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In Vitro Inhibition of Rabbit and Guinea Pig Erythrocyte Acetylcholinesterase by Soman
Toxicology in Vitro 13 (1999) 403±408 www.elsevier.com/locate/toxinvit
In Vitro Inhibition of Rabbit and Guinea Pig Erythrocyte Acetylcholinesterase by Soman J. KASSA* and J. BAJGAR Purkyne Military Medical Academy, PO Box 35/T, 500 01, Hradec KraÂloveÂ, Czech Republic (Accepted 8 October 1998) AbstractÐGuinea pig and rabbit erythrocytes were incubated with a high concentration of soman to achieve zero acetylcholinesterase activity. After washing with saline, the erythrocytes were incubated with horse plasma butyrylcholinesterase. The inhibition of plasma butyrylcholinesterase activity was observed because of inhibitor release from the erythrocytes. This release was quanti®ed and found to be the highest after 30 min of incubation. Our results con®rm that erythrocytes can serve as a depot for soman and make it possible to lengthen its toxic eects. # 1999 Elsevier Science Ltd. All rights reserved Keywords: guinea pig; rabbit; soman; erythrocyte acetylcholinesterase; plasma butyrylcholinesterase. Abbreviations: AChE = acetylcholinesterase; BuChE = butyrylcholinesterase.
INTRODUCTION
Soman (O-pinacolyl methylphosphono¯uoridate) belongs to the highly toxic cholinesterase inhibitors. Following the reaction of soman with acetylcholinesterase (AChE, EC 3.1.1.7), soman-inhibited AChE undergoes a chemical change (ageing process) that makes reactivation by oximes in vivo no longer possible (Bajgar, 1985; Dawson, 1994; Fleisher and Harris, 1965). This is probably the main reason for the diculty in treating soman poisoning. In addition, the releasing of soman from depot, where soman is stored after penetration into the organism, can also make the treatment of acute soman intoxication more dicult (Kadar et al., 1985; Shapira et al., 1990). Erythrocytes contain signi®cant amounts of AChE. The function of erythrocyte AChE is not known very well. It seems to hydrolyse acetylcholine in the spleen and cause the haemolysis of the old erythrocytes by lowering of pH (Torp, 1986). It was shown previously that physostigmine reduced the destruction of erythrocytes in the spleen by its inhibiting eect on AChE activity (Dacie, 1954). The aim of this study was to inhibit erythrocyte AChE to very low/zero activity using high concentrations of soman and to demonstrate the ability of inhibitor to release from the erythrocytes. *Corresponding author.
MATERIALS AND METHODS
Animals Male Pirbright-white guinea pigs (Dunkin± Hartley strain) weighing 250±300 g and male New Zealand White rabbits weighing 3200±3500 g were purchased from Velaz Prague (Czech Republic). The animals were maintained in an air-conditioned room (22 2 18C and 50 2 10% relative humidity, with light from 07.00 to 19.00), and were allowed free access to standard chow (BIOPO PozorÏ ice) and tap water. Experiments were performed under the supervision of the Ethics Committee of the Military Medical Academy. Enzymes and chemicals After exsanguination of anaesthetized animals by decapitation (ip injection of urethane 1.5 g/kg), the blood was centrifuged to obtain plasma and erythrocytes. Erythrocytes were used as a source of AChE. Freeze-dried horse plasma, a source of butyrylcholinesterase (BuChE, EC 3.1.1.8), was obtained from the Military Technical Institute, Brno (Czech Republic). Soman of 98.5% purity was purchased from the Military Facility, ZemianskeÂ, Kostolany (Slovakia). Inhibition of erythrocyte AChE Erythrocytes were incubated with soman (1 10ÿ6 M) in saline for 0.5, 4 and 24 hr at room temperature (22 2 28C). After incubation and separ-
0887-2333/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0887-2333(99)00009-0
404
J. Kassa and J. Bajgar
