Effects of 2-PAMCI and toxogonin of retinal and brain acetylcholinesterase inhibited by sarin

EUROPEANJOURNALOF PHARMACOLOGY14 (1971) 38--46. NORTH-HOLLANDPUBLISHINGCOMPANY

E F F E C T S O F 2-PAMCI A N D T O X O G O N I N O N R E T I N A L A N D B R A I N A C E T Y L C H O L I N E S T E R A S E I N H I B I T E D BY S A R I N * Larrel W. HARRIS, Joseph H. FLEISHER and Henry I. YAMAMURA Basic Medical Sciences Department, Medical Research Laboratory Research Laboratories, Edgewood Arsenal, Mayland 21010, USA

Received 24 August 1970

Accepted 15 October 1970

L.W. HARRIS, J.H. FLEISHER and H.I. YAMAMURA,Effects o f 2-PAMC1 and toxogonin on retinal and brain acetylcholinestemse inhibited by sarin, European J. Pharmacol. 14 (1971) 38-46. The acetylcholinesterase (ACHE) activity of retinal and cerebral tissue but not that of red blood cells (rbc) recovered from inhibition within 24 hr after giving satin to atropinized guinea pigs. Retinal ACHEdiffers from that in cerebral tissue in showing recovery in 3½ hr after poisoning when assayed in intact tissue. Intramuscular injection of 7.5 mg (0.044 mmole)/kg of 2-PAMCIone half hour after satin elevated ACHE 23.9% in intact retinas, 2.5% in sonicated retinas, and 3.7% in cerebral homogenates over values obtained in the corresponding tissues of satin controls. Substitution of an equimolar dose of toxognnin for 2-PAMCIyielded significantly less reactivation when measured in intact retinas and similar reactivation in sonicates or homogenates. Injection of monoisonitrosoacetone (MINA) at 0.088 mmole/kg reactivated inhibited AChE in retinal sonicates and cerebral homogenates. The influence of membrane barriers on enzyme reactivation in vitro and in vivo is discussed. Acetylcholinesterase, inhibition and reactivation Pyridinium aldoximes Retinal AChE

1. INTRODUCTION Oximes, when injected in amounts that raise the LDs o of an organophosphate, reactivate phosphorylated acetylcholinesterase (ACHE) in blood and peripheral tissue (Hobbiger, 1963). A similar therapeutic role in the central nervous system of intoxicated animals must be based in part on significant reactivation of the phosphorylated enzyme following treatment with oximes. Attempts to demonstrate such reactivation of brain AChE in animals poisoned with isopropyl methylphosphonofluoridate (sarin) and treated with

* In conducting the research described in this report, the investigators adhered to the Guide for Laboratory Animal Facilities and Care as promulgated by the Committee on the Guide for Laboratory Animal Resources, National Academy of Sciences, National Research Council.

Blood-retina and blood-brain barrier Cerebral AChe

pyridinium 2-aldoxime methiodide (Rutland, 1958; Cohen and Wiersinga, 1960) or with 1,1 '-trimethylene bis (4-formyl-pyridinium bromide dioxime (TMB-4) (Fleisher et al., 1960) yielded negative results. It was assumed, therefore, that the quaternary compounds were unable to pass through the blood-brain barrier in sufficient amounts to produce reactivation of AChE (Rutland, 1958). In the studies cited above, the enzymatic activity of the whole brain was used to assess reactivation of ACHE. Thus, reactivation in some brain areas with a higher permeability for quaternary ammonium compounds (Mayer and Bain, 1956) would have escaped detection. This possibility is supported by the different recoveries of AChE in various parts of the brain of rabbits given 0,0-diethyl 0-p-nitrophenylphosphate (paraoxon) and then treated with high doses of PAM intravenously (Rosenberg, 1960). Subsequently, Erdman (1965) reported that 1,1'-(oxydimethylene)

