarcoplasmic reticulum. The insecticides aldrin (A), DDT (D), and lindane (L) were added at nominal concentrations of 1O-4 M.
reticulum.
The
than in membranes, were used. In spite of these uncertainties, these data are quite adequate for our comparative purposes. On a practical basis, i.e., when the effects are related to the added concentrations of the compounds, the following order of effectiveness can be described: parathion > azinphos > DDT = aldrin > malathion = lindane. This order is very similar to that previously described for the effects of the insecticides on the permeability of lipid membranes (19) and also for perturbations of thermotropic transitions of pure lipid systems (20). These effects have been interpreted in terms of alterations of cooperative lipid interactions resulting in increased permeability to nonelectrolytes and to ion-ionophore complexes (19, 20). Similar perturbations may occur in the membranes of sarcoplasmic reticulum under the interaction of insecticide compounds. The interactions would presumably render the membrane with an appropriate fluidity or viscosity for improved activity of the Ca2+ pump. However, it must be taken into account that the compounds may as well affect lipid-protein interactions which provide the enzyme with a more suitable conformation for optimal activity. Direct
EFFECTS
OF
INSECTICIDES
ON
SARCOPLASMIC
RETICULUM
Cc?+
189
PUMP
TABLE 2 CaZ+IATP Ratio at the 15th set ajier Initiation of Ca2+-Pump Activity, as Affected by Organochlorinated insecticides Concentration (PM) 0 10 20 40 60 80 100
Aldrin
DDT
Lindane
-
CaZ+/ATP
% increase
Caz+/ATP
% increase
Ca2+/ATP
% increase
1.47 1.51 1.59 1.59 1.57 1.60 1.60
2.1 8.2 8.2 6.8 8.8 8.8
1.51 1.54 1.55 1.58 1.56 1.57 1.57
2.0 2.6 4.6 3.3 4.0 4.0
1.51 1.49 1.51 1.49 1.45 1.44 1.42
-1.3 -1.3 -4.0 -4.6 -6.0
interactions of the compounds with the enzyme by the formation of covalent complexes are not expected. Thus, it appears that only the oxon forms of phosphorothionates are active as serine acylating agents (1,21), but the common sulfur forms are not reactive, since the phosphorus atom is much less electrophilic in the latter forms. Studies are in progress to evaluate the perturbations on the microviscosity of the membrane and on lipid-protein interactions. Apparently, the coupling ratio of Ca2+ transport (Ca2+/ATP) is improved by some insecticides. These lipophilic compounds may induce alterations on lipid-protein interactions allowing the Ca2+-pump enzyme to acquire a more suitable conformation for optimized activity. In fact, recent findings on fluorescence energy transfer from protein tryptophyls to the lipid phase indicate that parathion and azinphos significantly perturb lipid- protein interactions. Effects similar to those described here for the insecticide compounds were recently described for diethyl ether which also significantly increases the activity of the Ca2+ pump (22). Our findings are in contrast to several reports of other investigators (23-25) who have shown apparent inhibitions of Ca2+ uptake and ATP hydrolysis induced by parathion and other insecticides. In these experiments, the radiotracer 45Ca was used in combination with a filtration procedure
through Millipore filters, instead of continuous recording methods. Recently, Salama and Scarpa (22), using continuous recording methods, have shown effects of diethyl ether opposite to those previously reported where filtration procedures were used. Scarpa and Salama pointed out that treated vesicles are trapped in the filters to a lesser extent that untreated membranes are. Also, the time required for some manipulations could have led to irreversible damage of sarcoplasmic reticulum. Similar situations may be responsible for the reported effects of insecticides, when filtration methods are used. Furthermore, it should be taken into account that shearing forces and pressure gradients generated during filtration may induce variable degree of damage in untreated and treated membranes . Our findings are highly reproducible and have the advantage of using direct and continuously recording methods, thus avoiding potential artifacts resulting from membrane damage induced by physical injuries and long incubation periods. The results unequivocally demonstrate that the insecticide compounds have the immediate effect of stimulating Ca2+ uptake and ATP hydrolysis promoted by the Ca2+-pump system of sarcoplasmic reticulum. It is proposed that the stimulation of sarcoplasmic reticulum Ca2+ pump promoted by insecticide compounds may account for some of the physiological effects observed
190
ANTUNES-MADEIRA
upon insecticide poisoning, namely, muscle paralysis (1, 2, 21). Although the concentrations used are not readily comparable with the toxic doses of the compounds, the amounts of insecticides bound to the membranes in vivo are unknown and, possibly, may reach significant levels, since the compounds are preferentially retained in lipophilic regions, namely, biomembranes. Furthermore, it is worthy of note that the effectiveness of the compounds parallels quite well with their toxicity in mammals (21). ACKNOWLEDGMENTS
AND
12.