ation of erythrocytes from soman solution by centrifugation (1000 g, 5 min), the erythrocytes were washed with saline three times to remove free soman. After separation of erythrocytes from saline by centrifugation (1000 g, 5 min), the erythrocytes were incubated with 1.5% horse plasma solution in saline in equal volume for various time intervals (from 5 to 60 min) at room temperature. At each time interval, erythrocytes were again separated from horse plasma by centrifugation (1000 g, 5 min) and activities of horse plasma BuChE as well as erythrocyte AChE were determined. Enzymatic analyses Erythrocyte AChE activity was determined in haemolysate (1:20) at room temperature by the method of Ellman et al. (1961) using acetylthiocholine as a substrate (10ÿ3 M). Horse plasma BuChE activity was determined by the same method using butyrylthiocholine as a substrate (10ÿ3 M). The results were expressed as arbitrary units (absorbance at 412 nm, spectrophotometer UVIKON 942, Kontron Instruments). Both cholinesterases were expressed as mmol acetylthiocholine hydrolysed/ litre/sec or mmol butyrylthiocholine hydrolysed/ litre/ sec and presented as percent of controls. Each determination was made in triplicate. Inhibition of BuChE at
Horse plasma BuChE was incubated with soman concentrations of 7.5 10ÿ9, 1 10ÿ8 and
1.5 10ÿ8 M and the activity was determined at dierent time intervals (1±60 min). Calculation of concentration
inhibition
constants
and
soman
The bimolecular rate constant of inhibition (ki) was calculated using the half-life of plasma BuChE inhibition (t0.5) with known concentrations of soman [I] according to the formula [1] as described elsewhere (Eto, 1976). ki
ln 2 I:t0:5
1
The calculation of soman concentration causing the inhibition of plasma BuChE activity after incubation with soman-treated erythrocytes was performed using the bimolecular rate constant obtained as described (ki) and half-life of inhibition (t0.5). Statistical evaluation Bimolecular rate constants and soman concentrations were obtained from linear regression analysis of the inhibition plots using nine points for each line (semilogarithmical transformation). Statistical signi®cance was determined using the paired Student's t-test. Dierences were considered to be signi®cant if P < 0.05. Statistical evaluation was performed with relevant programs using an ADT 4500 computer.
Fig. 1. Course of inhibition of plasma BuChE activity following in vitro incubation with known concentrations of somanÐsemilogarithmic transformation. The equations of lines are: y = ÿ 0.0325x + 1.873 for 7.5 10ÿ9 M; y = ÿ 0.0515x + 1.873 for 1 10ÿ8 M and y = ÿ 0.0904x + 1.873 for 1.5 10ÿ8 M.
In vitro inhibition of erythrocyte AChE by soman
405
Table 1. Changes of guinea pig and rabbit erythrocyte AChE activity following in vitro incubation with soman at a concentration of 10ÿ6 M Erythrocyte AChE activity (% of controls) Animals Control values Experimental values Time of incubation with soman
0.5 hr 4 hr 24 hr
Guinea pigs
Rabbits
21.3 (17.7±24.9) mkat/litre 100 (83.2±116.8)%
16.0 (13.7±13.83) mkat/litre 100 (85.8±114.2)%
Before 60 min incubation with horse plasma
After 60 min incubation with horse plasma
Before 60 min incubation with horse plasma
After 60 min incubation with horse plasma
1.5 (0.8±2.4) 0 0
0 0 0
4.1 (3.2±5.0) 5.7 (4.3±7.1) 3.6 (2.7±4.5)
0 0 0
RESULTS
Guinea pig and rabbit erythrocyte AChE activities after incubation with soman were demonstrated in Table 1. Guinea pig as well as rabbit erythrocyte AChE activity was practically totally inhibited. This table demonstrates a strong inhibiting eect of soman on erythrocyte AChE. Bimolecular rate constant of plasma BuChE inhibition with soman, calculated with the help of plasma BuChE incubation with known concentrations of soman (Fig. 1), is 1.1432 0.24, 107 Mÿ1 minÿ1.
Following 30 min incubation of guinea pig as well as rabbit erythrocytes with soman, the amount of soman released during incubation of soman-treated erythrocytes with horse plasma BuChE was very high. After 5 min of incubation of guinea pig soman-treated erythrocytes with horse plasma, the decrease in BuChE activity was achieved to 6.3% of control values (P < 0.05). Following 10 (30, 60) min incubation no plasma BuChE activity was observed (Fig. 2). This rate of inhibition corresponds to the inhibition produced by soman at a concentration of 4.31 10ÿ8 M (Fig. 3). Following
Fig. 2. Changes of plasma BuChE activity following in vitro incubation with guinea pig soman-treated erythrocytes. The value of plasma BuChE activity before incubation with soman-treated erythrocytes is 8 mkat/litre (100%). The results represent the mean (n = 8) and standard deviation. Statistical signi®cance between two compared values (u): ± P < 0.05.
