TOXICOLOGY AND APPLIED PHARMACOLOGY 98,128- 133 ( 1989)

The Relationship between Oxime-induced Reactivation of Carbamylated Acetylcholinesterase and Antidotal Efficacy against

Carbamate Intoxication’

LARREL W. HARRIS, BRIAN G. TALBOT, WILLARD J. LENNOX, AND DANA R. ANDERSON

U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010542S

Received March 28,1988; accepted November 18, I988

The Relationship between Oxime-Induced Reactivation of Carbamylated Acetylcholinester- ase and Antidotal Efficacy against Carbamate Intoxication. HARRIS, L. W., TALBOT, B. G., LENNOX, W. J., AND ANDERSON, D. R. (1989). Toxicol. Appl. Pharmacol. 98, 128-133. The efficacy ofthe oximes pyridinium-2-aldoxime methochloride (2-PAM) and I-[[[(rl-aminocarbo- nyl)pyridinio]methoxy]methyl]-2-[(hydrox~mino)methyl]p~dinium dichloride (HI-6), in combination with atropine (At), against lethality by either carbaryl (CA) or physostigmine (Phy) was investigated in rats. The protection by At, 8 mgjkg, iv, against CA intoxication was reduced by 2-PAM (22 mg/kg, iv) and HI-6 (50 mgjkg, iv) from a protective ratio (PR) of 6.6 to 3.5 and 2.3, respectively. However, in Phy-intoxicated rats, the administration, iv, of At alone, At + 2- PAM, or At + HI-6 at I min following Phy provided good protection and resulted in PRs of 7.2, 8.8, and 23.3, respectively. In experiments on decarbamylation of inhibited acetylcholinesterase (AChE), HI-6 and 2-PAM accelerated (p c 0.05) the decarbamylation of Phy-inhibited AChE in vitro, and HI-6 decreased (p < 0.05) the inhibition of whole blood AChE in Phy-intoxicated rats. These findings show that the protection was increased substantially by the use of either 2- PAM or HI-6 against Phy-induced lethality, whereas the use of oximes against carbaryl poison- ing was contraindicated. Furthermore, even though CA and Phy are both N-methyl carbamates, the data indicate that there is no adverse interaction between 2-PAM or HI-6 and Phy. o 1989

Academic Press. Inc

There is controversy in the literature on the use of pyridinium oximes (e.g., pyridinium- 2aldoxime methochloride (2-PAM)) as anti- dotes against carbamate intoxication (Car-

’ In conducting the research described in this report, the investigators adhered to the “Guide for the Care and Use of Laboratory Animals,” NIH Publication No. 85- 23, revised 1985. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. Part of this work was presented in August 1987 at the U.S. Army Medical Research and Development Command’s Sixth Medical Chemical Defense Bioscience Review at The Johns Hop- kins University Applied Physics Laboratory, Colum- bia, MD.

penter et al., 196 1; Natoff and Reiff, 1973; Reese, 1984). Although oximes, such as 2- PAM, are effective against organophospho- rus (OP) intoxication, they are not recom- mended for use against carbamate intoxica- tion (Reese, 1984). It has been reported that 2-PAM exacerbates the toxicity of carbaryl (1-naphthyl N-methylcarbamate). The ad- verse effect by 2-PAM in carbaryl poisoning has been observed in species other than the rat (Aleksashina, 1969) and may have con- tributed to the fate of a 39-year-old man (Far- ago, 1969). These observations may have contributed to the recommendation that, in general, Z-PAM be contraindicated as an an- tidote for carbamate insecticide poisoning

0041-008X/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

128

OXIME TREATMENT OF CARBAMATE INTOXICATION 129

(Reese, 1984). However, this contraindica- tion may apply to only a limited number of carbamates since, to our knowledge, poison- ing by carbaryl [an N-methylcarbamate like physostigmine (Phy)] is the only reported condition in which 2-PAM treatment poten- tiated the toxicity. In fact, we recently showed that l-[[[(4-aminocarbonyl)pyridinio]meth- oxylmethyl-2-[(hydroxyimino)methyl]pyri- dinium dichloride (HI-6) in conjunction with atropine was highly effective against pyridos- tigmine intoxication (Harris et al., 1987). The report and formal judgement by Reese ( 1984) have raised the question of whether carba- mate and oxime (e.g., 2-PAM) should be given at close intervals, as they have been ex- perimentally, in carbamate pretreatment and subsequent oxime and atropine therapy against nerve agent intoxication (Gordon et al., 1978; Lennox et al., 1985; Leadbeater, 1985). In an effort to answer this question, we studied the effects of 2-PAM and/or HI-6 on spontaneous decarbamylation of Phy-inhib- ited erythrocyte acetylcholinesterase (AChE) in vitro and in vivo and, in conjunction with atropine, on Phy- and carbaryl-induced tox- icity in rats.

