JOURNAL OF APPLIED TOXICOLOGY, VOL. 10(2), 87-91 (1990)

Oxime-Induced Reactivation of Acetylcholinesterase Inhibited by Organophosphinates*

D. W. Hanke,? M. S. Beckett, M. A. Overton, C. K. Burdick and C. N. Lieske US Army Medical Research Institute of Chemical Defense. Aherdeen Proving Ground. MD 21010-5425. USA

Key word5 organophosphinate, orgdnophosphonatc. harin, oxiiiieq ,icetylcholinesterasc. rexti\dtioii. pretrc'itment

The comparative potency of oximes for reactivation of inhibited eel acetylcholinesterase (AChE) in vitro is dependent on the organophosphinate inhibitor. Some of the data, dealing with a reference organophosphonate, support the conclusion of other investigators that the oxime potency order is also dependent on the inhibiting phosphonate. This work was done to identify more clearly the nature of phosphinylated AChE with regard to oxime reactivation potency and the potential of phosphinates as pretreatment drugs to protect AChE against organophosphonate poisoning. We have determined the reactivation potency of four oximes-2-PAM, HI-6, TMB-4 and toxogonin-against four phosphinates-4-nitrophenyl methyl(pheny1)phosphinate (PMP). 4- nitrophenyl chloromethyl(pheny1)phosphinate (CPMP), 4-nitrophenyl trifluoromethyl(phenyI) phosphinate and 4-nitrophenyl bis(2-thieny1)phosphinate. For comparison, the phosphonate sarin (GB, isopropyl methylphosphonofluoridate) was included. Incubation of the inhibited enzyme (I-AChE) at 25°C was with 0.30 p M oxime for PMP, 3.0 p M oxime for sarin and CPMP and 100 p M oxime for the two remaining phosphinates. AChE activity was assayed spectrophotometrically for 3.0 min at 272.5 nm at 25°C in 0.10 M MOPS buffer (pH 7.60) using phenyl acetate as substrate. When sarin was the inhibitor (0% spontaneous recovery after a 2-h incubation), the order of oxime reactivation was 2-PAM (46%) 3 toxogonin (33%) = TMB-4 (31%) > HI-6 (9%) after 2-h incubations. For PMP (12% spontaneous recovery after a 2-h incubation) the oxime order was toxogonin (67%) > TMB-4 (53%) > 2-PAM (40%) after 2-h incubations. For CPMP (8% spontaneous recovery after a 30-min incubation) the order was toxogonin (65%) = TMB-4 (59%) = HI- 6 (57%) > 2-PAM (12%) after 30-min incubations. For the trifluorophosphinate the order was toxogonin (18%) = TMB-4 (17%) 3 HI-6 (12%) 2 2-PAM (3%) after 5-min incubations. For the bis-thienylphosphinate the order was HI-6 (38%) > 2-PAM (20%) > TMB-4 (0.4%) > toxogonin (0%) after 5-min incubations. Despite some uncertainty in the oxime orders, these results support the conclusion that oxime potency is dependent on the organophosphinate used as the AChE inhibitor. The results also broaden the database that underpins the generally accepted conclusion, that for a given oxime its reactivation potency is dependent on the organophosphorous compound wed to inhibit AChE.

INTRODUCTION

I t is well documented that acetylcho1inestr:rase (AChE, EC' 3.1.1.7) is inhibited at varying ratcs by a wide variety of organophosphonates'.' and organophos- phinates."~" There is also a considerable body of cvitience that identifies numerous oximes that restore AChE activity after the enzyme has been inhibited by a n y one of a variety of organophosphorus (OP) compounds. However, oxime selection in the treatment of OP poisoning is more complicated when one considers that the comparative potency of oximes

1 his work was presented in part at the 19x5 and 19x6 Society of Iosicology meetings in San Diego. CA and Ncw Orleans. LA, t-c\pectivcly. Thc opinions or asscrtions contained herein are thc piivatc views o f the authors and are n o t to he coilstrued as official ( 1 1 as rcHectiny the views 01 the Department of thc Army o r the 1)cpartment of Deiense.