406
J. Kassa and J. Bajgar
Fig. 3. Course of plasma BuChE inhibition following in vitro incubation with guinea pig soman-treated erythrocytes-semilogarithmic transformation. The equations of lines are: y = ÿ 0.00176x + 2.0 for 3.55 10ÿ10 M; y = ÿ 0.0162x + 2.0 for 3.45 10ÿ9 M and y = ÿ 0.212x + 2.0 for 4.31 10ÿ8 M.
incubation of rabbit soman-treated erythrocytes with plasma BuChE, its activity decreased to 5,9% of control values within 5 min (P < 0. 05). After 10 (30, 60) min incubation, no plasma BuChE activity was determined (Fig. 4). This rate of inhibition corresponds to the inhibition produced by soman at a concentration of 4.64 10ÿ8 M (Fig. 5). Following 4 hr of incubation of both animal erythrocytes with soman, lesser amounts of soman were released to inhibit plasma BuChE activity in comparison with 30 min of incubation (Figs 2 and 4). In the case of guinea pig erythrocytes, half of the plasma BuChE activity was inhibited by released soman within 17.5 min (Fig. 3). This rate of inhibition corresponds to the inhibition produced by soman at a concentration of 3.45 10ÿ9 M. In the case of rabbit erythrocytes, half of the plasma BuChE activity was inhibited by released soman within 30 min (Fig. 5). This rate of inhibition corresponds to the inhibition produced by soman at a concentration of 2.01 10ÿ9 M. Following 24 hr of incubation of both animal erythrocytes with soman, the smallest inhibiting eect of released soman was observed (Figs 2 and 4). This rate of inhibition corresponds to the inhibition produced by soman at a concentration of 3.55 10ÿ10 M in the case of guinea pig erythrocytes, 2.62 10ÿ10 M in the case of rabbit erythrocytes, respectively (Figs 3 and 5).
DISCUSSION
It is generally accepted that soman inhibits erythrocyte AChE and the enzyme is unreactivatable within tens of minutes, that is, the level of inhibition remains stable without spontaneous reactivation (Dawson, 1994; Fleisher and Harris, 1965; Marrs, 1993). However, it was clearly demonstrated as a release of inhibitor in our experiments with horse plasma although the erythrocytes were washed several times to remove free soman before these experiments. It is not known whether it is soman or its analogue that is able to inhibit BuChE activity. BuChE inhibition is dependent on the time of soman incubation with guinea pig as well as rabbit erythrocytes. We quanti®ed soman release from erythrocytes with the help of calculation of inhibition rate of BuChE activity in horse plasma during incubation with soman-treated erythrocytes. The inhibiting eect of soman-treated erythrocytes is much higher after 30 min of incubation with soman in comparison with 4 and 24 hr of incubation. These data correspond with our results in vivo. A signi®cant decrease in AChE or BuChE activity in the plasma, diaphragm and liver following transfusion of soman-treated erythrocytes was found in rabbits when the erythrocytes were previously incubated with soman for 30 min (before transfusion). On the other hand, no changes of
In vitro inhibition of erythrocyte AChE by soman
Fig. 4. Changes of plasma BuChE activity following in vitro incubation with rabbit soman-treated erythrocytes. The value of plasma BuChE activity before incubation with soman-treated erythrocytes is 8 mkat/litre (100%). For symbols, see Fig. 2.
Fig. 5. Course of plasma BuChE inhibition following in vitro incubation with rabbit soman-treated erythrocytesÐsemilogarithmic transformation. The equations of lines are: y = ÿ 0.00128x + 2.0 for 2.62 10ÿ10 M; y = ÿ 0.0088x + 2.0 for 2.0 10ÿ9 M and y = ÿ 0.233x + 2.0 for 4.64 10ÿ8 M.
