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The effects of pretreatment with soman simulator in the skeletal muscle: Direct interactions with acetylcholinesterase
Chem.-Biol. Interactions, 87 (1993) 253-257
253
Elsevier Scientific Publishers Ireland Ltd.
THE EFFECTS OF PRETREATMENT WITH SOMAN SIMULATOR IN THE SKELETAL MUSCLE: DIRECT INTERACTIONS WITH ACETYLCHOLINESTERASE
ZORAN GRUBIC, MARTINA BRANK and MIRO BRZIN
Institute of Pathophysiology, School of Medicine, University of Ljubljana, 61105 Ljubljana (Slovenia)
SUMMARY
Soman simulator PDP is a compound that has a chemical structure identical to soman, except that the fluorine atom is replaced by a methyl group which makes PDP unable to bind covalently to the AChE active center. In rats, late mortality observed after treatment with high doses of soman could be prevented by PDP pretreatment. Such pretreatment has been much less efficient in primates. The effect of PDP in rats has been explained by blocking the deposition of soman in so-called soman depots in which soman is stored intact and subsequently released. In this paper we demonstrate that in the presence of PDP, inhibition of rat muscle AChE by soman is reduced in rat but not in human muscle homogenates. This result suggests that at least part of the beneficial effects of PDP pretreatment in rat might be due to the direct interaction of PDP with AChE resulting in reduced AChE phosphorylation by soman.
Key words: Soman simulator -- PDP - Soman -- HI-6 - Acetylcholinesterase -- Skeletal muscle
INTRODUCTION
Rats treated with high doses of soman ( 6 - 8 x LD50) survived initial toxic effects if treated with oxime reactivator HI-6; however, after 4 - 5 h their conditions deteriorated and they finally died [1]. A generally accepted explanation of this phenomenon is that a part of applied soman is stored and remains intact in a depot from where it is gradually released causing subsequent reinhibition of the previously reactivated acetylcholinesterase (ACHE; EC 3.1.1.7.). Such delayed mortality could be prevented by pretreatment with pinacolyl dimethylphosCorrespondence to: Zoran Grubi~, Institute of Pathophysiology, School of Medicine, 61105 Ljubljana, Zalos'ka 4, Slovenia. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
254
phinate -- PDP, a compound with chemical structure identical to soman except that the fluorine atom is replaced by a methyl group, which makes PDP unable to bind covalently to the serine in the AChE active center [2]. Beneficial effect has been explained by occupation of soman depot sites by PDP, which prevents formation of soman depots. Such depots were reported to be located also in the skeletal muscle proper [3]. In our opinion an additional explanation of reduced mortality of PDP-pretreated rats is possible. Having almost identical chemical structure as soman, PDP can occupy soman binding sites on the AChE molecule, interfering in this way with the enzyme inhibition by soman. Having the fluorine atom replaced by a methyl group, PDP cannot react in nucleophylic substitutions on the phosphorous atom. PDP is therefore not an irreversible inhibitor and is more stable than soman, which is quickly hydrolysed and irreversibly bound to various esterases in tissues and plasma. PDP therefore outlives soman in the organism and protects AChE against irreversible inhibition by soman released from the depot. In order to test our hypothesis we first tested the influence of PDP on the ACh hydrolysis in muscle homogenates. Then we determined percent of AChE inhibition after phosphorylation of AChE by soman in muscle homogenates in the presence and in the absence of PDP. HI-6 reactivation of soman-inhibited AChE of muscle homogenates was also determined in the absence and in the presence of PDP. Since differences in PDP effects in different species were reported [4] our study was carried out on both rat and human muscle homogenates. METHODS