13. 14.
15.
This work was supported by grants from INIC and JNICT. REFERENCES
1. M. Eto, “Organophosphorus Pesticides: Organic and Biological Chemistry,” pp. 123-231, CRC Press, Cleveland, 1974. 2. R. D. O’Brien, The biochemistry of toxic action of insecticides, in “Biochemistry of Insects” (M. Rockstein, Ed.), pp. 515-539, Academic Press, New York, 1978. 3. R. J. Kuhr and H. W. Dorough, “Carbamate Insecticides: Chemistry, Biochemistry and Toxicology,” pp. 41-70, CRC Press, Cleveland, 1976. 4. G. T. Brooks, ‘Chlorinated Insecticides,” Vol. II, pp. 130- 144, CRC Press, Cleveland, 1974. 5. T. Narahashi and H. G. Haas, DDT: Interaction with nerve membrane conductance changes, Science
157, 1438 (1967).
6. T. Narahashi and H. G. Haas, Interaction of DDT with the components of lobster nerve membrane conductance, J. Gen. Physiol. 51, 177 (1968). 7. A. Martonosi, Biochemical and clinical aspects of sarcoplasmic reticulum function, Curr. Top. Membr. Transp. 3, 83 (1972). 8. A. Martonosi, J. R. Donley, A. G. Pucell, and R. A. Halpin, Sarcoplasmic reticulum. XI. The mode of involvement of phospholipids in the hydrolysis of ATP by sarcoplasmic reticulum membranes, Arch. Biochem. Biophys. 144,529 (1971). 9. W. Fiehn and W. Hasselbach, The effect of phospholipase A on the calcium transport and the role of unsaturated fatty acids in ATPase activity of sarcoplasmic vesicles, Eur. J. Biochem. 13, 510 (1970). 10. R. The and W. Hasselbach, Unsaturated fatty acids as reactivators of the calcium-dependent ATPase of delipidated sarcoplasmic membranes, Eur. J. Biochem. 39, 63 (1973). 11. J. P. Bennett, K. A. McGill, and G. B. Warren,
MADEIRA
The role of lipids in the functioning of a membrane protein: The sarcoplasmic reticulum calcium pump, Curr. Top. Membr. Transp. 14, 127 (1980). A. P. Carvalho and A. M. Mota, The role of ATP and of a bound phosphoryl group acceptor on Ca binding and exchangeability in sarcoplasmic reticulum, Arch. Biochem. Biophys. 142, 201 (1971). V. M. C. Madeira, Subunits of the calcium ionpump system of sarcoplasmic reticulum, Biochim. Biophys. Acta 464, 583 (1977). V. M. C. Madeira, A rapid and ultrasensitive method to measure Ca++ movements across biological membranes, Biochem. Biophys. Res. Commun. 64, 870 (1975). V. M. C. Madeira, M. C. Antunes-Madeira, and A. P. Carvalho, Activation energies of the ATPase activity of sarcoplasmic reticulum, Biochem.
16. 17. 18. 19. 20.
21.
22.
23.
24.
25.
Biophys.
Res.
Commun.