407
408
J. Kassa and J. Bajgar
AChE or BuChE activity in plasma, diaphragm and liver following transfusion of soman-treated erythrocytes were observed in rabbits when erythrocytes were previously incubated with soman for 24 hr (before transfusion) (Kassa et al., 1997). Therefore we can conclude that the amount of released soman falls if the time of erythrocyte incubation with soman increases. Thus, erythrocytes can serve as a depot for soman and possibly lengthen its toxic eects. These ®ndings support a hypothesis that soman is able to leave some depot places where it is stored and continue its toxic eects for many hours (Kadar et al., 1985; Shapira et al., 1990). This fact can make the ecacy of antidotal treatment of soman acute poisoning very low. AcknowledgementsÐThe authors would like to thank to Mrs M. Zechovska and Mrs M. KaraÂskova for their skilled technical assistance. This study was supported by the grant of IGA MZ, No 4074-2. REFERENCES
Bajgar J. (1985) Poisoning with organophosphorus inhibitors of cholinesteraseÐeect, diagnosis and treatment (in Czech). In News in Medicine, ed. R. Samkova, pp. 5±40. Avicenum, Prague. Dacie J. V. (1954) The Haemolytic Anaemias, pp. 270. J. & A. Churchill, London.
Dawson R. M. (1994) Review of oximes available for treatment nerve agent poisoning. Journal of Applied Toxicology 14, 317±331. Ellman G. L., Courtney D. K., Andres V., Jr and Featherstone R. M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7, 88±95. Eto M. (1976) Organophosphorus Pesticides: Organic and Biological Chemistry, Vol. 320. CRC Press, Cleveland, OH. Fleisher J. H. and Harris L. W. (1965) Dealkylation as a mechanism for aging of cholinesterase after poisoning with pinacolyl methylphosphono¯uoridate. Biochemical Pharmacology 14, 641±648. Kadar T., Raveh L., Cohen G., Oz N., Baraness I., Balan A., Ashani Y. and Shapira S. (1985) Distribution of 3Hsoman in mice. Archives of Toxicology 58, 45±49. Kassa J., Bajgar J. and Fusek J. (1997) Changes of cholinesterase activity in erythrocytes, plasma, diaphragm, liver and various parts of the brain in the rabbit following transfusion of erythrocytes with soman-inhibited acetylcholinesterase. Acta Medica (Hradec KraÂloveÂ) 40, 37±39. Marrs T. C. (1993) Organophosphate poisoning. Pharmacology and Therapeutics 58, 51±66. Shapira S., Kadar T., Cohen G., Chapman S. and Raveh L. (1990) Eects of CBDP and MEPQ on the toxicity and distribution of 3H-soman in mice. Archives of Toxicology 64, 663±668. Torp H. E. (1986) Haemolytic eect of the enzyme system acetylcholine erythrocyteÐcholinesterase. Scandinavian Journal of HaematologyÐSuppl. 36, 1±11.
In Vitro Inhibition of Rabbit and Guinea Pig Erythrocyte Acetylcholinesterase by Soman J. KASSA* and J. BAJGAR Purkyne Military Medical Academy, PO Box 35/T, 500 01, Hradec KraÂloveÂ, Czech Republic (Accepted 8 October 1998) AbstractÐGuinea pig and rabbit erythrocytes were incubated with a high concentration of soman to achieve zero acetylcholinesterase activity. After washing with saline, the erythrocytes were incubated with horse plasma butyrylcholinesterase. The inhibition of plasma butyrylcholinesterase activity was observed because of inhibitor release from the erythrocytes. This release was quanti®ed and found to be the highest after 30 min of incubation. Our results con®rm that erythrocytes can serve as a depot for soman and make it possible to lengthen its toxic eects. # 1999 Elsevier Science Ltd. All rights reserved Keywords: guinea pig; rabbit; soman; erythrocyte acetylcholinesterase; plasma butyrylcholinesterase. Abbreviations: AChE = acetylcholinesterase; BuChE = butyrylcholinesterase.
INTRODUCTION
Soman (O-pinacolyl methylphosphono¯uoridate) belongs to the highly toxic cholinesterase inhibitors. Following the reaction of soman with acetylcholinesterase (AChE, EC 3.1.1.7), soman-inhibited AChE undergoes a chemical change (ageing process) that makes reactivation by oximes in vivo no longer possible (Bajgar, 1985; Dawson, 1994; Fleisher and Harris, 1965). This is probably the main reason for the diculty in treating soman poisoning. In addition, the releasing of soman from depot, where soman is stored after penetration into the organism, can also make the treatment of acute soman intoxication more dicult (Kadar et al., 1985; Shapira et al., 1990). Erythrocytes contain signi®cant amounts of AChE. The function of erythrocyte AChE is not known very well. It seems to hydrolyse acetylcholine in the spleen and cause the haemolysis of the old erythrocytes by lowering of pH (Torp, 1986). It was shown previously that physostigmine reduced the destruction of erythrocytes in the spleen by its inhibiting eect on AChE activity (Dacie, 1954). The aim of this study was to inhibit erythrocyte AChE to very low/zero activity using high concentrations of soman and to demonstrate the ability of inhibitor to release from the erythrocytes. *Corresponding author.