Preparation of muscle homogenates Female Wistar strain albino rats weighing 150 - 200 g were anaesthetized with ether and perfused with saline through the inferior vena cava. Hemidiaphragms were isolated, weighed and frozen in liquid nitrogen until used. Homogenates (1.45 mg/10 #l of phosphate Ringer's solution of the following compositions: 138 mM NaC1, 4 mM KC1, 1 mM MgS04, 20 mM phosphate buffer, pH = 7.4) were prepared in hand-operated glass homogenizers kept on ice. Human muscle preparations were obtained at the Institute of Legal Medicine of the Medical Faculty, Ljubljana. Intercostal muscles were isolated within five hours after death from the victims of traffic accidents. Homogenates were prepared in the same way as described for rat muscle. The determination of the effects of PDP on the phosphorylation by soman and on HI-6 reactivation of AChE in rat and human muscle homogenates Muscle homogenates were incubated for 5 min with the following combinations of soman (0.2 nM final conc.), PDP (2 nM final conc.)' and HI-6 (0.5 mM final conc.): (I) in control experiments (100°7o activity) 20 ill of phosphate Ringer's solution were incubated with 20 ~1 homogenate. (II) for determining baseline inhibition of AChE by soman 20 ~1 of soman was mixed with 20 ~1 homogenate. (III) the effect of PDP on AChE phosphorylation by soman was tested by mixing 10 ~1 of PDP and 10 ~1 of soman with 20 ~1 of the homogenate. The effect of PDP
255
on HI-6 reactivation of soman-inhibited AChE was tested by determining reactivation by HI-6 in the presence and in the absence of PDP. Therefore 20 ul of muscle homogenate were incubated with (IV) 10 ~l of soman, 5 ~l of HI-6 and 5 ~l of Ringer's solution or (V) 10 ~l of soman, 5 ~l of HI-6 and 5 ~l of PDP. After 5-min incubation at room temperature, mixtures I, II, III, IV and V were preincubated for 30 min by iso-OMPA in order to block non-specific ChE and then proceeded for radiometric determination of AChE activity as described before [5]. The effects of PDP on ACh hydrolysis by AChE was tested as follows: 20 ul of homogenate was incubated with 10 ~l Ringer's solution and 10 ~l of PDP, which was also introduced into the preincubation mixture with iso-OMPA and into the incubation mixture with ACh. PDP concentration was kept at 2 nM throughout the procedure. ACh concentration was 3 mM. RESULTS AND DISCUSSION
The results are given in Fig. la for rat muscle homogenate and in Fig. lb for human muscle homogenate. At the concentration of soman used, AChE was subtotally inhibited (Fig. la and lb -- II). Higher inhibition of AChE in rat in comparison to human muscle homogenate is probably due to the higher consumption rate of soman in human muscle homogenate, which had higher specific activity of AChE (2.54 nmol ACh/mg/min in human vs. 1.22 nmol ACh/mg/min in rat muscle homogenate) and probably of other soman-binding enzymes. However the percentage of AChE activity could not be increased significantly in human AChE either by HI-6 (IV) or by PDP (III) and also not by the combination of HI-6 and PDP (V) added together with soman to the homogenate. Rat muscle AChE behaved in a different manner. PDP alone significantly (P < 0.01, paired t-test) reduced inhibition by soman (Fig. la -- III); however, reactivation by HI-6 alone gave a better recovery (IV). In the presence of PDP, HI-6 recovery of AChE was no better than obtained by HI-6 alone (V). The effects of PDP must be due to the direct interactions with the AChE molecule since ACh hydrolysis was reduced by about 50% in the presence of 2 nM PDP in both rat and human muscle homogenate. Our results demonstrate a protective effect of PDP on AChE phosphorylation by soman. Better survival of soman-injected rats pretreated by PDP [1] could therefore partly be explained by this PDP effect and not only by the occupation of a soman depot. Observed differences in PDP effects between rat and human muscle accord well with the experiments in which PDP pretreatment prevented formation of soman depot in rat but not in primate muscle [4]. However, inhibition of electric eel AChE was used in these experiments for the registration of soman released in vitro from the pectoralis muscles isolated after treatment of animals in vivo, which means that in this case our interpretation could not explain species specificity observed. Our results therefore do not negate the existence of a soman depot. They call the attention to the fact that using soman simulator PDP for demonstration of a soman depot one should consider the possibility of a direct protection effect of this compound based on attenuation of soman phosphorylation of ACHE.
