Toxicology Letters, 62 (1992) 127-l 37 0 1992 Elsevier Science Publishers B.V. All rights reserved 0378-4274/92/S 05.00

127

TOXLET 02750

Studies of the amplification of carbaryl toxicity by various oximes*

Claire N. Lieske, James H. Clark, Donald M. Maxwell, Lamar Dean Zoeffel and Walter E. Sultan US Army Medical Research Insritute of Chemical Defense, Aberdeen Proving Ground, MD (USA)

(Received 11 February 1992) (Accepted 3 April 1992) Key words: Carbaryl; Oximes; Acetylcholinesterase;

Human serum cholinesterase

SUMMARY The administration of 2-pyridine aldoxime methyl chloride (2-PAM Cl) is a standard part of the regimen for treatment of human overexposure to many organophosphorus pesticides and nerve agents. However, some literature references indicate that poisoning by carbaryl (I-naphthyl N-methyl carbamate), an insecticide in everyday use, is aggravated by the administration of 2-PAM Cl. This effect has been reported in the mouse, rat, dog and man. We have found that the inhibition of both eel acetylcholinesterase (eel AChE, EC 3.1.1.7) and human serum cholinesterase (human BuChE, EC 3.1.1.8) by carbaryl was enhanced by several oximes. Based on 95% confidence limits the rank order of potentiation with eel AChE was TMB-4 = Toxogonin > HS-6 = HI-6 > 2-PAM Cl. By the same criterion, the rank order of potentiation with human BuChE was TMB-4 > Toxogonin > HS-6 = 2-PAM Cl. Carbaryl-challenged mice also reflected a potentiation since TMB-4 exacerbated the toxicity more than 2-PAM Cl. Our hypothesis is that certain oximes act as allosteric effecters of cholinesterases in carbaryl poisoning, resulting in enhanced inhibition rates and potentiation of carbaryl toxicity.

INTRODUCTION

Attempts to understand the physico-chemical inhibition relationships between enzymes and their inhibitors and the therapeutic manipulations of these relationships Correspondence to: Claire N. Lieske, Applied Pharmacology Branch, USAMRICD, Aberdeen Proving Ground, MD 21010, USA. *In conducting the research described in this report, the investigators adhered to the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (NIH Publication No. 86-23, 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.

128

have been the focus of many researchers for nearly 50 years. One group of reactivators of inhibited choline&erase developed during the decade of the 1950s was the oxime series. In 1955, Wilson and Ginsburg [l] in the United States and Childs et al. [2] in England researched and published independently on the efficacy of the compound 2-pyridine aldoxime methyl iodide (ZPAM I) as a reactivator of phosphylated cholinesterases. This compound still remains the model upon which new antidotes are designed [3,4]. Today 2-pyridine aldoxime methyl chloride (2-PAM Cl), a more soluble 2-PAM salt, is used as a standard part of the regimen for treatment of overexposure to many insecticides and for nerve agent poisoning in the Armed Forces of the United States [5,6]. The structure of this oxime, which is the only acetylcholinesterase reactivator currently available for general use in the United States, is included in Figure 1. However, there are at least five literature reports that poisoning by the carbamate carbaryl (1-naphthyl N-methyl carbamate) is exacerbated by the administration of 2-PAM [7-l 11.It has also been reported that the toxicity of carbaryl is significantly increased by the oxime Toxogonin (l,l’-(oxydimethylene)bis(4-formylpyridinium) dioxime dichloride) [9,10,12]. This oxime is widely used in the European medical community. In our work we addressed two questions concerning ~-PAM’S enhancement of the toxicity of carbaryl. First, as proposed by Sterri et al. [12], does the phenomenon result from a carbamylated oxime produced by a 2-PAM/carbaryl reaction? Second, does the phenomenon result from a cholinesterase perturbation? Limited studies on the former produced no evidence suggesting formation of a carbamylated oxime. We did find, however, that the in vitro inhibition of both eel acetylcholinesterase (eel AChE, EC 3.1.1.7) and human serum cholinesterase (human BuChE, EC 3.1.1.8) by carbaryl was enhanced by several oximes. Concurrent with our enzymatic investigations the effects of 2-PAM and TMB-4 on carbaryl toxicity in mice were ascertained. Our findings in these in vitro and in vivo studies, and their possible relationship to the enhancement of carbaryl toxicity by 2-PAM, are the subject of this paper. MATERIALS AND METHODS

General Zn vitro studies.