~ :2uthor to whom correspondence should he addressed.

is dependent on the particular phosphonate inhibitor AChE.3.13-IS.lh Our data on nerve agent poisoning

and oxime reactivation of AChE are in agreement with Bregovec er a l . " Furthermore, we reported previously in abstract form'.' that the oxime potency order is also dependent upon the inhihiting phosphinate. For the current study we used AChE from the freshwater teleost, Elecfroplzorits electricits (electric eel). to test the oximes (N-methylpyridinium-2-hydroyiminomethyl chloride (2-PAM.CI), bis-l.3-(4-hydroxyiminomethyl- 1-pyridino)propane dibromide (TMB-4.2Br). his-l,3- (4-hydroxyiminomethyI-l-pyridino)-2-oxapropane di- chloride (toxogonin.2CI) and 1-(2-hydroxyiminorn- ethyl- 1 -pyridino)-3- (4-carbanioyl- I -pyridino)-2-oxa- propane dichloride (HI-6.2CI) against a representative phosphonate-sarin (GB, isopropyl methylphosphono- fluoridate)-and four phosphinates-4-nitrophenyl methyl (pheny1)phosphinate (PMP), 4-nitrophenyl chloromethyl(pheny1)phosphinate (CPMP), 4-nitro- phenyl trifluoromethyl(pheny1)phosphinate (tri-F- PMP) and h i t rophenyl bis(2-thieny1)phosphinate (bis- thienyl-PMP). See Fig. 1 for inhibitor structures.

'lliis article was authored by B US Government employee and is IhL,refore not suhject to copyright protection.

88 D. W. HANKE E T A L .

CH3

4-Nitrophenyl rnethyl(pheny1)phosphinote (PMP)

oy- 0 0 NO2

CHzCl

4 -Nitrophenyl chlorornethyl(phenyl)phosphinate (CPMP)

CH3

4-Nitrophenyl 4-trifluoromethyIphenyl(methyI)phosphinate (tri-F-PMP)

(@ !- 0

2

4-Nitrophenyl bis(2-thieny1)phosphinote (bjs-thienyl-PMP)

Y 3 s H - C - 0 - P - F

LH3 I

H3C

Isopropyl rnethylphosphmfluoridote (sorin or GB)

Figure 1. Inhibitor structures.

~~ ~

EXPERIMENTAL

Materials The buffer, 0.10 M 3-(N-morpholino)propanesulfonic acid (MOPS), with additives 0.01 M MgCI,, 0.0296 sodium azide (wh) and 0.01% bovine serum albumin (HSA, wiv), was adjusted to pH 7.60. All chemicals were reagent grade. Unless specified, chemicals were obtained from Sigma Chemical Company. Throughout this paper, MOPS buffer and its additives will be referred to as the 'buffer'.

Stock solutions of the four phosphinates (obtained from Ash Stevens Co. on Government contract) were prepared weekly in ethanol at 14.0 mM. Due to variable solubilities, the inhibiting phosphinate concen- trations in buffer were 1.40 mM for PMP and CPMP, 1.07-1.20 pM for tri-F-PMP and 24.6-31.2 FM for his- thienyl-PMP. Neat sarin (96.6%) was diluted with 0.9% NaCl to 2.0 mg ml-', and 5.0-ml aliquots of these stock solutions were stored frozen at -70°C until needed. During the experiment, all inhibitor solutions were kept on ice.

The stock oxime solutions of 2-PAM.CI, TMB- 4.2Br, toxogonin.2CI and HI-6.2CI were prepared fresh daily at 3.0 mM in water and diluted with buffer to a final concentration of 0.30. 3.0 or 100 pM in the incubation with the respective inhibited enzyme (I- AChE).

Stock electric eel AChE solutions were prepared from commercially available (Sigma Chemical Co.) AChE (EC 3.1.1.7) at 1355 units mg-' by solubilizing with buffer to 0.5 units p1-I. Working solutions of AChE were prepared by taking 20.0-pl aliquots of the

enzyme stock solution and adding them to 2.0 ml of 0°C buffer. This produced a working solution of 4.95 units ml-I.

A 1.58 M stock solution of phenyl acetate in aceton- itrile was prepared by diluting 2.0 ml of phenylacetate (Eastman White label or equivalent) to 10.0 ml with acetonitrile.