METHODS

Male Sprague-Dawley rats (200-280 g) from Charles River Laboratories were used. The animals were housed singly in temperature/humidity-controlled quarters and were maintained on a 12-hr light-dark cycle with lights on at 0600. Food and water were provided ad libitum.

Therapeutic drugs were obtained from the repository at Walter Reed Army Institute of Research. The original sources and purities of the drugs were atropine sulfate, Sigma Chemical (St. Louis, MO), 99.0%; 2-PAM Cl, Ay- east Laboratory (St. Laurent, Quebec), 99.4%; and HI-6, SRI International (Menlo Park, CA), 99.8%. Physostig- mine salicylate was obtained from O’Neill, Jones, and Feldman (St. Louis, MO) and carbaryl, technical grade (assay 99.0%), was obtained from Union Carbide Corp. (New York, NY). Polyethylene glycol200 (PEG 200) was purchased from Sigma. Physostigmine and the antidotal drugs were dissolved in 0.9% NaCl (saline) solution. Car- baryl was dissolved in PEG 200.

To assess the effects of oximes on decarbamylation, heparinized rat whole blood (5 ml) was incubated with

Phy ( IOe6 M, final concentration), carbaryl(5 X 10m6 M,

final concentration), or saline for 30 min at 37°C. Excess inhibitor was removed by washing erythrocytes four times (12 ml/wash) with cold, heparinized saline. Ali- quots (0.5 ml) of packed erythrocytes were added to I .O ml of phosphate buffer preparation (PBP), (pH 7.3) alone, or with a final oxime (2-PAM or HI-6) concentra- tion of 2 X 10m4 M. These mixtures were incubated at 37°C and aliquots were assayed after 0, 15, 30, 45, and 60 min of incubation for AChE activity using the mdio- metric method (Siakotos et al., 1969) and acetyl-B-meth- ylcholine Cl as the substrate. Details of the preparation of PBP and ofthe methodology for oxime-induced decar- bamylation were recently reported (Harris et al., 1987). The data were analyzed using least squares regression techniques to obtain half-times for decarbamylation. The maximum F ratio test (Bliss, 1967) was applied to aid in judging acceptability for pooling of variances of half-time estimates within treatment groups. A least sig- nificant difference (Bliss, 1967) was computed and used to judge differences among mean half-times for treat- ment groups.

For in viva studies on decarbamylation, rats were in- jected with Phy [0.075 mg/kg, intravenously (iv)] and then administered HI-6 (50 mg/kg, iv) or vehicle (twice distilled water, 1 ml/kg, iv) 9.5 min later. Whole blood AChE activity was monitored (as described above) before and at 5,9, 15,20,40,60, and 80 min after Phy adminis- tration. The AChE activity at 9 min was subtracted from the AChE activity at subsequent observation times and the resulting changes (decreases) in percentage whole blood AChE inhibition were plotted as a function of time. The difference between results for vehicle- and ox- ime-treated animals at each observation time was tested for significance using Student’s t test.

In initial antidotal efficacy studies in rats, range finding experiments were conducted to determine the intramus- cular (im) doses of Phy and carbaryl to use in full-scale efficacy studies. These experiments revealed that because of the low toxicity and solubility of carbaryl, it would be necessary to administer carbaryl intraperitoneally (ip); to maintain consistency, Phy was administered by the same route. In the full studies, dosages of carbamate were spaced at 0.15 log intervals to bracket the LD50. Ther- apy, consisting of atropine (8 mg/kg, iv) alone and to- gether with 2-PAM Cl (22 mg/kg, iv) or HI-6 (50 mg/kg, iv), was administered 1 min after carbamate injection. The iv administration of therapy was chosen because it is the route most often used in hospitals in the treatment of insecticide poisoning. The lethality data were analyzed by probit analysis (Finney, I97 I), using both parallel and nonparallel line procedures. The logs of the LDSO values (one pair per treatment group) generated in the study of Phy were subjected to a one-way analysis of variance (ANOVA). The square root of the error mean square in the ANOVA was used in subsequent significance testing