256
a) rat diaphragm % A C h E activity lOO%
87%
100% 80%
86%
+
50% 50%
40%
26%
A
20%
0%
I b) h u m a n
II
III
IV
V
intercostal muscle
% A C h E aclivi 100% 100% - -
;++ +
80%--
60%--
61%
65%
61%
III
IV
V
40%-I
20%-0%
I
II
Fig. 1. (a) Rat muscle homogenate; % AChE activity recovered after the procedures described in Methods. Roman numerals under the columns correspond to the same numerals used in Methods: I -- control, II -- inhibition by soman, III -- inhibition of soman in the presence of PDP, IV -- inhibition by soman in the presence of HI-6, V -- inhibition by soman in the presence of PDP and HI-6. Standard deviations are indicated by bars (n = 5). (b) Human muscle homogenate; the same as in (a) (n = 8).
257 REFERENCES 1
2
3 4
5
O.L. Wolthuis, H.P. Bennschop and F. Berends, Persistence of the anticholinesterase soman in rats; antagonism with a nontoxic simulator of this organophosphate, Eur. J. Pharmacol., 69 (1981) 379-383. H.P.M. Van Helden, H.P. Benschop and O.L. Wolthuis, The prophylactic efficacy of various simulators against intoxication with the organophosphate soman: structure-activity studies, J. Pharm. Pharmacol., 38 (1986) 19-23. H.P.M. Van Helden and O.L. Wolthuis, Evidence for an intramuscular depot of the cholinesterase inhibitor soman in the rat, Eur. J. Pharmacol., 89 (1983) 271-274. H.P.M. Van Helden, H.J. Van der Wiel and O.L. Wolthuis, Retention of soman in rats, guineapigs and marmosets: species-dependent effects of the soman simulator, pinacolyl dimethyl phosphinate (PDP), J. Pharm. Pharmacol., 40 (1988) 35-41. Z. Grubi~ and A. Tomaz~, Mechanism of action of HI-6 on soman inhibition of acetylcholinesterase in preparations of rat and human skeletal muscle; comparison to SAD-128 and PAM-2, Arch. Toxicol., 63 (1989) 68-71.
253
Elsevier Scientific Publishers Ireland Ltd.
THE EFFECTS OF PRETREATMENT WITH SOMAN SIMULATOR IN THE SKELETAL MUSCLE: DIRECT INTERACTIONS WITH ACETYLCHOLINESTERASE
ZORAN GRUBIC, MARTINA BRANK and MIRO BRZIN
Institute of Pathophysiology, School of Medicine, University of Ljubljana, 61105 Ljubljana (Slovenia)
SUMMARY
Soman simulator PDP is a compound that has a chemical structure identical to soman, except that the fluorine atom is replaced by a methyl group which makes PDP unable to bind covalently to the AChE active center. In rats, late mortality observed after treatment with high doses of soman could be prevented by PDP pretreatment. Such pretreatment has been much less efficient in primates. The effect of PDP in rats has been explained by blocking the deposition of soman in so-called soman depots in which soman is stored intact and subsequently released. In this paper we demonstrate that in the presence of PDP, inhibition of rat muscle AChE by soman is reduced in rat but not in human muscle homogenates. This result suggests that at least part of the beneficial effects of PDP pretreatment in rat might be due to the direct interaction of PDP with AChE resulting in reduced AChE phosphorylation by soman.