58,
897
(1974). - H. H. Taussky and E. Shorr, A microcolorimetric method for the determination of inorganic phosphorus, J. Biol. Chem. 202,675 (1953). A. Leo, C. Hansch, and D. Elkins, Partition coefficients and their uses, Chem. Rev. 71, 525 (1971). K. A. Connors, “A Text Book of Pharmaceutical Analysis,” pp. 277-294, Wiley, New York, 1967. M. C. Antunes-Madeira and V. M. C. Madeira, Interaction of insecticides with lipid membranes, Biochim. Biophys. Acta 550,384 (1979). M. C. Antunes-Madeira, A. P. Carvalho, and V. M. C. Madeira, Effects of insecticides on thermotropic lipid phase transitions, Pestic. Biochem. Physiol. 14, 161 (1980). R. L. Metcalf, The chemistry and biology of pesticides, in “Pesticides in the Environment”, (R. White-Stevens, Ed.), Vol. I, Part I, pp. 1-144, Dekker, New York, 1971. G. Salama and A. Scarpa, Enhanced Ca++ uptake and ATPase activity of sarcoplasmic reticulum in the presence of diethyl ether, J. Biol. Chem. 255, 6525 (1980). H. Huddart, M. Greenwood, and A. J. Williams, The effect of some organophosphorus and organochlorine compounds on calcium uptake by sarcoplasmic reticulum isolated from insect and crustacean skeletal muscle, J. Comp. Physiol. 93, 139 (1974). N. Binder, E. J. Landon, L. Wecker, and W.-D. Dettbam, Effect of parathion and its metabolites on calcium uptake activity of rat skeletal muscle sarcoplasmic reticulum, in vitro, Biochem. Pharmacol. 25, 835 (1976). H. Huddart, The effect of some organophosphorus and organochlorine insecticides on contractility, membrane potential and calcium regulation on insect skeletal muscle, Comp. Biochem. Physiol. 58C, 91 (1977).
BIOCHEMISTRY
AND
Interaction
PHYSIOLOGY
17, 185-190 (1982)
of Insecticides with the Ca2+-Pump Sarcoplasmic Reticulum
Activity
of
M. C. ANTUNES-MADEIRA AND VITOR M. C. MADEIRA Centro
de Biologia
Celular,
Universidade
de Coimbra,
Coimbra,
Portugal
Received September 24, 1981; accepted December 30, 1981 The organophosphorus insecticides, parathion and azinphos (10-5- 10m4M), significantly stimulate the Ca*+-pump activity of sarcoplasmic reticulum, while malathion has a limited effect. The rates of Ca2+ translocation and ATP hydrolysis are both stimulated and, apparently, the CaZ+/ATP ratio is improved. Parathion and azinphos maximally increase this ratio by 26 and 14%, respectively. The organochlorine compounds, DDT and aldrin, also stimulate the Caz+ pump, and lindane has a reduced effect. These effects are smaller than those observed for parathion and azinphos. The order of effectiveness is similar to the toxicity of the compounds to mammals and can be described as follows: parathion > azinphos > DDT = aldrin > malathion = lindane.