Solvents and reagents were analytical grade. The zwitterionic buffer 3-(N-morpholino)propanesulfonic acid (MOPS) was used in the pH 7.60 inhibition studies. The buffer was 0.10 M and contained 0.01 M Mg’*, 0.002% NaN,, and 0.01% bovine serum albumin. Each pH determination was made at 25.O”C with a Beckman Model 70 digital pH meter. Oximes were obtained from the Drug Assessment Division of USAMRICD. Carbaryl (1-naphthyl N-methylcarbamate) (Lot No. 6-l 39) was purchased from Chem Service, Inc., West Chester, PA. It had a purity of >98% by NMR. In vivo studies. 2-PAM Cl, USP grade, was obtained from Ayerst Laboratories, Inc., New York, NY. 1,1’-(Trimethylene)bis(4-formylpyridinium)dioxime dichloride (TMB-4 Cl) (Lot No. 040857) was obtained from Aldrich Chemical Company, Mil-

129

waukee, WI. Atropine sulfate (Lot No. 352) was acquired from C.H. Boehringer & Son, Ingelheim, Germany. The carbaryl source was as noted above. Eel acetylcholinesterase for inhibition studies Eel acetylcholinesterase (eel AChE, EC 3.1.1.7) was purchased from Sigma Chemical Company. It was obtained as a lyophilized powder, 1130 units/mg solid, containing approx. 15% buffer salts. Enzyme concentrate was prepared by dissolving 4.5 mg of this powder in 0.80 ml of a previously boiled solution containing 0.225 M KCl, 0.10% gelatin, and 0.02% sodium azide. Enzyme stock solutions were prepared by adding 10.0 ,ul of the enzyme concentrate to 2.0 ml of the pH 7.60 MOPS buffer. An enzyme control solution was made by a dilution of 0.33 ml of the stock solution into a 25.0 ml volumetric flask which was brought up to volume with MOPS buffer. A representative activity of such a preparation was 0.442 absorbance units/min at 272.5 nm when assayed at 25.O”C with the substrate phenyl acetate [13]. Assay concentration of the phenyl acetate substrate was 3.9 x 10e3 M. Human serum cholinesterase for inhibition studies Human serum cholinesterase (human BuChE, EC 3.1.1.8) was purchased from Sigma Chemical Company. It was obtained as a lyophilized powder, 5 units/mg protein, or 3.7 units/mg solid. Enzyme stock solution was prepared by dissolving 30 mg in 10.0 ml (volumetric flask) of pH 7.60 MOPS buffer. The enzyme stock solution was diluted 1: 10 in MOPS buffer to provide the working enzyme concentration. A representative working solution showed an activity of 0.300 absorbance units/min at 412 nm when assayed at 25.O”C with the substrate S-butyrylthiocholine iodide (Ellman procedure) [14]. Assay concentration of the substrate was 5.5 x lo-‘M. Inhibition of eel acetylcholinesterase with carbaryl or oxime/carbaryl combinations Enzymatic activities were determined at 272.5 nm (25.O”C) using a Beckman 5270 spectrophotometer. For these assays, the substrate was 3.9 x 10m3M phenyl acetate. In each case, a 25.0 ml volume of enzyme-buffer solution was equilibrated at 25.O”C for 15 min before the addition of carbaryl. At zero time 50.0 ~1 of 2.5 x 10m4M carbaryl in acetonitrile were added to the enzyme-buffer solution to yield an inhibitor concentration of 5 x lo-’ M. Aliquots (1 .OOml) of the inhibited enzyme were removed from the flask and placed in 1 ml quartz cuvettes at 5.0-min intervals and assayed with phenyl acetate substrate. Absorbance change was recorded on the spectrophotometer using a chart speed of 5.0 in/min. The natural logs of the activity at the 5.0-min intervals were plotted against time. A linear regression calculation was performed on the linear part of the data to give the tX for inhibition. When the assay used an oxime in conjunction with carbaryl, the oxime of choice was added to the enzyme-buffer solution 10 min prior to the carbaryl. Each oxime was made up in doubly distilled water in stock concentrations of either 2.5 x lo-’ M or 2.5 x 10e3 M. The final concentration of the oxime in the enzyme-buffer solution was either 5 x lo-’ or 5 x 10m6M. Results of these studies are shown in Table I.