Methods A jacketed glass column (1.6 cm inner diameter), as modified from Lieske et d.,' was packed to a height of 20 cm with Sephadex G-25 (coarse, Pharmacia) prepared in 500 ml of buffer. The column temperature was controlled by circulating 0°C water-methanol (9O:lO) through the jacket from an external pump (Lauda) to retard spontaneous reactivation of I-AChE while on the column. The buffer was the eluant and the buffer reservoir was kept on ice. Buffer flow was 1.0 ml min-' through the column, maintained by a nitrogen gas-driven reciprocating pump. A fresh col- umn was prepared after each I-AChE sample had been run to avoid any possible contamination of the next enzyme fraction with free inhibitor remaining on the column. A 0.33 ml sample loop contained 1.63 units of either control AChE or I-AChE, which was injected onto the column.

After the first AChE sample was injected onto the column, the first 25.0 ml of eluent were collected directly into a 25.0 ml graduated cylinder kept on ice. This 25.0 ml volume included the void volume and the enzyme fraction volume (ca. 5 ml) as predetermined using blue dextran. The I-AChE sample was injected onto the column to separate inhibitor-bound enzyme from free inhibitor. The first 25.0 ml of eluate (composed of the void and fraction volumes) contained only inhibitor-bound AChE, since the smaller-molecul- ar-weight unreacted inhibitor and leaving group, released from the inhibitor, remained on the column. As a test for contamination of this eluate with unreacted or free inhibitor, samples of the I-AChE eluate were spiked with AChE. There was no depression in AChE activity as compared to the control AChE spike. The free inhibitor on the column was detoxified by pouring the column contents into a container holding 2 N NaOH. Time-zero for all incubations was defined as that moment when the 0°C 25.0 ml column fraction was divided into equal aliquots for either spontaneous or oxime-induced reactivation and placed into a 25°C water-bath.

A control sample of 0.33 ml of the working solution of AChE was applied to the column in a fume hood, and the 25.0 ml fraction was collected on ice. Aliquots of the divided column fraction were assayed for AChE activity after various times of incubation: 2 h for PMP and sarin experiments; 30 min for CPMP experiments; 5 min for tri-F-PMP and his-thienyl-PMP experiments.

A 20.0 pI aliquot of 0°C working strength inhibitor was added to 1 .0 ml of the working enzyme solution and mixed to yield the inhibiting concentration. Five minutes later a 0.33 ml sample was applied to the column, as described above for the control AChE, and the 25.0 ml fraction was collected on ice. Aliquots of this 25.0 ml fraction were assayed to determine spontaneous reactivation of I-AChE.

OXIME-INDUCED REACTIVATION OF ACETYLCHOLINESTERASE 89

Spertrophotometric assay procedure. For each exper- iment. incubations or replicates were assayed for AChE activity at 272.5 nm and 25°C in 0.10 M buffer (pH 7.60) after the appropriate time of incubation. To start the assay, 2.5 FI of phenyl acetate were added to 1 . 0 nil of incubation medium in a cuvette. Absorbance redings were taken every 30 s for 3.0 min. A 1.0 ml sample of a typical control incubation medium showed a n activity corresponding to an increase of 0.628 (20.047 SD) absorbance units min-’.

To determine the per cent recovery of control AChE applied to the column, the 25.0 ml volume collected off the column was assayed for AChE activity (three 1 .0 ml samples) as described above.

When the oxime concentration was 100 pM, the degree of oxime inhibition of AChE was determined and taken into account when assigning per cent reactivation values to the oximes. The oxinie control was prepared by adding an aliquot of the working oxime solution to a portion of the AChE column control fraction to give the appropriate oxime incubation concentration. The oxime control was assayed then as detailed above.

The I-AChE column fraction (25.0 ml) was handled as described for oxime controls. I-AChE was incubated for 3 h with the 0.30 pM oxime for PMP and 3.0 pM for sarin experiments. I-AChE was incubated for 30 inin with 3.0 pM oxime for CPMP and for 5 min with 100 pM oxime for tri-F-PMP and bis-thienyl-PMP experiments. Just prior to assay of AChE activity, the 100 pM oxime incubations were diluted to 5 FM oxime.

The data were reported as the mean per cent (k SD) of the rate of substrate hydrolysis by .4ChE for spontaneous recovery or oxime-induced reactivation.