L. W.Harris, Oximes on sarin-inhibited acetylcholinesterase

bis[4-formyl pyridinium chloride] dioxime (toxogonin) produced marked reactivation of AChE in the cerebella of rats previously injected with paraoxon. In confirmation, Hobbiger and Vojvodic (1966) found that 0.1 mmole/kg of either toxogonin or TMB-4 administered intraperitoneally to paraoxon-poisoned rats reactivated the AChE of cerebellar homogenates by 28%. Later, they reported that the same dose of TMB-4 injected intraperitoneally into rats intoxicated with diisopropyl phosphorofluoridate (DFP) reactivated only 3% of the AChE in cerebellar homogenates (Hobbiger and Vojvodic, 1967). From these observations it is apparent that AChE activity in the central nervous system of organophosphate-intoxicated animals following treatment with pyridinium aldoximes will be influenced in part by the anticholinesterase, the dosage and route of administration of the oxime used for treatment, and the particular tissues of the central nervous system used for assay. This laboratory has recently reported upon the antagonism of sarin intoxication by atropine and the relatively low doses of pyridinium 2-aldoxime methochloride (2-PAMC1) and toxogonin compatible with intramuscular administration (Fleisher et al., 1970). The present paper reports an investigation into the reactivation of AChE in the central nervous system after such low doses of oximes are given intramuscularly to sarin-poisoned guinea pigs. As part of this study, the retina, which embryologically as well as functionally is a part of the central nervous system (Francis, 1953), was used to investigate the penetration of the retinal portion of the blood-brain barrier by quaternary oximes. Changes in the AChE activity of brain homogenates and red blood cells of sarinintoxicated animals treated with oximes were also studied for purposes of comparison.

2. MATERIALS AND METHODS 2.1. Materials

Acetyl-1-14C~-methylcholine iodide (2.35 mCi[ (mmole), and acetyl-l-14C-choline iodide (2.4 mCi/ mmole) were purchased from New England Nuclear Corporation, Boston, Massachusetts. Amberlite CG120 resin, sodium form, 200-400 mesh analytical grade was a product of Mallinckrodt Company, St.

39

Louis, Missouri. Naphthalene was obtained from Eastman Organic Chemicals, Rochester, New York, 2-5-diphenyloxazole (PPO) and 1,4-bis[2-5-phenyloxazolyl] benzene (POPOP) were obtained from Packard Instruments Company, Downers Grove, Illinois. Lubrol WX was obtained from I.C.I. Organics Incorporated, Stamford, Connecticut. 2.1. Methods

2.2.1. Radiometric assay for (ACHE) activity The method used, recently reported by Siakotos et al. (1969) is based upon the adsorption of unreacted substrates as their acid-l- 14C choline ester on Amberlite CG-120 resin suspended in dioxane. The supernatant solution containing the product of hydrolysis, the free acid-1-~4C, is counted in a liquid scintillation spectrometer. Details concerning preparation of resin and the scintillation cocktail (fluor) are given by Siakotos et al. (1969). 2.2.2. Preparation of a standard curve relating radioactivity to hydrolysis of substrate. 30.5 mg of acetyl-1-14C-~-methylcholine iodide (2.35 mCi/mmole) was dissolved in 1 mM acetate buffer at pH 4.5. Sufficient nonlabelled acetyl~-methylcholine was added to give a concentration of 0.03 mmole/ml. This stock solution was subdivided and stored at -20°C. For preparation of a standard for assay of AChE activity of retina and rbc the stock was diluted with dioxane to contain 6 nmoles/ml. Aliquots of this dilution, varying from 0.1 to 5 ml with the difference in volume compensated with pure dioxane, were added to 12 ml of modified Bray's cocktail (Siakotis et al., 1969). The samples were counted in a scintillation spectrometer (Packard Model 3375) with an efficiency of 66% at the optimized 14C setting. The radioactivity in cpm was plotted against the quantity of substrate. For studies with brain homogenates, a similar curve was prepared from acetyl-1-l 4C choline iodide (2.4mCi/mmole) and non-labelled acetylcholine chloride. After removal of unhydrolyzed substrate with dioxane resin, the radioactivity in 5 ml of dioxane supernatant was measured, and the AChE activity estimated by interpolation. 2.2.3. Preparation of tissues and enzyme assay. Guinea pigs (Hartley strain) of mixed sex and