130

Inhibition of human serum cholinesterase with carbaryl or oximekarbaryl combinations Enzymatic activities were determined at 412 nm (25.O”C) using a Beckman 5270 spectrophotometer and the Ellman procedure [ 141.The Ellman’s reagent consisted of 1.OOml of S-butyrylthiocholine iodide (0.7225 g in 25.0 ml of 0.10 M MOPS buffer), 2.0 ml of 5.5’-dithio-bis(2-nitrobenzoic acid) (0.1945 g in 50.0 ml of 0.10 M MOPS buffer), and 52.0 ml of 0.10 M MOPS buffer of pH 7.60. The solution was freshly prepared every 4 h since it is unstable. For the inhibition studies a 1:lO dilution of human BuChE stock (kept frozen) solution was made. At zero time 20.0 ,ul of 2.5 x 10e3 M carbaryl (in acetonitrile) were added to the 10.0 ml of the enzyme-buffer solution (25.O”C) to yield an inhibitor concentration of 5 x 10m6M. At 5.0-min intervals, aliquots (0.10 ml) of the enzyme-buffer solution were removed from the flask and added to cuvettes containing 2.9 ml of Ellman’s reagent (25.O”C). Absorbance change was recorded on the spectrophotometer using a chart speed of 5.0 in/min. The natural logs of the activity at the 5.0-min intervals were plotted against time. A linear regression calculation was performed on the linear part of the data to give the tyzfor inhibition. When the assay used an oxime in conjunction with carbaryl, the oxime of choice was added to the enzyme-buffer solution 10 min prior to the carbaryl. Each oxime was made up in doubly distilled water in stock concentrations of 2.5 x 10m3M. The final concentration of the oxime in the enzyme-buffer solution was 5 x 10m6M. Results of these studies are shown in Table II. Mouse handling and housing Healthy male Crl:CD-1 (1CR)BR Swiss mice (Mus musculus) weighing 18 to 25 g were used in this protocol. The mice were housed in groups of ten in polycarbonate shoebox cages (Lab Products, Maywood, NJ) on hardwood chip contact bedding (Beta-Chip; Northeastern Products Corp., Warrensburg, NY) with complete cage changes twice a week. They were provided rodent ration (Zeigler Bros., Gardners, PA), as appropriate, and tap water ad libitum. The mice rooms were maintained at 20_22”C, relative humidity of 50 + lo%, with at least ten complete air changes per hour of 100% conditioned fresh air. All animals were on a 12-h light/dark full spectrum lighting cycle with no twilight. The mice were quarantined and acclimated for 5 days prior to experimental use. At the conclusion of the experiments the surviving mice were euthanized with CO,. Carbaryl LD,, determination in mice The approximate intraperitoneal (ip) toxicity was found in a range-finding study. For the definitive study ten mice at each of six logarithmically spaced dosage levels were administered carbaryl solutions in volumes corresponding to 5.0 ml/kg. The 24-h LD,, estimate and the 95% confidence limits were determined by the moving average method of Thompson and Weil [15]. Results of this study are included in Table III.

131

Orthodox therapeutic regimens and carbaryl toxicity in mice

Male mice weighing between 18-25 g were used. Carbaryl toxicity (ip) as modified by orthodox treatment compounds administered intravenously in dosage volumes of 10.0 ml/kg was determined. The compounds and doses were atropine sulfate (11.2 mg/kg), 2-PAM Cl (25.1 mg/kg), TMB4 Cl (12.6 mg/ml), or combinations of the oximes with atropine. The compounds were administered 5 min after carbaryl challenge (ip). The carbaryl challenges ranged from 25 to 200 mg/kg (approx. l/4 to 2.0 LD,,). Mice were assigned a particular challenge level and therapy based on a randomized block design. They were placed in individually numbered 6-inch2 cubicles on an observation platform. After injections were completed they were returned to their cubicles for observation over the next 24 h. Food and water were available ad libitum. The 24-h LD,, estimate and the 95% confidence limits were determined by the moving average method of Thompson and Weil [15]. Results of these studies are shown in Table III. RESULTS AND DISCUSSION