Key words: Soman simulator -- PDP - Soman -- HI-6 - Acetylcholinesterase -- Skeletal muscle
INTRODUCTION
Rats treated with high doses of soman ( 6 - 8 x LD50) survived initial toxic effects if treated with oxime reactivator HI-6; however, after 4 - 5 h their conditions deteriorated and they finally died [1]. A generally accepted explanation of this phenomenon is that a part of applied soman is stored and remains intact in a depot from where it is gradually released causing subsequent reinhibition of the previously reactivated acetylcholinesterase (ACHE; EC 3.1.1.7.). Such delayed mortality could be prevented by pretreatment with pinacolyl dimethylphosCorrespondence to: Zoran Grubi~, Institute of Pathophysiology, School of Medicine, 61105 Ljubljana, Zalos'ka 4, Slovenia. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
254
phinate -- PDP, a compound with chemical structure identical to soman except that the fluorine atom is replaced by a methyl group, which makes PDP unable to bind covalently to the serine in the AChE active center [2]. Beneficial effect has been explained by occupation of soman depot sites by PDP, which prevents formation of soman depots. Such depots were reported to be located also in the skeletal muscle proper [3]. In our opinion an additional explanation of reduced mortality of PDP-pretreated rats is possible. Having almost identical chemical structure as soman, PDP can occupy soman binding sites on the AChE molecule, interfering in this way with the enzyme inhibition by soman. Having the fluorine atom replaced by a methyl group, PDP cannot react in nucleophylic substitutions on the phosphorous atom. PDP is therefore not an irreversible inhibitor and is more stable than soman, which is quickly hydrolysed and irreversibly bound to various esterases in tissues and plasma. PDP therefore outlives soman in the organism and protects AChE against irreversible inhibition by soman released from the depot. In order to test our hypothesis we first tested the influence of PDP on the ACh hydrolysis in muscle homogenates. Then we determined percent of AChE inhibition after phosphorylation of AChE by soman in muscle homogenates in the presence and in the absence of PDP. HI-6 reactivation of soman-inhibited AChE of muscle homogenates was also determined in the absence and in the presence of PDP. Since differences in PDP effects in different species were reported [4] our study was carried out on both rat and human muscle homogenates. METHODS
Preparation of muscle homogenates Female Wistar strain albino rats weighing 150 - 200 g were anaesthetized with ether and perfused with saline through the inferior vena cava. Hemidiaphragms were isolated, weighed and frozen in liquid nitrogen until used. Homogenates (1.45 mg/10 #l of phosphate Ringer's solution of the following compositions: 138 mM NaC1, 4 mM KC1, 1 mM MgS04, 20 mM phosphate buffer, pH = 7.4) were prepared in hand-operated glass homogenizers kept on ice. Human muscle preparations were obtained at the Institute of Legal Medicine of the Medical Faculty, Ljubljana. Intercostal muscles were isolated within five hours after death from the victims of traffic accidents. Homogenates were prepared in the same way as described for rat muscle. The determination of the effects of PDP on the phosphorylation by soman and on HI-6 reactivation of AChE in rat and human muscle homogenates Muscle homogenates were incubated for 5 min with the following combinations of soman (0.2 nM final conc.), PDP (2 nM final conc.)' and HI-6 (0.5 mM final conc.): (I) in control experiments (100°7o activity) 20 ill of phosphate Ringer's solution were incubated with 20 ~1 homogenate. (II) for determining baseline inhibition of AChE by soman 20 ~1 of soman was mixed with 20 ~1 homogenate. (III) the effect of PDP on AChE phosphorylation by soman was tested by mixing 10 ~1 of PDP and 10 ~1 of soman with 20 ~1 of the homogenate. The effect of PDP
255
on HI-6 reactivation of soman-inhibited AChE was tested by determining reactivation by HI-6 in the presence and in the absence of PDP. Therefore 20 ul of muscle homogenate were incubated with (IV) 10 ~l of soman, 5 ~l of HI-6 and 5 ~l of Ringer's solution or (V) 10 ~l of soman, 5 ~l of HI-6 and 5 ~l of PDP. After 5-min incubation at room temperature, mixtures I, II, III, IV and V were preincubated for 30 min by iso-OMPA in order to block non-specific ChE and then proceeded for radiometric determination of AChE activity as described before [5]. The effects of PDP on ACh hydrolysis by AChE was tested as follows: 20 ul of homogenate was incubated with 10 ~l Ringer's solution and 10 ~l of PDP, which was also introduced into the preincubation mixture with iso-OMPA and into the incubation mixture with ACh. PDP concentration was kept at 2 nM throughout the procedure. ACh concentration was 3 mM. RESULTS AND DISCUSSION