40

L. It.Harris, Oximes on sarin-inhibitedacetylcholinesterase

weighing 325 to 375 g were used throughout. Unless designated otherwise, each of the studies described below was carried out with tissues obtained from eight or more animals. i) Intact retinas: Guinea pigs were anesthetized with ether. In each eye the conjunctiva was reflected and the major extraocular muscles were cut. The eye was enucleated, and incised circumferentially at the equator. The anterior portion was discarded along with the attached vitreous humor. The posterior portion was placed in cold saline. Holding the scleral coat with forceps, the retina was separated from the choroid and cut from the optic nerve at its point of attachment. For assay, the retina was washed with saline into small glass vials. Excess saline was removed by vacuum, and 1.8 ml of a Ringer-glucose solution (Umbreit et al., 1949) buffered with 0.01 M tris at pH 7.8 was added. 0.2ml of O.03M acetyl 1-14C/3-methylcholine iodide was added and the preparation incubated at 37°C. Aliquots of 0.05 ml were taken into 4.95 ml of dioxane resin. The samples were made to 10 ml with dioxane, mixed and centrifuged; 5 ml of the supernatant was transferred to 12 ml of the fluor and counted. Blanks for the corresponding time intervals were always run without tissue in this and other assays described below. The corrected readings for intact retinal AChE activity were linear for forty minutes. ii) Retinal sonciates: The contralateral retina, prepared as described above, was sonicated with a Bronwill Biosonik at 20% of maximum setting for 2 min at 0°C. 0.9 ml of the clear sonicate was used for enzymatic assay. 0.1 ml of 0.03 M substrate was added and the preparation incubated at 37°C. Aliquots of the incubation mixture were processed as described above for the intact retina. The AChE activity was linear for twenty minutes. iii) Red blood cell hemolysates: Guinea pigs were anesthetized with ether and blood was taken with a heparinized syringe by cardiac puncture. The sample was brought to 12 ml with heparinized saline and centrifuged at 3000 rpm for 5 min. The supernatant was discarded and the packed cells washed with 12 ml of cold saline followed by centrifuging as above. The procedure of washing, centrifugation and removal of supernatant was repeated. 0.1 ml of packed red blood cells was hemolyzed by transfer to 4.9 ml of H20. An equal volume of 0.1 M sodium phosphate buffer at

pH 7.8 containing 0.3 M NaC1 and 1% (w/v) Lubrol WX was then added. 0.1 ml of 3 × 10-a M acetyl1-14C/3-methylcholine was incubated with 0.2 ml of 1% hemolysate at 37°C. Hydrolysis was stopped by the addition of dioxane-resin. The AChE activity was linear with time for 16 min. Routinely, samples of blood hemolysates were incubated for 12 min and the reaction stopped with dioxane resin. iv) Brain homogenates: Guinea pigs were anesthetized, the thoracic cavity was opened, and heparinized saline was injected into the heart. The blood vessels of the brain were washed free of blood by perfusion with 0.9% NaC1 before the brain was removed from the cranial cavity. 10% homogenates of the cerebral hemispheres were prepared in 0.1 M sodium phosphate buffer at pH 7.8 containing 0.3 M NaCI and 1% Lubrol WX. Further dilution to 1% in the same buffered medium was made prior to enzyme assay. 0.2 ml of 0.03 M 14C_acetylcholine was added to 1.8 ml of a 1% cerebral homogenate in the phosphate buffered medium at 37°C. 0.2 ml samples of the mixture were transferred into 4.8 ml of dioxane resin at 4,8,12 and 16 min. The readings corrected for spontaneous hydrolysis were linear throughout the 16 rain.

3. RESULTS

3.1. Acetylcholinesterase activity of tissues from normal guinea pigs AChE activity with 95% confidence limits for twenty-eight intact retinas was 303.0 (272.8-332.2) nmoles per retina per hour. The mean percent difference for the AChE activity between left and right retinas from 16 animals was 4.8 -+ 0.8% (standard error). The mean AChE values with their 95% confidence limits for the other preparations were found to be: retinal sonicates, 3130.0 (2900-3360.0) nmoles per retina per hour; red blood cells, 20.2 (15.8-25.6) nmoles/2/al rbc/min; cerebral homogenates, 12.31 (11.65-13.01) nmoles/min/mg of tissue wet weight. 3.2. Effect of 2-PAMCI in vitro on AChE activity of retinas of satin-in toxicated guinea pigs Guinea pigs were injected with 16 mg/kg of atropine i.m. followed 5 - 1 0 min later by subcutaneous