INTRODUCTION
Organophosphorus and carbamate insecticides are known to be powerful inhibitors of acetylcholinesterase (1 - 3). However, some of their effects, viz., chronic toxicity, remain unexplained. Organochlorine compounds of DDT type may exert their actions by poisoning the axonic membranes (4-6), and cyclodiene compounds may interact at the level of synapse (2, 4). In all cases, the precise biochemical mechanisms are not completely understood. However, since all the compounds are lipophilic, it can be predicted that they are incorporated in biomembranes where they may induce chemical and physical changes which alter their native properties. Therefore, the investigation of the effects in well-defined membrane mechanisms is of great interest to clarify the mode of action of insecticide compounds. The simplicity of sarcoplasmic reticulum membranes makes the intrinsic Ca2+-pump system an attractive and useful model for studying the interactions of physiologically active compounds. The activity of the Ca2+-pump system intrinsically associated with the membrane is modulated by membrane lipids (7-10). This modulation probably occurs through lipid-protein interac-
tions (11). The lipophilic insecticide compounds may be easily retained in lipid moieties of biomembranes. Therefore, these compounds may induce alterations which perturb the normal membrane mechanisms and, presumably, also the Ca2+pump system of sarcoplasmic reticulum. It is shown that the insecticide compounds specifically modulate the Ca”+pump activity of sarcoplasmic reticulum, in the sense that the Ca2+ transiocation and ATP hydrolysis are both stimulated. MATERIALS
AND
METHODS
Fragmented sarcoplasmic reticulum was obtained from white muscles of rabbits, as described elsewhere (12). The purity of preparations was routinely checked by electrophoretic estimation of the Caz+ATPase enzyme which amounted to about 80% of the total protein (13). The Ca2+ uptake activity was determined as previously described (14), with a Ca2* electrode of neutral carrier type generously supplied by Dr. W. Simon (Laboratorium fur Organische Chemie, Zurich). The reaction media contained 50 mit4 KCl, 5 mill MgC12, 60 PM CaCl,, 5 mM Tris-Cl, pH 6.9, and 0.12 mg of membrane protein/ml. The reactions monitored at 20°C were initi18.5 0048-3575/82/020185-06$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
186
ANTUNES-MADEIRA
AND MADEIRA I
I
I
1
I
1 -0
I 100
..C”
-4oa
/
s
-50
FIG. 1. Effect of parathion (A) and azinphos (B) on the Caz+-pump activity of sarcoplasmic reticulum. The activities were recorded with the aid of electrodes. The insecticides were added at concentrations @M) shown on the traces. The effect of X-537A addition (20 pM) is shown for one of the curves. Note the response of the Ca 2+ electrode to the addition of 20 nmol CaZ+. The reaction media contained 50 mM KCI, 5 mM M&l,, 5 mM Tris -Cl, pH 6.9.60 pM CaC&, 0.2 mM ATP, and 0.3 mg of protein in 2.5 ml total volume. For technical reasons, the Ca2+ uptake was Gtitiated by adding Mg-ATP and the ATP hydrolysis by adding CaCl,.
ated by addition of 0.2 mA4 Mg-ATP. The Ca2+-dependent ATP hydrolysis was recorded as previously described (15) by following the production of protons from ATP splitting. These data were normalized by comparison with liberation of inorganic phosphate detected by the method of Taussky and Shorr (16). The kinetics of Ca2+ translocation and ATP hydrolysis were normalized to the same scale and the molar ratio of Ca2+ taken up per ATP hydrolyzed (Ca2+/ATP) was calculated 15 set after initiation of the reactions. The insecticides under test were delivered from ethanolic solutions to the membrane suspensions 1 min before starting the reactions. The small volumes of added ethanol were without effect on the measured activities. The fraction volume of membranes was
estimated taking 800 A for vesicular radius, 55 A for membrane thickness, and 5 ~1 of vesicular volume/mg of protein. RESULTS
AND DISCUSSION
The organophosphorus insecticides, parathion and azinphos,’ at concentrations as low as 10e5 M (Fig. l), significantly increase the activities of Ca2+ translocation and ATP hydrolysis promoted by the Ca2+ pump of sarcoplasmic reticulum, while malathion is much less effective (Fig. 2). As 1 Abbreviations used: azinphos, o,o-diethyl-S-[4oxo-1,2,3-benzotriazin-3-(4H)-ylmethyllphosphorothioate; parathion, o,o-diethyl-o-p-nitrophenyl phosphorothioate; malathion, o,o-dimethyl phosphorodithioate of diethyl mercaptosuccinate; aldrin, 1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro-1,4-endo, S,S-exo-dimethanonaphthalene; p,p’DDT, l,l,l-trichloro-2,2-bis(p-chlorophenyl)ethane; lindane, 1,2,3,4,5,6-hexachlorocyclohexane.