The results are given in Fig. la for rat muscle homogenate and in Fig. lb for human muscle homogenate. At the concentration of soman used, AChE was subtotally inhibited (Fig. la and lb -- II). Higher inhibition of AChE in rat in comparison to human muscle homogenate is probably due to the higher consumption rate of soman in human muscle homogenate, which had higher specific activity of AChE (2.54 nmol ACh/mg/min in human vs. 1.22 nmol ACh/mg/min in rat muscle homogenate) and probably of other soman-binding enzymes. However the percentage of AChE activity could not be increased significantly in human AChE either by HI-6 (IV) or by PDP (III) and also not by the combination of HI-6 and PDP (V) added together with soman to the homogenate. Rat muscle AChE behaved in a different manner. PDP alone significantly (P < 0.01, paired t-test) reduced inhibition by soman (Fig. la -- III); however, reactivation by HI-6 alone gave a better recovery (IV). In the presence of PDP, HI-6 recovery of AChE was no better than obtained by HI-6 alone (V). The effects of PDP must be due to the direct interactions with the AChE molecule since ACh hydrolysis was reduced by about 50% in the presence of 2 nM PDP in both rat and human muscle homogenate. Our results demonstrate a protective effect of PDP on AChE phosphorylation by soman. Better survival of soman-injected rats pretreated by PDP [1] could therefore partly be explained by this PDP effect and not only by the occupation of a soman depot. Observed differences in PDP effects between rat and human muscle accord well with the experiments in which PDP pretreatment prevented formation of soman depot in rat but not in primate muscle [4]. However, inhibition of electric eel AChE was used in these experiments for the registration of soman released in vitro from the pectoralis muscles isolated after treatment of animals in vivo, which means that in this case our interpretation could not explain species specificity observed. Our results therefore do not negate the existence of a soman depot. They call the attention to the fact that using soman simulator PDP for demonstration of a soman depot one should consider the possibility of a direct protection effect of this compound based on attenuation of soman phosphorylation of ACHE.
256
a) rat diaphragm % A C h E activity lOO%
87%
100% 80%
86%
+
50% 50%
40%
26%
A
20%
0%
I b) h u m a n
II
III
IV
V
intercostal muscle
% A C h E aclivi 100% 100% - -
;++ +
80%--
60%--
61%
65%
61%
III
IV
V
40%-I
20%-0%
I
II
Fig. 1. (a) Rat muscle homogenate; % AChE activity recovered after the procedures described in Methods. Roman numerals under the columns correspond to the same numerals used in Methods: I -- control, II -- inhibition by soman, III -- inhibition of soman in the presence of PDP, IV -- inhibition by soman in the presence of HI-6, V -- inhibition by soman in the presence of PDP and HI-6. Standard deviations are indicated by bars (n = 5). (b) Human muscle homogenate; the same as in (a) (n = 8).
257 REFERENCES 1
2
3 4
5
O.L. Wolthuis, H.P. Bennschop and F. Berends, Persistence of the anticholinesterase soman in rats; antagonism with a nontoxic simulator of this organophosphate, Eur. J. Pharmacol., 69 (1981) 379-383. H.P.M. Van Helden, H.P. Benschop and O.L. Wolthuis, The prophylactic efficacy of various simulators against intoxication with the organophosphate soman: structure-activity studies, J. Pharm. Pharmacol., 38 (1986) 19-23. H.P.M. Van Helden and O.L. Wolthuis, Evidence for an intramuscular depot of the cholinesterase inhibitor soman in the rat, Eur. J. Pharmacol., 89 (1983) 271-274. H.P.M. Van Helden, H.J. Van der Wiel and O.L. Wolthuis, Retention of soman in rats, guineapigs and marmosets: species-dependent effects of the soman simulator, pinacolyl dimethyl phosphinate (PDP), J. Pharm. Pharmacol., 40 (1988) 35-41. Z. Grubi~ and A. Tomaz~, Mechanism of action of HI-6 on soman inhibition of acetylcholinesterase in preparations of rat and human skeletal muscle; comparison to SAD-128 and PAM-2, Arch. Toxicol., 63 (1989) 68-71.