MATERIALS AND METHODS
Animals Male Pirbright-white guinea pigs (Dunkin± Hartley strain) weighing 250±300 g and male New Zealand White rabbits weighing 3200±3500 g were purchased from Velaz Prague (Czech Republic). The animals were maintained in an air-conditioned room (22 2 18C and 50 2 10% relative humidity, with light from 07.00 to 19.00), and were allowed free access to standard chow (BIOPO PozorÏ ice) and tap water. Experiments were performed under the supervision of the Ethics Committee of the Military Medical Academy. Enzymes and chemicals After exsanguination of anaesthetized animals by decapitation (ip injection of urethane 1.5 g/kg), the blood was centrifuged to obtain plasma and erythrocytes. Erythrocytes were used as a source of AChE. Freeze-dried horse plasma, a source of butyrylcholinesterase (BuChE, EC 3.1.1.8), was obtained from the Military Technical Institute, Brno (Czech Republic). Soman of 98.5% purity was purchased from the Military Facility, ZemianskeÂ, Kostolany (Slovakia). Inhibition of erythrocyte AChE Erythrocytes were incubated with soman (1 10ÿ6 M) in saline for 0.5, 4 and 24 hr at room temperature (22 2 28C). After incubation and separ-
0887-2333/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0887-2333(99)00009-0
404
J. Kassa and J. Bajgar
ation of erythrocytes from soman solution by centrifugation (1000 g, 5 min), the erythrocytes were washed with saline three times to remove free soman. After separation of erythrocytes from saline by centrifugation (1000 g, 5 min), the erythrocytes were incubated with 1.5% horse plasma solution in saline in equal volume for various time intervals (from 5 to 60 min) at room temperature. At each time interval, erythrocytes were again separated from horse plasma by centrifugation (1000 g, 5 min) and activities of horse plasma BuChE as well as erythrocyte AChE were determined. Enzymatic analyses Erythrocyte AChE activity was determined in haemolysate (1:20) at room temperature by the method of Ellman et al. (1961) using acetylthiocholine as a substrate (10ÿ3 M). Horse plasma BuChE activity was determined by the same method using butyrylthiocholine as a substrate (10ÿ3 M). The results were expressed as arbitrary units (absorbance at 412 nm, spectrophotometer UVIKON 942, Kontron Instruments). Both cholinesterases were expressed as mmol acetylthiocholine hydrolysed/ litre/sec or mmol butyrylthiocholine hydrolysed/ litre/ sec and presented as percent of controls. Each determination was made in triplicate. Inhibition of BuChE at
Horse plasma BuChE was incubated with soman concentrations of 7.5 10ÿ9, 1 10ÿ8 and
1.5 10ÿ8 M and the activity was determined at dierent time intervals (1±60 min). Calculation of concentration
inhibition
constants
and
soman
The bimolecular rate constant of inhibition (ki) was calculated using the half-life of plasma BuChE inhibition (t0.5) with known concentrations of soman [I] according to the formula [1] as described elsewhere (Eto, 1976). ki
ln 2 I:t0:5
1
The calculation of soman concentration causing the inhibition of plasma BuChE activity after incubation with soman-treated erythrocytes was performed using the bimolecular rate constant obtained as described (ki) and half-life of inhibition (t0.5). Statistical evaluation Bimolecular rate constants and soman concentrations were obtained from linear regression analysis of the inhibition plots using nine points for each line (semilogarithmical transformation). Statistical signi®cance was determined using the paired Student's t-test. Dierences were considered to be signi®cant if P < 0.05. Statistical evaluation was performed with relevant programs using an ADT 4500 computer.
Fig. 1. Course of inhibition of plasma BuChE activity following in vitro incubation with known concentrations of somanÐsemilogarithmic transformation. The equations of lines are: y = ÿ 0.0325x + 1.873 for 7.5 10ÿ9 M; y = ÿ 0.0515x + 1.873 for 1 10ÿ8 M and y = ÿ 0.0904x + 1.873 for 1.5 10ÿ8 M.