EFFECTS
OF
INSECTICIDES
ON
SARCOPLASMIC
TIME (S) 2. Comparative effects of parathion (P), azinphos (A), and malathion (M) on the Ca2+-pump activity of sarcoplasmic reticulum. The concentrations of added insecticides were 10M4 M. The experimental conditions were identical to those of Fig. 1. FIG.
shown in Fig. 1, experimental artifacts can be ruled out, since the recordings of Ca2+ translocation can be completely reversed upon addition of the ionophore, X-537A. Furthermore, it is shown in Fig. 1 that the response of the Ca2+ electrode is fast enough to record the kinetics of Ca2+ translocation at 20°C. In addition to the pump activities, also the Ca2+/ATP ratio is increased by the organophosphorus compounds (Table 1). This ratio at the 15th set after initiation of
Ca’+lATP
Ca2+
187
PUMP
the reactions, is increased by 26 and 10% when parathion and azinphos (10e4 M) are added, respectively, and 20 and 14%, respectively, at 4 X 10e5 M. It appears that the effects of parathion and azinphos are rather specific since the added concentrations produce mild accumulations in the membrane. Thus, membrane concentrations up to 14 and 7 mM are expected for parathion and azinphos, respectively, as it can be predicted from the partition coefficients (17) and the fractions taken up by the organic phase (18), assuming a fraction volume of membranes of 1.2 x 10p4. These concentrations of insecticides produce molar ratios, lipid to insecticide, up to 33 and 62 for parathion and azinphos, respectively, when the nominal concentrations in the medium are 1O-4 M. The organochlorine compounds, DDT and aldrin, also activate the Caz+ pump (Fig. 3) and lindane has a limited effect (Fig. 4). The effects of these compounds are comparatively smaller than those described for parathion and azinphos, regarding the activities of the pump and also the Ca”+/ ATP ratio (Table 2). The relative effects of the various compounds can be more accurately compared by taking into account the membrane concentrations estimated from the partition coefficients. For nominal concentrations of 10m4M of chlorinated compounds, the estimated membrane concentrations are 476. 474, and 95 mA4 for aldrin, DDT, and lindane, respectively. These concentrations
TABLE 1 Ratio at the 15th set after lnitiation ofCa’+-Pump Organophosphorus
Concentration (PM) 0 10 20 40 60 80 100
RETICULUM
Activity,
as Aficted
by
Insecticides
Azinphos
Parathion
Malathion
Ca=+/ATP
% increase
Ca2+/ATP
% increase
1.60 1.68 1.78 1.92 1.90 1.93 2.02
5.0 11.2 20.0 18.8 20.6 26.3
1.60 1.73 1.82 1.81 1.81 1.76
8.1 13.8 13.1 13.1 10.0
Ca’+IATP 1.65 1.70 1.70 1.73
-
% increase 3.0 3.0 4.8
188
ANTUNES-MADEIRA
, I 0
I 10
I 20 TIME
AND
1
30
(S)
FIG. 3. Effect of aldrin (A) and DDT experimental conditions were identical
1 0
MADEIRA
I 10
I 20
1
3
TIME (S)
(B) on the Caz+-pump activity of sarcoplasmic to those described for Fig. I.
produce molar ratios, lipid to insecticide, of 0.95, 0.97, and 4.77, respectively. These fraction values are much higher than those estimated for the organophosphorus compounds. Therefore, the relative effects of chlorinated compounds are considerably lower than those of parathion and azinphos. It should be noted that the values given above are probably rough estimates since the partition coefftcients in hexane, rather
FIG. 4. Comparative effects of organochlorine insecticides on the Ca2+-pump activity of sarcoplasmic reticulum. The insecticides aldrin (A), DDT (D), and lindane (L) were added at nominal concentrations of 1O-4 M.
reticulum.