L. W.Harris, Oximes on satin-inhibited acetylcholinesterase

injection of 50ttg/kg of sarin (approximately 1.3 LDso). At ½ hr after poisoning, the animals were sacrified, the retinas were excised, washed, and incubated with 10-4M 2-PAMC1 in tris-Ringer-glucose medium at pH 7.8 for 90 min at 37°C. The 2-PAMC1 solution was removed and the retina washed with additional cold saline which was then replaced with 1.8 ml of buffered Ringer medium. After equilibration at 37°C, substrate was added and the AChE activity determined. Some 2-PAMCl-treated preparations were sonicated just before assay; other retinas obtained from sarin-intoxicated guinea pigs were sonicated prior to incubation with 2-PAMC1. The AChE activity of these sonicates was compared with that of sonicates from unpoisoned

41

animals assayed in 10-4 M 2-PAMCI. Results are shown in table 1. AChE activity of intact retinas incubated in buffered Ringer medium for 90 minutes (group 2) was significantly higher than that in the retinas of group 1 assayed immediately after sacrifice. Enzyme activity in intact retinas incubated with 2-PAMC1 (group 3) was 43.0% of normal compared to 9.4% for those incubated in buffer alone (group 2). Intact retinas incubated with 2-PAMC1 and then assayed as a sonicate (group 5) showed 23.9% of the AChE activity of unpoisoned retinal sonicates, compared to 2.9% for sonicates of inhibited retinas not treated with oxime (group 4). The largest increase occurred following direct addition of 2-PAMC1 to the sonicate of sarin-inhibited retinal tissue (group 7).

Table 1 Effects of 2-PAMC1 in vitro on the AChE activity of retinas of sarin-intoxicated guinea pigs. Group

Condition studied

No. of prepn,

AChE activity (nmoles of substrate per retina per hr (95% confidence limits)

Percent of control*

(1)

Sarin; assayed immediately, intact

6

3.3 (0.5-6.1)

1.1

(2)

Sarin; incubated with buffer; in-

6

28.3 (18.2-38.3)

9.4

(3)

Sarin; incubated with 2-PAMCI;intact

6

130.3 (100.7 - 159.9)

43.0

(4)

Sarin; incubated with buffer intact; then sonicated

8

90.0 (59.2-120.8)

2.9

(5)

Sarin; incubated with 2-PAMC1intact; then sonicated

8

746.0 (595.0-897.0)

23.9

(6)

Satin; sonicated incubated with buffer

8

161.4 (35.4-287.4)

5.2

(7)

Sarin; sonieated, incubated with 2-PAMCI**

8

1630.4 (1621.6-1650.4)

60.4**

* When referred to control values of 303.0 and 3130 nmoles per retina/hr for intact and sonicated preparations, respectively. ** Corrected for approximately 17.0% inhibition in control sonicates incubated with 10-4 M 2-PAMCI.

42

L. W.Harris, Oximes on sarin.inhibited acetyicholinesterase

3.3. AChE recovery in the absence o f oxime treatment Guinea pigs were given 16 mg/kg of atropine intramuscularly and 5 - 1 0 m i n later injected subcutaneously with 50/ag/kg of sarin. At ½ hr, 3½ hr and 24 hr after receiving sarin, animals were anesthetized, blood samples were obtained by cardiac puncture, the brains were perfused and excised, and the retinas removed. As part of this study, the presence of uncombined inhibitor in the tissues of sarin-intoxicated guinea pigs was tested in four animals sacrificed ½ hr after poisoning. Inhibited retinal tissue was sonicated, and cerebral tissue homogenized in controls from the corresponding tissues of normal animals. However, 5 times as much experimental tissue was used as was present in the controls, so that if free inhibitor were present significant inhibition of the ChE activity of the control would have occurred during homogenization. No inhibition of the control retinal and cerebral AChE activity was found indicating absence of uncombined anticholinesterase. Changes in retinal AChE were obtained by assay of one retina as an intact preparation, and the contralateral retina as a sonicated preparation. The results are shown in table 2. The AChE in retinal and cerebral tissue showed a significant return of activity in 24 hr following poisoning; red blood cell AChE did