Carbaryl was introduced in 1956 under the trade name Sevin, and is the most widely used carbamate today. It is a wide-spectrum insecticide that is used to control 100-I 50 species of insects. In this regard it is fortunate that carbaryl has a low mammalian toxicity. The acute oral LD,, in female rats is about 500 mg/kg [16]. For comparison purposes the acute oral LD,, for DDT is 118 mg/kg, and for parathion it is 3.6 mg/kg. Interest in carbaryl continues to the present time. Recent papers focus on environmental considerations [ 171, biochemical interactions [ 181, and behavioral effects [ 191. As early as 1961 Carpenter and co-workers reported that the protective effectiveness of atropine against carbaryl poisoning in the canine species was reduced by 2-PAM [7]. In 1969 Farago reported the death of an adult man who had ingested 0.5 liter of carbaryl [8]. During the course of treatment atropine was administered, but atropinization was not observed. Upon administration of 250 mg of 2-PAM the patient’s condition deteriorated rapidly. In 1973 Natoff and Reiff reported that the therapeutic administration of P2S (Zpyridinium aldoxime methanesulfonate) markedly increased the toxicity of carbaryl in rats [9]. Three years later Boskovic and co-workers reported the same effect in mice [IO]. The phenomenon has also been observed with several other oximes, including Toxogonin and TMB4 [9,10,12,20]. A caution on the indiscriminate administration of either 2-PAM or Toxogonin in case of cholinesterase poisoning was published in 1978 in the South African Medical Journal [21]. A similar view was expressed by Reese in 1984 [22]. Speaking as a physician from a clinical point of view, he said that 2-PAM Cl was contraindicated in carbamate poisoning. We initially addressed the carbaryl/2-PAM question by asking whether the phenomenon results from a carbamylated oxime produced by a carbaryV2-PAM reaction. Such a species might well be a more potent inhibitor of cholinesterases than

132 TABLE I INHIBITION OF EEL AChE, 5 x 1O-7M CARBARYL (pH 7.60) 0.10 M MOPS BUFFER (25.O”C) WITH OR WITHOUT OXIMES, n = 3 Oxime

2-PAM Cl 2-PAM Cl 2-PAM MSb TMA-Cl’ TMA-Cl TMB4 Cl“ HI-6 Cl’ HS-6 Cl’ Toxogonin CP

Concentration (M)

tllz(min)

95% confidence limits

_

35.1 25.2 36.3 24.9 40.4 36.6 14.9 31.1 26.6 16.4

33.7-31.1 23.0-21.4 35.9-36.1 23.3-26.5 38842.0 36.5-36.7 13.4-16.4 28.3-33.9 24.9-28.3 16.3-16.5

5x 5x 5x 5x 5x 5x 5x 5x 5x

lo-5 lo-6 lo-5 1o-5 1o-6 lo-” lo-6 1om6 lo-6

a 2-Pyridine aldoxime methyl chloride; b2-pyridine aldoxime methanesulfonate; ‘tetramethylammonium chloride; “1,1’-(trimethy1ene)-bis(4-formy1pyridinium)dioxime dichloride; ‘I-(2-hydroxyiminomethyl-lpyridinio)-3-(4-carbamoyl-l-pyridinio)-2-oxapropanedichloride; ‘l-(2-hydroxyiminomethyl-l-pyridinio)3-(3-carbamoyl-1-pyridinio)-2-oxapropane dichloride; g1,l’-(oxydimethylene)~bis(4-formylpyridinium)dioxime dichloride.

carbaryl. Numerous industrial and academic studies have provided a variety of oxime carbamates with a broad range of insecticidal activity. Registered compounds belonging to this series include Methomyl and Aldicarb. In this regard it is worthy of note that phosphylated oximes are recognized as a problem in the therapy of organophosphate poisoning [23]. Phosphylated oximes are generally unstable compounds that hydrolyze to give an oxime and an organophosphorus acid, but they can also act as anticholinesterases where the oxime constitutes the leaving group. For example, the product from the interaction of paraoxon with Toxogonin has been found to be lo-times stronger an anticholinesterase than paraoxon [24]. Our efforts, however, to obtain evidence for the formation of a covalent bond from the reaction of carbaryl and 2-PAM were unsuccessful.* For subsequent studies of the carbaryl/2-PAM question we selected cholinesterase inhibition kinetics as our investigative tool. Our cholinesterase inhibition studies were directed at ascertaining whether oximes in general, and 2-PAM in particular, would perturb the inhibition rate by carbaryl. It is known that 2-PAM and other quaternary oximes used in therapy for organophosphorous intoxication are, in the simplest sense, reversible inhibitors of cholinesterases [25-281. Not unexpectedly, a number of earlier workers have studied irreversible inhibition in the presence of reversible inhibitors. *The interaction of 2-PAM Cl and carbaryl at pH 7.60 in 0.05 M phosphate buffer (25.O”C) was examined using high-pressure Iiquid chromatography. Good separation of the reactants and products was achieved using a 3/8” x 30 cmpBondapak C,, column with an eluent of the following composition: 94.5% water, 5% ethanol, and 0.5% acetic acid. None of the product peaks isolated was an inhibitor of eel AChE.