In vitro inhibition of erythrocyte AChE by soman
405
Table 1. Changes of guinea pig and rabbit erythrocyte AChE activity following in vitro incubation with soman at a concentration of 10ÿ6 M Erythrocyte AChE activity (% of controls) Animals Control values Experimental values Time of incubation with soman
0.5 hr 4 hr 24 hr
Guinea pigs
Rabbits
21.3 (17.7±24.9) mkat/litre 100 (83.2±116.8)%
16.0 (13.7±13.83) mkat/litre 100 (85.8±114.2)%
Before 60 min incubation with horse plasma
After 60 min incubation with horse plasma
Before 60 min incubation with horse plasma
After 60 min incubation with horse plasma
1.5 (0.8±2.4) 0 0
0 0 0
4.1 (3.2±5.0) 5.7 (4.3±7.1) 3.6 (2.7±4.5)
0 0 0
RESULTS
Guinea pig and rabbit erythrocyte AChE activities after incubation with soman were demonstrated in Table 1. Guinea pig as well as rabbit erythrocyte AChE activity was practically totally inhibited. This table demonstrates a strong inhibiting eect of soman on erythrocyte AChE. Bimolecular rate constant of plasma BuChE inhibition with soman, calculated with the help of plasma BuChE incubation with known concentrations of soman (Fig. 1), is 1.1432 0.24, 107 Mÿ1 minÿ1.
Following 30 min incubation of guinea pig as well as rabbit erythrocytes with soman, the amount of soman released during incubation of soman-treated erythrocytes with horse plasma BuChE was very high. After 5 min of incubation of guinea pig soman-treated erythrocytes with horse plasma, the decrease in BuChE activity was achieved to 6.3% of control values (P < 0.05). Following 10 (30, 60) min incubation no plasma BuChE activity was observed (Fig. 2). This rate of inhibition corresponds to the inhibition produced by soman at a concentration of 4.31 10ÿ8 M (Fig. 3). Following
Fig. 2. Changes of plasma BuChE activity following in vitro incubation with guinea pig soman-treated erythrocytes. The value of plasma BuChE activity before incubation with soman-treated erythrocytes is 8 mkat/litre (100%). The results represent the mean (n = 8) and standard deviation. Statistical signi®cance between two compared values (u): ± P < 0.05.
406
J. Kassa and J. Bajgar
Fig. 3. Course of plasma BuChE inhibition following in vitro incubation with guinea pig soman-treated erythrocytes-semilogarithmic transformation. The equations of lines are: y = ÿ 0.00176x + 2.0 for 3.55 10ÿ10 M; y = ÿ 0.0162x + 2.0 for 3.45 10ÿ9 M and y = ÿ 0.212x + 2.0 for 4.31 10ÿ8 M.
incubation of rabbit soman-treated erythrocytes with plasma BuChE, its activity decreased to 5,9% of control values within 5 min (P < 0. 05). After 10 (30, 60) min incubation, no plasma BuChE activity was determined (Fig. 4). This rate of inhibition corresponds to the inhibition produced by soman at a concentration of 4.64 10ÿ8 M (Fig. 5). Following 4 hr of incubation of both animal erythrocytes with soman, lesser amounts of soman were released to inhibit plasma BuChE activity in comparison with 30 min of incubation (Figs 2 and 4). In the case of guinea pig erythrocytes, half of the plasma BuChE activity was inhibited by released soman within 17.5 min (Fig. 3). This rate of inhibition corresponds to the inhibition produced by soman at a concentration of 3.45 10ÿ9 M. In the case of rabbit erythrocytes, half of the plasma BuChE activity was inhibited by released soman within 30 min (Fig. 5). This rate of inhibition corresponds to the inhibition produced by soman at a concentration of 2.01 10ÿ9 M. Following 24 hr of incubation of both animal erythrocytes with soman, the smallest inhibiting eect of released soman was observed (Figs 2 and 4). This rate of inhibition corresponds to the inhibition produced by soman at a concentration of 3.55 10ÿ10 M in the case of guinea pig erythrocytes, 2.62 10ÿ10 M in the case of rabbit erythrocytes, respectively (Figs 3 and 5).