not. Retina ACHE, unlike that in cerebral homogenates, showed a significant recovery within 3½ hr after inhibition by sarin. 3.4. In vivo effects o f toxogonin on the AChE activity o f red blood cells and intact retinas o f sarin-poisoned guinea pigs Atropinized guinea pigs were given sarin as described above. At ½ hr after intoxication, one group of animals received 34 mg (0.088 mmole/kg of toxogonin intramuscularly. A second group was anesthetized and 20 #1 of a solution of 5 mM toxogonin (38/ag) in 0.9% NaC1 was injected into the vitreous humor of one eye with a micro-syringe equipped with a 30 gauge needle; 0.9% saline alone was injected into the contralateral eye. Unpoisoned controls that received only saline or oxime were run concurrently. After 3 hr, blood and intact retinal samples were obtained and AChE was assayed. Results are given in table 3. Intramuscular injection of 34 mg/kg of toxogonin produced marked reactivation of red blood cell AChE but only a small, albeit significant, reactivation of retinal ACHE. The reverse was found when toxogonin was injected directly into the vitreous humor. In this instance, the AChE of the intact retina was largely restored while a very small increase in rbc AChE activity occurred. Direct comparison with the results

Table 2 Recovery of AChE in vivo in some tissues of the guinea pig after sarin intoxication. Tissue 1/2 hr

AChE activity Percent of normal (95% confidence limits)* 3-1/2 hr 24 hr

Intact retina

3.44 ( 1.42-5.45)

11.8 (8.5 - 15.1)

35.4 (25.2 -42.2)

Sonicated retina

3.26 (2.10-4.42)

8.6 (8.3 -8.9)

19.9 (18.0-21.8)

Red blood cells

1.19 (0.59-1.78)

2.57 (1.93-3.22)

2.48 (0.79-3.27)

Cerebral homogenates

8.8 (5.8-11.7)

7.4 (6.2-8.5)

16.6 (15.-18.0)

* Relative recoveries are obtained from mean values of 12.31 nmoles/mg/min for cerebral homogenates; 3130.0 nM/retina/hr for retina homogenates; 303.0 nmole/retina/hr intact retina and 20.2 nmoles/2/J1 rbc/min in tissues from unpoisoned animals.

L. W.Harris, Oximes on sarin-inhibitedacetylcholinesterase

43

Table 3 Effects of Toxogonin on the AChE activity of red blood cells and intact retinas of satin-poisoned guinea pigs. AChE activity nmoles of t4C'acetyl-#-methylcholine RBC

(95% confidence limits) Retina

No satin toxogonin intravitreous

20.2(15.8-24.6)

389.0(289.6-488.4)

Sarin, no treatment

0.5(0.4-0.7)

35.7(25.7-45.7)

Satin toxogonin, i.m.

10.5(9.6-11.4)

65.2(55.0-74.0)

Sarin, toxogonin intravitreous

1.2(1.0-1.4)

Conditions

236.0(193.4-278.6)

obtained by intramuscular injection cannot be made because the dose of oxime given by intravitreous injection was a minute fraction (approximately 0.25%) of that given systematically. 3.5. In vivo reactivation of retinal and cerebral AChE following administration of oximes to sarin-poisoned guinea pigs Guinea pigs were given 16 mg/kg of atropine intra-

muscularly, then 50/~g/kg of sarin subcutaneously. One-half hour after poisoning, 0.044 mmole/kg of 2-PAMC1 or toxogonin (equivalent to 7.5 mg/kg and 17.0 mg/kg respectively) was injected intramuscularly. For purposes of comparison, 0.044 mmole/kg and 0.088 mmole/kg of the tertiary oxime, monoisonitrosoacetone (MINA) was also injected into the sarinpoisoned guinea pigs. The animals were sacrificed 3 hr after treatment with the oximes and the AChE of

Table 4 Reaction of retinal and cerebral AChE followingadministration of oximes to sarin-inotxicated guinea pigs. Oxime used

Dose (mmoles/kg)

2-PAMCI

0.044

Toxogonin

0.044

MINA

None

0.088

Intact retina

AChE activity Percent of normal* (95% confidence limits) Sonicated Cerebral retina homogenate

35.7 (30.3-41.1)

11.1 (10.3-11.9)

11.1 (10.6-11.6)

21.5

11.3

10.4

(18.1-24.5)

( 10.2 - 12.4)

(8.9-11.8)

17.7 (I 3.8- 21.6)

1 I.I (9.6 - 12.6)

13.7 (I 2.7 - 14.8)

11.8

8.6

7.4

(8.5 - 15.1 )

(8.3 - 8 . 9 )

(6.2-8.5)

* When referred to control values of 303.0 nmoles per intact retina/hr, 3130 nmoles per sonicated retina/hr and 12.31 nmoles/ min/mgof cerebral tissue.