133

P-PAM

Cl

\ cl-

H 4

L=N-OH

0:

Cl0

H

H

&N-O”

h=N-OH

2 Cl0 CM 2 -

CH2 -

CH2

0

‘;‘o CH2 -

H HO-N=;

Toxogonin

H ;=N-OH

Ct 2 Cl0

Fig. I. Oxime structures and abbreviations.

The most obvious effect that a reversible inhibitor would have on irreversible inhibition would be to slow it by competing with the irreversible inhibitor for the active site. However, in 1963 Metzger and Wilson reported that TEA (the tetraethyl ammonium ion) accelerates the rate of carbamylation of eel AChE by dimethylcarbamy1 fluoride by a factor of fourteen [29]. The tetramethy1 a~onium ion (TMA) did not affect the rate of reaction. Investigations by Iverson [30] in 1971 led him to conclude that the tetraethyl ammonium ion may bind to the anionic portion of the active site of eel acetylcholinesterase and/or to an allosteric site [31]. The oximes used in our research are shown in Figure 1. A summary of our results on the inhibition of eel AChE by carbary1 in the presence and absence of various oximes is shown in Table I. As is evident from Table I, in the presence of 5 x 10e5 M 2-PAM CI the lli,for carbaryl inhibition is reduced to 25.2 min

134

and in the presence of 5 x lo-’ M 2-PAM methanesulfonate it is reduced to 24.9 min. Since the tli, for the carbamylation reaction in the absence of oxime is 35.4 min, it is apparent that the approx. 30% increase in rate observed is independent of the anion associated with the oxime. The two most effective accelerators are TMB4 and Toxogonin. As a frame of reference the reversible inhibitor tetramethylammonium chloride (TMA Cl) was included in our studies. This compound appears to act as a standard reversible inhibitor. That is, at 5 x 10m5M it retards carbamylation, and at 5 x 10e6 M it has no observable effect. The studies with the additional oximes listed in Table I show a role for each similar to that of 2-PAM. To explain the observed acceleration in carbamylation we propose that an allosteric site and/or sites are involved. From an allosteric site the pyridinium aldoximes may induce a conformational change in the active site such that either the binding of carbaryl (KJ or the carbamylation step (k2) is enhanced. The proposed allosteric site may or may not be identical with that postulated by Berry [32], Dawson and Poretski [33], or Harris et al. [34] who invoked allosteric sites to explain increases in decarbamylation rates. In any case, when the concentration of 2-PAM chloride is reduced to 5 x 10m6M there is no detectable perturbation in the carbaryl inhibition rate. A summary of our results from the experiments on the inhibition of human BuChE by carbaryl in the presence of selected oximes is shown in Table II. As was true in the case of eel enzyme at 5 x 1O-6M, neither 2-PAM chloride nor the tetramethylammoniurn ion accelerated the inhibition reaction. The two most effective accelerators were the oximes TMB4 and Toxogonin. The data shown in Table II are in concert with the earlier studies by Boskovic et al. [lo]. Using horse serum cholinesterase they found that 2-PAM Cl, TMB-4 Cl, Toxogonin, HS-3, HS-6, and HS-7 all decreased the concentration of carbaryl required for an I,,. For a 2-fold reduction in carbaryl LD,, the oxime concentration ranged from 2.1 x 10M6M for TMB4 Cl to 3.9 x 10e4 M for 2-PAM Cl. TABLE II INHIBITION OF HUMAN BuChE, 5 x lO-5 M CARBARYL (250°C) WITH OR WITHOUT OXIMES, n = 4.

(pH 7.60) 0.10 M MOPS BUFFER

Perturbing agent

Concentration (M)

2%(min)

95% confidence limits

_

_

2-PAM Cl” TMA-Clb TMB4 Cl HS-6 Cld Toxogonin C1’

5x 5x 5x 5x 5x

42.7 41.0 41.6 18.8 41.7 22.6

38.946.5 37.344.7 39.1-13.5 16.6-21 .O 40.4-43.0 21.5-23.7

lo-6 10-e 1o-6 10-e 1o-6

a 2-Pyridine aldoxime methyl chloride; ?etramethylammonium chloride; ‘l,l’-(trimethylene)bis(4formylpyridinium)dioxime dichloride, dl-(2-hydroxyiminomethyl-l-pyridinio)-3-(3-carbamoyl-l-pyridino)-2-oxapropane dichloride; ‘1,l’-(oxydimethylene)bis(4-formylpyridinium)dioxime dichloride.