130 HARRIS ET AL.

TABLE 1

SPONTANEOUS AND OXIME-INDUCED DECARBAMY- LATION OF PHYSOSTIGMINE-INHIBITED RAT ERYTHRO- CYTE ACETYLCHOLINESTERASE

Half-time (mm)” Treatment N Mean (95% confidence limits)

Control 5 45.0 (36.0-55.0) 2-PAM 5 28.0 (19.0-36.0) HI-6 5 15.0 (8.9-22.0)

Op < 0.05 between any two groups.

of differences between the mean values of log LD50 for the treatment groups; it was also used in setting the 95% confidence limits for each mean log LDSO. Protective ra- tios (PR = LD50 with therapy/LDSO without therapy) were determined from the LD50 values.

RESULTS

The effects of HI-6 and 2-PAM on sponta- neous decarbamylation of Phy-inhibited erythrocyte AChE are indicated in Table 1. Clearly, both oximes accelerated decarbamy- lation, but the effect was more pronounced with HI-6. The half-times for spontaneous decarbamylation of carbaryl-inhibited eryth- rocyte AChE (not shown) were variable and extremely long, suggesting that residual car- baryl might be present in the incubation me- dia; however, addition of 2-PAM virtually stopped decarbamylation.

The data in Fig. 1 illustrate the effective- ness of HI-6 in accelerating spontaneous decarbamylation in Phy-intoxicated rats. Clearly, the blood AChE activity was signifi- cantly (p < 0.05) elevated in Hid-treated rats when compared with corresponding controls.

The effectiveness of atropine and oxime therapy against intoxication by Phy is indi- cated in Table 2. The data show that atropine sulfate provided outstanding protection (PR = 7.2) against Phy intoxication and that, in combination with 2-PAM, some additional protection may have been provided as indi- cated by the 22% increase in PR. Moreover,

ml

*p < 0.05 COMPARED To WATER l’Nf3.m CONTROL

0 20 r) 40 50 Bo 70 80

TIME (MINUl’ES)

FIG. 1. Effects of HI-6 on whole blood AChE activity in physostigmine-treated rats. Rats received Phy (0.075 mg/kg, iv) followed by HI-6 or vehicle 9.5 min later. The AChE activity at 9 min was subtracted from the AChE activity at subsequent observation times and the result- ing changes (decreases) in percentage whole blood AChE inhibition were plotted as a function of time.

when HI-6 was combined with atropine ther- apy, the PR was further elevated to 23.3.

The data in Table 3 show that the LD50 values for carbaryl and for atropine plus car- baryl were less (i.e., lethality was increased) in the presence of 2-PAM treatment. Further- more, the antidotal benefit from atropine was

TABLE 2

THE EFFJXHVENESS OF ATROPINE AND OXIME THERAPY IN RATS INTOXICATED WITH F%YSOSTIGMINE

Treatment LD50, mg/kg, ip Protective

(95% confidence limits)” ratio

None (vehicle) 2.39 (1.66-3.49) 1.0 Atropine (At) 17.2 (11.8-25.1)* 7.2 At + 2-PAM 21.0 (14.4-30.7)” 8.8 At + HI-6 55.7 (38.2-81.3)’ 23.3

a Antilog of mean log LD50 f A, where A = t X SE; t = 2.7764 for 4 degrees of freedom and 2p = 0.05, and the SE is the square root of the error mean square from a simple one-way ANOVA of the set of log LD50 values.

*p < 0.05 compared with vehicle. ‘p < 0.05 compared with all others.

OXIME TREATMENT OF CARBAMATE INTOXICATION 131

TABLE 3

EFFECTIVENFSS OF ATROPINE AND OXIME THERAPY AGAINST CARBARYL INTOXICATION IN RATS

Treatment LD50, mg/kg, ip Protective

(95% confidence limits)“ ratio

None (vehicle) 69.9 (50.0-105.0) 1.0 2-PAM 39.4 (28.6-57.1)b 0.6 Atropine (At) 460.0 (335.0-669.0)’ 6.6 At + 2-PAM 244.0 ( 180.0-339.0)d 3.5 At + HI-6 164.0 ( 119.0-22 1 .O) 2.3

” Each LD50 value utilized 25 animals (5 groups X 5 animals/group). Probit analysis assuming parallel lines (equal regression coefficients) was used.

b Probit analysis assuming parallel lines was used for testing for statistical significance; Q < 0.05 compared with vehicle.