44

L. W.Harris, Oximes on sarin-inhibited acetylcholinesterase

intact and sonicated retinas and homogenates of the cerebral hemisphere were assayed. Controls not receiving oxime treatment were assayed concurrently. No reactivation of inhibited AChE was obtained following treatment with 0.044 nmole/kg of MINA. Other results, presented in table 4, show 2-PAMC1 to be more effective than Toxogonin in reactivation of sarin-inhibited AChE in the intact retina. However, 0.044 mmole of 2-PAMC1 appeared no more effective than the molar equivalent dose of toxogonin when reactivation was assayed in retinal sonicates or cerebral homogenates. AChE in these preparations showed a very small but statistically significant increase (p = 0.05) over that of untreated poisoned controls. Table 4 also shows that the percentage recovery of AChE in retinal sonicates approximately parallels that in cerebral homogenates. Injection of MINA at a molar equivalent dosage two-fold greater than that used for the pyridinium aldoximes reactivated inhibited AChE in retinal sonicates and cerebral homogenates.

4. DISCUSSION Recovery of AChE in nerve tissue after inhibition by sarin in vivo may result from dephosphorylation leading to regeneration of the original enzyme (Fleisher et al., 1970) and by synthesis of new enzyme (Blaber and Creasey, 1960). Both mechanisms may have contributed to the return of enzyme activity in the tissue of our experimental animals. A major aim in our experiments was comparison of enzyme activity in intact retina with sonicates of the contralateral retina and cerebral homogenates of animals undergoing recovery from sarin intoxication. In this regard, the lower values found for recovery of AChE in retinal sonicates and cerebral homogenates than were obtained for intact retinal tissue (tables 2 and 4) might have resulted from he presence of uncombined sarin during homogenization. However, retinal and cerebral tissues from animals sacrificed ½ hr after poisoning, when the possibility of free inhibitor was greatest yielded no evidence for the presence of uncombined sarin. Therefore the greater reactivation found in intact compared to sonicated retinas (table 4) may mean that the quaternary oxime is more effective in reactivating

superficially situated AChE than that located within the interior of the tissue (tables 1 and 4). Sonication leading to the determination of the AChE of the total tissue yields a value affected by the lesser degree of reactivation of the internally situated enzyme. Conversely, disintegration of membrane barriers before incubation with oxime should facilitate reactivation in vitro. This was confirmed by our finding that maximal reactivation of inhibited retinal enzyme resulted from addition of 2-PAMC1 to the inhibited sonicated preparation (table 1, group 7).Furthermore, the greater reactivation of inhibited AChE shown in the intact retina after treatment with 2-PAMC1 in vivo than in animals treated with an equimolar dose of toxogonin was not evident when enzyme activity was measured in sonicates or cerebral homogenates (table 4). This suggests that assay of enzyme activity in intact tissue may be more useful than assay of sonicated or homgenized tissue for discriminating the effectiveness of quaternary oximes for the reactivation of inhibited AChE following administration of these compounds to animals poisoned with organophosphates. Of the three in vitro procedures under which reactivation by 2-PAMC1 was studied, the one in which oxime was added to inhibited intact retinas, followed by removal of oxime by washing, and assay of enzyme activity in intact tissue (group 3, table 1) most closely resembles the conditions prevailing in tissues of the surviving guinea pig. The similarity between the values for reactivation obtained under this protocol (table 1) and those for intact retinas from sarin-intoxicated animals treated with 2-PAMC1 in vivo (table 4) confirms this expectation. In this connection, earlier studies showed that treatment of intact organophosphate-inhibited muscle tissue with quaternary oximes followed by assay of enzyme activity in the intact preparation yielded values for AChE recovery that were more closely correlated with a return toward normal neuromuscular function than those obtained with homogenized muscle (Fleisher et al., 1958, 1960). Rose and Glow (1965) previously reported that intravitreous injection of 1 mg of TMB-4 yielded almost complete protection, while intramuscular administration of 50 mg/kg of the oxime yielded no protection against inactivation of retinal AChE by DFP. In our study, 0.02ml of 5 mM (38/.tg) of