135 TABLE III EFFECTS OF ORTHODOX THERAPEUTIC BARYL TOXICITY IN MICE

1

2 3 4 5 6

COMPOUNDS

ON INTRAPERITONEAL

Regimen

LD,a (mg/kg)

Protective ratio

None (water) 2-PAM Cl” TMB4 Clb Atropine Atropine + 2-PAM Cl Atropine + TMB-4 Cl

109 (833143)* 47 (35p 64) 16( 4 65) >158** 107 (62-185) 79 (499127)

1.0 0.43 0.14

CAR-

0.98 0.72

a 2-Pyridine aldoxime methyl chloride; “1, I’-(trimethylene)bis(4-formylpyridinium)dioxime *95% confidence limits *‘Insufficient mortality for accurate computation; three deaths out of 50 animals.

dichloride.

To examine the in vivo relevance of our in vitro enzymatic studies, we measured the effect of 2-PAM and TMB4 on carbaryl toxicity in mice. Table III is a summation of our five in vivo therapeutic regimens. These regimens were (i) 2-PAM Cl alone (25.1 mg/kg), (ii) TMB4 Cl alone (12.6 mg/kg), (iii) atropine sulfate alone (11.2 mg/kg), (iv) atropine and 2-PAM Cl (11.2/25.1 mg/kg), and (v) atropine and TMB-4 Cl (11.2/12.6 mgikg). Assigning a protective ratio (PR) of 1.Oto no therapy, the PR was reduced to 0.43 for 2-PAM Cl therapy and to 0.14 for TMB-4 Cl therapy. These results clearly identify two oximes whose use is contraindicated in carbaryl poisoning in mice.

TABLE IV IN VITRO AND IN WV0 CARBARYUOXIME Toxicity, mice”

Toxogonin’ II TMB-4d HS-6’ 2-PAM’

INTERACTIONS

Inhibition, eel AChEb

D E C R E A s I N ‘/ G

E F F E c T

TMB-4 Toxogonin HS-6 2-PAM

Inhibition, human serum ChEb D E C R E A s I N i G

E F F E c T

TMB4 Toxogonin 2-PAM II HS-6

“From B. Boskovic et al. [lo], based on 95% confidence limits; bThis study (based on 95% confidence limits); ‘1,l’-(oxydimethylene)bi(4-formylpyridinium)dioxime dichloride; dl,l’-(trimethylene)bis(4formylpyridinium)dioxime dichloride; ‘1-(2-hydroxyiminomethyl-l-pyridinio)-3-(3-carbamoyl-l-pyridinio)-2-oxapropane dichloride; ‘2-pyridine aldoxime methyl chloride.

136

It should be noted that Boskovic et al. reported similar findings in their study on the effects of various oximes on carbaryl poisoning in mice [lo]. Both sexes were used in their study. The LD,, of carbaryl (i.p. administration) was 63 mg/kg; with TMB4 chloride it was 11.6 mg/kg; with Toxogonin it was 11.5 mg/kg; and with HS-6 it was 15.4 mg/kg. The rank order of Boskovic’s findings on these oximes relative to their effect on carbaryl toxicity in the mouse is given in Table IV. In addition, our in vitro enzyme results are ranked on the effectiveness of these oximes in accelerating the carbamylation of eel AChE and human BuChE. It is apparent that these rankings are very similar. If future in vivo studies correlate with enzymatic in vitro studies, it may be possible to readily identify from in vitro studies carbamates that are incompatible with specific ‘therapeutic’ oximes. Minimizing animal use without decreasing the validity of the conclusions is a notable goal in any research area. Our results, coupled with those of Boskovic et al., cast considerable doubt on the explanation advanced by Sterri et al. in 1979 [ 121 that the adverse effects of oximes result from the formation of a carbamylated oxime that is a more potent acetylcholinesterase inhibitor than carbaryl itself. Our alternative hypothesis is that certain oximes act as allosteric effecters of cholinesterases in carbaryl poisoning. The net results are enhanced inhibition rates and potentiation of carbaryl toxicity. Our in vitro enzyme kinetic studies support our hypothesis and offer a potentially useful tool for future studies in this area.

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