‘p < 0.05 compared with all others. dp i 0.05 compared with 2-PAM, vehicle, and at-

ropine.

also adversely affected by HI-6 administra- tion, because the PR was reduced by HI-6 from 6.6 (atropine alone) to 2.3.

DISCUSSION

Pytidine-2-aldoxime is commonly used in combination with atropine as an antidote for the treatment of organophosphorus intoxica- tion (Kewitz et al., 1956; Harris et al., 1969). In recent years, atropine and 2-PAM therapy has been combined with carbamate pretreat- ment to protect against multiple lethal doses of soman (Lennox et al., 1985). However, since 2-PAM has been reported to reduce the protection provided by atropine sulfate against carbaryl poisoning in dogs, oximes have generally been excluded as antidotes for carbamate insecticide intoxication (Kuhr and Dorough, 1976). The reported adverse interaction between carbaryl and 2-PAM has raised questions of whether carbamates (e.g., pyridostigmine or Phy) should be given at close intervals with oximes as pretreatment/ therapy against OP intoxication. This is espe-

cially critical for Phy since, like carbaryl, it is a N-methylcarbamate, and the inhibited AChE would be expected to retain a cova- lently bound moiety that is identical to that of carbaryl-inhibited AChE (Wilson et al., 1960: O’Brien, 1965, 1967, 1968). Thus, assuming that both carbamates are toxic due to their anti-AChE activity, the intoxicated animals should respond similarly to conventional therapy for AChE inhibition. Contrary to what might be expected, carbaryl- and Phy- intoxicated animals respond differently to at- ropine/oxime therapy (compare Tables 2 and 3). Atropine therapy is highly, and equally, effective against intoxication by both carba- mates. In contrast, the effectiveness of atro- pine is augmented by oxime against Phy-in- duced intoxication (Table 2) and reduced by oxime against intoxication by carbaryl (Table 3). These findings, suggesting an adverse in- teraction between carbaryl and oxime, are consistent with those in the literature (Car- penter et al., 196 1; Farago, 1969; Boskovic et al., 1976). It is not presently known why 2- PAM and HI-6 potentiate the toxicity of car- baryl. Clark et al. ( 1983) attempted to eluci- date the mechanism and found that it was not due to the formation of a carbamylated ox- ime; they did find that several oximes, includ- ing 2-PAM and HI-6, enhanced the inhibi- tion of both eel and human serum cholines- terase. Some oximes may act as allosteric effecters of the AChE in carbaryl poisoning. Another possibility is that 2-PAM might re- duce the rate of spontaneous decarbamyla- tion of the inhibited AChE (Harris et al., 1986).

Oximes have been reported to be ineffec- tive in accelerating the rate of decarbamyla- tion (Wilson et a/., 1960; O’Brien, 1968). However, in our studies (Table 1) both 2- PAM and HI-6 were effective (p < 0.05) in accelerating decarbamylation of Phy-inhib- ited rat erythrocyte AChE. These findings also agree with those reported earlier on ox- ime-induced decarbamylation of human erythrocyte AChE inhibited by pyridostig-

132 HARRIS ET AL.

mine (Harris et al., 1985) and with those in a recently published report by Dawson and Poretski ( 1985), who used oximes to acceler- ate the decarbamylation of neostigmine-in- hibited AChE. The precise mechanism of ox- ime-induced decarbamylation is not pres- ently known. However, by analogy with the well-documented mechanism for the reacti- vation of phosphylated cholinesterases (Wil- son and Ginsburg, 1955; Eto, 1974), it may be that the nucleophilic oxime is directly dis- placing the carbamyl moiety at the active site of the inhibited enzyme. Also, binding to an ancillary site on the AChE and exerting allo- steric effects is a possibility, since structurally similar compounds, which are devoid of a nucleophilic oxime group, are capable of ac- celerating decarbamylation (Dawson and Poretski, 1985).

In summary, the toxicity data in rats show that atropine sulfate is an extremely effective antidote against intoxication by carbaryl or Phy. Also, the data show that oximes are ca- pable of accelerating decarbamylation of Phy-inhibited erythrocyte AChE. Further- more, the data suggest that 2-PAM and HI-6 are contraindicated and should not be used in cases of intoxication by carbaryl. However, no evidence was found that either 2-PAM or HI-6 increases the toxicity of physostigmine. Finally, the experimental evidence gathered provides a rational basis for the use of carba- mate pretreatment in conjunction with the standard therapy against powerful OPs (e.g., soman) while taking into account the contra- indicated use of 2-PAM and HI-6 in poison- ing by carbaryl.

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