DISCUSSION
It is generally accepted that soman inhibits erythrocyte AChE and the enzyme is unreactivatable within tens of minutes, that is, the level of inhibition remains stable without spontaneous reactivation (Dawson, 1994; Fleisher and Harris, 1965; Marrs, 1993). However, it was clearly demonstrated as a release of inhibitor in our experiments with horse plasma although the erythrocytes were washed several times to remove free soman before these experiments. It is not known whether it is soman or its analogue that is able to inhibit BuChE activity. BuChE inhibition is dependent on the time of soman incubation with guinea pig as well as rabbit erythrocytes. We quanti®ed soman release from erythrocytes with the help of calculation of inhibition rate of BuChE activity in horse plasma during incubation with soman-treated erythrocytes. The inhibiting eect of soman-treated erythrocytes is much higher after 30 min of incubation with soman in comparison with 4 and 24 hr of incubation. These data correspond with our results in vivo. A signi®cant decrease in AChE or BuChE activity in the plasma, diaphragm and liver following transfusion of soman-treated erythrocytes was found in rabbits when the erythrocytes were previously incubated with soman for 30 min (before transfusion). On the other hand, no changes of
In vitro inhibition of erythrocyte AChE by soman
Fig. 4. Changes of plasma BuChE activity following in vitro incubation with rabbit soman-treated erythrocytes. The value of plasma BuChE activity before incubation with soman-treated erythrocytes is 8 mkat/litre (100%). For symbols, see Fig. 2.
Fig. 5. Course of plasma BuChE inhibition following in vitro incubation with rabbit soman-treated erythrocytesÐsemilogarithmic transformation. The equations of lines are: y = ÿ 0.00128x + 2.0 for 2.62 10ÿ10 M; y = ÿ 0.0088x + 2.0 for 2.0 10ÿ9 M and y = ÿ 0.233x + 2.0 for 4.64 10ÿ8 M.
407
408
J. Kassa and J. Bajgar
AChE or BuChE activity in plasma, diaphragm and liver following transfusion of soman-treated erythrocytes were observed in rabbits when erythrocytes were previously incubated with soman for 24 hr (before transfusion) (Kassa et al., 1997). Therefore we can conclude that the amount of released soman falls if the time of erythrocyte incubation with soman increases. Thus, erythrocytes can serve as a depot for soman and possibly lengthen its toxic eects. These ®ndings support a hypothesis that soman is able to leave some depot places where it is stored and continue its toxic eects for many hours (Kadar et al., 1985; Shapira et al., 1990). This fact can make the ecacy of antidotal treatment of soman acute poisoning very low. AcknowledgementsÐThe authors would like to thank to Mrs M. Zechovska and Mrs M. KaraÂskova for their skilled technical assistance. This study was supported by the grant of IGA MZ, No 4074-2. REFERENCES
Bajgar J. (1985) Poisoning with organophosphorus inhibitors of cholinesteraseÐeect, diagnosis and treatment (in Czech). In News in Medicine, ed. R. Samkova, pp. 5±40. Avicenum, Prague. Dacie J. V. (1954) The Haemolytic Anaemias, pp. 270. J. & A. Churchill, London.
Dawson R. M. (1994) Review of oximes available for treatment nerve agent poisoning. Journal of Applied Toxicology 14, 317±331. Ellman G. L., Courtney D. K., Andres V., Jr and Featherstone R. M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7, 88±95. Eto M. (1976) Organophosphorus Pesticides: Organic and Biological Chemistry, Vol. 320. CRC Press, Cleveland, OH. Fleisher J. H. and Harris L. W. (1965) Dealkylation as a mechanism for aging of cholinesterase after poisoning with pinacolyl methylphosphono¯uoridate. Biochemical Pharmacology 14, 641±648. Kadar T., Raveh L., Cohen G., Oz N., Baraness I., Balan A., Ashani Y. and Shapira S. (1985) Distribution of 3Hsoman in mice. Archives of Toxicology 58, 45±49. Kassa J., Bajgar J. and Fusek J. (1997) Changes of cholinesterase activity in erythrocytes, plasma, diaphragm, liver and various parts of the brain in the rabbit following transfusion of erythrocytes with soman-inhibited acetylcholinesterase. Acta Medica (Hradec KraÂloveÂ) 40, 37±39. Marrs T. C. (1993) Organophosphate poisoning. Pharmacology and Therapeutics 58, 51±66. Shapira S., Kadar T., Cohen G., Chapman S. and Raveh L. (1990) Eects of CBDP and MEPQ on the toxicity and distribution of 3H-soman in mice. Archives of Toxicology 64, 663±668. Torp H. E. (1986) Haemolytic eect of the enzyme system acetylcholine erythrocyteÐcholinesterase. Scandinavian Journal of HaematologyÐSuppl. 36, 1±11.