The spontaneous or oxime-induced reactivation of acetylcholinesterase inhibited by sarin, PMP, CPMP, tri-F-PMP or bis-thienyl-PMP is shown in Table 1. The oxime potency order is nearly the same for PMP and CPMP, which may be a reflection of their close structural similarity. There are significant differences among the oxime potency orders when comparing either PMP or CPMP to bis-thienyl-PMP or to sarin. The absolute oxime per cent reactivations may be compared directly with each other only for a given inhibitor, because the experimental conditions for testing the inhibitors are different. The differences in experimental conditions are due to the relative rates of spontaneous reactivation, as well as the relative buffer solubility and toxicity to AChE of the phosphi- nates tested. There is some uncertainty in the oxime orders where the SD values are large. Against sarin, 2-PAM was the most effective oxime to induce recovery of enzyme activity, while HI-6 was the least effective. Thew was n o significant difference between toxogonin and TMB-4 for the sarin, tri-F-PMP or his-thienyl- PMP experiments. All of the oximes tested significantly increased the rate of recovery of enzyme activity over the rates of spontaneous recovery tested.

DISCUSSION

As a class of organophosphorus compounds, the phosphinates have been considered as pretreatment compounds for prophylaxis against nerve agent poison- ing. The basis for this consideration is that many phosphinates are inhibitors of AChE, although to a lesser degree (about 100 times) than the nerve agents (phosphonates). The rationale for use of the phosphi- nates as pretreatment compounds is based, partially, on some general features common to the class. First, the phosphinates have two phosphorus-to-carbon bonds (in opposition to only one such bond in phosphonates) and, therefore, do not exhibit aging of I-AChE’ as does soman and, to a lesser extent, sarin. Additionally, in contrast to quaternary carbamates, e.g. pyridostig- mine, the uncharged phosphinates may have an advan- tage in that they may penetrate the endothelial blood-brain barrier more readily, thus providing pro- tection for central nervous system (CNS) AChE against organophosphonates. A difference in lipid solubility between pyridostigmine and the phosphinate PMP, for example, may make PMP the ‘drug of choice’ to protect the CNS from OP poisoning. Another import- ant feature of phosphinates in contrast to the phosphon- ates is that AChE exhibits measurable and significant spontaneous recovery from phosphinate inhibition, whereas there was no spontaneous recovery of AChE from sarin inhibition (Table 1).

Spontaneous recovery is also thought to be the basis for the efficacy of carbamates against the phosphonate nerve agents. Spontaneous recovery of AChE from PMP inhibition (Table 1) is slower than that from pyridostigmine inhibition. l7-Iy A final feature common to phosphinates, but not to carbamates. 17.20 (Hanke, unpublished observations in purified human erythro- cyte AChE), is that their inhibitory effects on AChE are readily reversible with the administration of oximes. Our initial experiments with PMP were designed to see how it might compare with pyridostigmine as a possible pretreatment compound that was not refrac- tory to oxime-induced reactivation of I-AChE. After testing four phosphinates we conclude, despite some uncertainty in the oxime orders, that oxime reactivation of AChE inhibited by phosphinates is variable, just like oxime reactivation of AChE inhibited by phosphonates. For prophylactic use we would recommend PMP, which has a significant spontaneous rate of recovery and is also sensitive to reactivation by oximes.

When sarin was the inhibitor, we were surprised initially to find that 2-PAM was the most effective reactivator of I-AChE after the 2 h incubation, since Steinberg et a/.” reported that the rate constant for reactivation of sarin-inhibited eel AChE by TMB-4 was nearly twice as large (1.90) as the rate constant for 2-PAM. However, his calculations were based on samples taken during 3.0 min incubations with 100 pM TMB-4, in contrast to our calculations that were based on 120 min incubations with 3.0 pM TMB-4. To address this apparent difference, in three separate experiments we measured the kinetics of oxime-induced recovery of sarin-inhibited eel AChE at 30, 60 and 120 min. From our kinetic data, we confirmed Stein- berg’s report using our results from the 30 min determi-

90 D. W. HANKE ET A L .

Table 1. Spontaneous or oxime-induced reactivation of acetylcholinesterase inhibited by sarin, 4-nitrophenyl methyl(pheny1)phosphinate (PMP), 4-nitrophenyl chloromethyl(pheny1)phosphinate (CPMP), 4-nitrophenyl trifluoromethyl(pheny1)phosphinate (tri-F-PMP) or 4-nitrophenyl bi5(2- thieny1)phosphinate (bis-thienyl-PMP)”.h

Per cent reactivation of acetylcholinesteraseb(t SD)