L.W.Harris, Oximes on satin-inhibited acetylcholinesterase

toxogonin injected into the vitreous humor resulted in a recovery of sarin-inhibited retinal AChE from 11.8% to 60.7% of normal (table 3). In contrast, 34 mg (0.088 mmole)/kg of the same oxime administered intramuscularly increased retinal AChE less than two-fold but brought about a more than 20-fold increase in the red blood cell enzyme activity (table 3). A blood-retinal barrier limiting passage of the quaternary reactivator is clearly indicated by these findings. Less reactivation of AChE was found in intact retinas of sarin-intoxicated animals given 0.088 mmole/kg of MINA than in those given half as much pyridinium aldoximes (table 4). This was unexpected, since MINA as an uncharged compound should have penetrated the blood-retinal barrier more readily than the quaternary ammonium reactivators. The observation by Davies and Green (1956) that the reactivation rate constant of MINA for sarin-phosphonylated AChE is only one-tenth that of 2-PAMC1 may explain the discrepancy. AChE in retinal tissue appeared to undergo a greater reduction after sarin intoxication than AChE in cerebral tissue (table 2). However, the percent recovery from sarin inhibition of retinal sonicates closely approximated that obtained with cerebral homogenates (table 2). The results suggest that the recovery of AChE activity in sonicates of retinal tissue may reflect that in cerebral homogenates. This possibility is also supported by the very close agreement between the percent recovery in sonicated retinas and cerebral homogenates from sarin-intoxicated animals treated with pyridinium aldoximes (table 4). Appreciable recovery of AChE in intact retina occurred within 3-½ hr after poisoning with sarin (table 2). This time interval approximates that noted by Meeter and Wolthuis (1968) for spontaneous recovery of respiration and neuromuscular transmission in sarin-poisoned rats. Unlike the enzyme in retinal and cerebral tissue, inhibited red blood cell AChE showed no recovery within 24 hr after sarin intoxication (table 2), a result which appears not to be representative of that in the tissues of the surviving animals. The greater reactivation of AChE obtained in intact retinas from sarin-intoxicated guinea pigs treated with 2-PAMCI than in those treated with toxo-

45

gonin (table 4) implies that the former oxime might be the better antidote for sarin poisoning in this species. Recently, we observed the opposite to be true (Fleisher et al., 1970) suggesting that toxogonin may counteract the toxic effects of the anticholinesterase by actions other than reactivation of inhibited ACHE. This interpretation is consistent with the anti-acetylcholine property shown by the closely related bisquaternary oxime, TMB-4, (Hobbiger and Sadler, 1959; Fleisher et al., 1965). These findings, and the very small increase in the AChE activity of cerebral tissue homogenates following treatment with the pyridinium aldoximes (table 4), indicate that the correlation between reactivation of the inhibited enzyme in the central nervous system and the antidotal potency of the quaternary oximes is poor. The report that intracisternal injection of N-methylpyridinium 2-aldoxime methane sulphonate (P2S), which acts like 2-PAMCI, fails to reverse the respiratory paralysis in dogs produced by sarin (Brown, 1960), further supports this conclusion.

ACKNOWLEDGEMENTS The authors wish to express their appreciation to Dr. George M. Steinberg and to Mrs. Marion P. Royston for their comments on the manuscript.

REFERENCES Blaber, L.C. and N.H. Creasey, 1960, The mode of recovery of ChE activity in vivo after organophosphorus poisoning. II. Brain ChE, Biochem. J. 77,597. Brown, R.V., 1960, The effects of intracisternal satin and pyridine 2-aldoxime methyl methanesulphonate in anesthetized dogs, Brit. J. Pharmacol. 15,170. Cohen, E.M. and H. Wiersinga, 1960, Oximes in the treatment of nerve gas poisoning. II, Acta Physiol. Pharmacol. Need. 9, 276. Davies, D.R. and A.L. Green, 1956, The kinetics of reactivation, by oximes, of cholinesterase inhibited by organophosphorus compounds, Biochem. J. 63,529. Erdman, W.D., 1965, Vergleichende Untersuchungen uber das Penetrationsvermogen einiger esterasereaktivierender Oxime in das zentrale Nervensystem, ArzneimitteI-Forsch. 15,135. Fleisher, J.H., J.P. Corrigan and J.W. Howard, 1958, Potentiation of the response of frog rectus muscle to acetylcholine by isopropyl methylphosphonofluoridate and its

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L. l¢.Harris, Oximes on satin-inhibited acetylcholinesterase

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