The
than in membranes, were used. In spite of these uncertainties, these data are quite adequate for our comparative purposes. On a practical basis, i.e., when the effects are related to the added concentrations of the compounds, the following order of effectiveness can be described: parathion > azinphos > DDT = aldrin > malathion = lindane. This order is very similar to that previously described for the effects of the insecticides on the permeability of lipid membranes (19) and also for perturbations of thermotropic transitions of pure lipid systems (20). These effects have been interpreted in terms of alterations of cooperative lipid interactions resulting in increased permeability to nonelectrolytes and to ion-ionophore complexes (19, 20). Similar perturbations may occur in the membranes of sarcoplasmic reticulum under the interaction of insecticide compounds. The interactions would presumably render the membrane with an appropriate fluidity or viscosity for improved activity of the Ca2+ pump. However, it must be taken into account that the compounds may as well affect lipid-protein interactions which provide the enzyme with a more suitable conformation for optimal activity. Direct
EFFECTS
OF
INSECTICIDES
ON
SARCOPLASMIC
RETICULUM
Cc?+
189
PUMP
TABLE 2 CaZ+IATP Ratio at the 15th set ajier Initiation of Ca2+-Pump Activity, as Affected by Organochlorinated insecticides Concentration (PM) 0 10 20 40 60 80 100
Aldrin
DDT
Lindane
-
CaZ+/ATP
% increase
Caz+/ATP
% increase
Ca2+/ATP
% increase
1.47 1.51 1.59 1.59 1.57 1.60 1.60
2.1 8.2 8.2 6.8 8.8 8.8
1.51 1.54 1.55 1.58 1.56 1.57 1.57
2.0 2.6 4.6 3.3 4.0 4.0
1.51 1.49 1.51 1.49 1.45 1.44 1.42
-1.3 -1.3 -4.0 -4.6 -6.0
interactions of the compounds with the enzyme by the formation of covalent complexes are not expected. Thus, it appears that only the oxon forms of phosphorothionates are active as serine acylating agents (1,21), but the common sulfur forms are not reactive, since the phosphorus atom is much less electrophilic in the latter forms. Studies are in progress to evaluate the perturbations on the microviscosity of the membrane and on lipid-protein interactions. Apparently, the coupling ratio of Ca2+ transport (Ca2+/ATP) is improved by some insecticides. These lipophilic compounds may induce alterations on lipid-protein interactions allowing the Ca2+-pump enzyme to acquire a more suitable conformation for optimized activity. In fact, recent findings on fluorescence energy transfer from protein tryptophyls to the lipid phase indicate that parathion and azinphos significantly perturb lipid- protein interactions. Effects similar to those described here for the insecticide compounds were recently described for diethyl ether which also significantly increases the activity of the Ca2+ pump (22). Our findings are in contrast to several reports of other investigators (23-25) who have shown apparent inhibitions of Ca2+ uptake and ATP hydrolysis induced by parathion and other insecticides. In these experiments, the radiotracer 45Ca was used in combination with a filtration procedure
through Millipore filters, instead of continuous recording methods. Recently, Salama and Scarpa (22), using continuous recording methods, have shown effects of diethyl ether opposite to those previously reported where filtration procedures were used. Scarpa and Salama pointed out that treated vesicles are trapped in the filters to a lesser extent that untreated membranes are. Also, the time required for some manipulations could have led to irreversible damage of sarcoplasmic reticulum. Similar situations may be responsible for the reported effects of insecticides, when filtration methods are used. Furthermore, it should be taken into account that shearing forces and pressure gradients generated during filtration may induce variable degree of damage in untreated and treated membranes . Our findings are highly reproducible and have the advantage of using direct and continuously recording methods, thus avoiding potential artifacts resulting from membrane damage induced by physical injuries and long incubation periods. The results unequivocally demonstrate that the insecticide compounds have the immediate effect of stimulating Ca2+ uptake and ATP hydrolysis promoted by the Ca2+-pump system of sarcoplasmic reticulum. It is proposed that the stimulation of sarcoplasmic reticulum Ca2+ pump promoted by insecticide compounds may account for some of the physiological effects observed
190
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upon insecticide poisoning, namely, muscle paralysis (1, 2, 21). Although the concentrations used are not readily comparable with the toxic doses of the compounds, the amounts of insecticides bound to the membranes in vivo are unknown and, possibly, may reach significant levels, since the compounds are preferentially retained in lipophilic regions, namely, biomembranes. Furthermore, it is worthy of note that the effectiveness of the compounds parallels quite well with their toxicity in mammals (21). ACKNOWLEDGMENTS
AND
12.
13. 14.
15.
This work was supported by grants from INIC and JNICT. REFERENCES
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