Inhibitor Sarin PMP CPMP Tri-F-PMP bis-Thienyl-PMP

Oxime concentration 3.0 pM 0.30 p M 3.0 p M 100 p M 100 pM Oxime incubation 2 h 2 h 30 rnin 5 rnin 5 min Replicates 10 9 6 4 4

Spontaneous 0 12P) 8 (2) - -

2-PAM.CI 46 (13) 40 (3) 12 (5) 3 (4) 20 (4) Toxogonin.2CI 33 (4) 67 (6) 65 (22) 18 (14) 0 TMB-4.2Br 31 (8) 53 (5) 59 (21) 17 (2) 0.4 (.8) HI-6.CI 9 12) - 57 (22) 12 (6) 38 (3)

a Acetylcholinesterase (AChE) was exposed to 0.140 p M sarin (GB, isopropyl methylphosphonofluoridate), 1.40 m M PMP, 1.40 m M CPMP, 1.07-1.20 pM tri-F-PMP and 24.6-31.2 p M bis-thienyl-PMP. Saturation of the active site serine was achieved within 5 min at 0°C in 0.10 M MOPS (pH 7.60). bThe inhibited-AChE (I-AChE) was incubated for 2 h with 3.0 pM or 0.30 )LM oxime for sarin or PMP experiments, respectively. I-AChE was incubated for 30 rnin with 3.0 p M oxime for CPMP experiments. I-AChE was incubated for 5 rnin with 100 pM oxime for tri-F-PMP and bis-thienyl-PMP experiments. All incubations were conducted at 25°C in 0.10 M MOPS buffer (pH 7.60). After the incubation with oxime, AChE activity was measured spectrophotometrically at 272.5 nm and 25°C in 0.10 MOPS buffer (pH 7.60) for 3.0 min using phenyl acetate as substrate; generation of phenol was measured. AChE activity or the reaction velocity of substrate turnover was determined as the mean slope of its absorbance curve i- SD. Recovery of AChE activity was tabulated as a mean per cent 2 SD of the appropriate control rate of substrate turnover. Spontaneous recovery of phosphinylated AChE (I-AChE) was determined as a per cent of the AChE control rate of substrate hydrolysis. Per cent oxime reactivation of I-AChE was based on individual oxime controls for the 100 p M oxime experiments and corrected for spontaneous recovery where necessary. A dash (-1 means the parameter was not measured.

nations of per cent oxime-induced recovery of I-AChE activity. The per cent I-AChE recovery induced by TMB-4 in our experiments was 1.60 times greater than that for 2-PAM at 30 min-30.2% vs. 18.9%, respectively. However, at 60 rnin 2-PAM was at least equivalent to TMB-4 in per cent oxime-induced recov- ery of I-AChE activity-32.9YO vs. 25.3%, respectively. Furthermore, by 120 rnin there was a clear reversal in rank position. and recovery induced by ‘-PAM was 1 .S1 times greater than TMB-4-47.5’70 vs. 31.5%, respectively. A modest net increase in the per cent oxirne-induced recovery over the 120 rnin incubation was seen with toxogonin and H1-6-9.3% and 8.7%, respectively-when compared to 28.6% for 2-PAM. The net increase for TMB-4 was only 1.3%. A significant difference in oxime potency as a conse- quence of the tirne-frame for measuring reactivation was also seen by Harvey et ril.,” where they tested P2S and HS6 against sarin. A possible explanation for this reversal in rank position over time between TMB-

4 and 2-PAM is that 2-PAM may be more stable than TMB-4 in solution, and at low oxime concentrations the differential rates of oxime degradation are inversely proportional over time in the per cent oxime-induced reactivations of I-AChE. Steinberg also alluded to ‘complex’ kinetics at low oxime concentrations. We were surprised to find that HI-6 restored so little inhibited eel AChE activity, since inhibited mammalian AChE responds very well to H I 4 treatment.’.’’ An explanation for this is that the two forms of AChE inhibited by organophosphorus compounds may differ sufficiently in their tertiary structures to respond differently to oxime-induced reactivation.

Acknowledgement

The author gratefully acknowledges the generous and expert assistancc o f Cpt W . W . Jcderberg in the writing of computer programs for data analysis.

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OXIME-INDUCED REACTIVATION OF ACETYLCHOLINESTERASE 91

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