oxime-induced reactivation of carboxylesterase inhibited by organophosphorus compounds

428 Chem. Res. Toxicol. 1994, 7, 428-433

Oxime-Induced Reactivation of Carboxylesterase Inhibited by Organophosphorus Compounds

Donald M. Maxwell,* Claire N. Lieske, and Karen M. Brecht United States Army Medical Research, Institute of Chemical Defense,

Aberdeen Proving Ground, Maryland 2101 0-5425

Received August 3, 199P

A structure-activity analysis of the ability of oximes to reactivate rat plasma carboxylesterase (CaE) that was inhibited by organophosphorus (OP) compounds revealed that uncharged oximes, such as 2,3-butanedione monoxime (diacetylmonoxime) or monoisonitrosoacetone, were better reactivators than cationic oximes. Cationic oximes that are excellent reactivators of OP-inhibited acetylcholinesterase, such as pyridine-2-aldoxime or the bis-pyridine aldoximes, HI-6 and TMB- 4, produced poor reactivation of OP-inhibited CaE. The best uncharged reactivator was 2,3- butanedione monoxime, which produced complete reactivation a t 0.3 mM in 2 h of CaE that was inhibited by phosphinates, alkoxy-containing phosphates, and alkoxy-containing phosphonates. Complete reactivation of CaE could be achieved even after inhibition by phosphonates with highly branched alkoxy groups, such as sarin and soman, that undergo rapid aging with acetylcholinesterase. CaE that was inhibited by phosphonates or phosphates that contained aryloxy groups were reactivated to a lesser extent. The cause of this decreased reactivation appears to be an oxime-induced aging reaction that competes with the reactivation reaction. This oxime-induced aging reaction is accelerated by electron-withdrawing substituents on the aryloxy groups of phosphonates and by the presence of multiple aryloxy groups on phosphates. Thus, reactivation and aging of OP-inhibited CaE differ from the same processes for OP- inhibited acetylcholinesterase in both their oxime specificity and inhibitor specificity and, presumably, in their underlying mechanisms.

Introduction Organophosphorus (OP)’ compounds produce their

toxicity by inhibition of acetylcholinesterase (AChE; EC 3.1.1.7), an enzyme that terminates the action of acetylcholine by catalyzing its hydrolysis (1 ). Inhibition of AChE produces a variety of toxic effects culminating in death, usually due to respiratory failure. Since the discovery of pyridine-2-aldoxime (PAM), oximes in combination with atropine have become the standard therapy against the toxicity of OP compounds (1, 2) . Reactivation of OP- inhibited AChE by oximes regenerates active AChE (3-5) in order to restore adequate cholinergic activity. The search for better oximes resulted in the development of bis-pyridine aldoximes, such as N,N’-trimethylenebis- (pyridine-4-aldoxime) (TMB-4), which were clearly superior to PAM as reactivators of OP-inhibited AChE (6). The most extensively studied of the new bis-pyridine aldoximes is N,N’-(oxydimethylene) (pyridine-Ccarboxa- mide)(pyridine-2-aldoxime) (HI-6), which produced a significant improvement in protection against pinacolyl methylphosphonofluoridate (soman), the most refractory of the highly toxic OP compounds (7).

The greatest contributor to the refractoriness of OP compounds is the aging of OP-inhibited AChE, a time-

* Author to whom correspondence should be addressed. @ Abstract published in Advance ACS Abstracts, April 1, 1994. 1 Abbreviations: OP, organophosphorus; AChE, acetylcholinesterase;

PAM, pyridine-2-aldoxime; TMB-4, N,N’-trimethylenebis(pyridine-4- aldoxime); HI-6, N,N’-(oxydimethylene)(pyridine-4-carboxamide) (pyridine-2-aldoxime); BuC hE, butyrylcholinesterase; CaE, c.arboxylesterase; sarin, isopropyl methylphosphonofluoridate; soman, pinacolyl methylphosphonofluoridate; GF, cyclohexyl methylphosphonofluoridate; dichlorvos, 2,2-dichlorovinyl dimethyl phosphate; paraoxon, 4-nitrophenyl diethyl phosphate; DFP, diisopropyl fluorophosphate; MINA, monoisonitrosoacetone; DAM (diacetylmonoxime), 2,3-butanedione monoxi me; INAP, isonitrosoacetophenone.

dependent process in which an OP-inhibited enzyme becomes progressively more resistant to oxime reactivation ( I ) . The resistance to reactivation results from the loss of a second substituent from the phosphorus atom of the OP-inhibited enzyme, which can occur by a cationic dealkylation (i.e., acid-catalyzed) mechanism or a hydrolytic (i.e., base-catalyzed) mechanism depending on the enzyme and OP inhibitor (8). The aging and reactivation of an OP-inhibited enzyme occur simultaneously, and the final level of enzyme reactivation is determined by the relative rates of these two competing reactions.

In addition to reactivation of AChE, oximes can also reactivate other enzymes that are inhibited by OP compounds, such as butyrylcholinesterase (BuChE; EC 3.1.1.8) and carboxylesterase (CaE; EC 3.1.1.1). Oxime- induced reactivation of OP-inhibited AChE and BuChE have been extensively studied and recently reviewed (9, 10). However, reactivation of OP-inhibited CaE has been studied in only a few papers (4, 11, 12). These studies have established that in vivo oxime-induced reactivation of OP-inhibited plasma carboxylesterase provides protection against soman and sarin in rats. However, no structure-activity analysis for oxime-induced reactivation of OP-inhibited CaE has been reported.

The purpose of this study was (1) to measure the in uitro oxime-induced reactivation of CaE that had been inhibited by a series of OP compounds (see Table l), (2) to evaluate the ability of several types of oximes to reactivate OP-inhibited CaE, and (3) to examine the aging of OP-inhibited CaE.

Materials and Methods Chemicals. Methyldichlorophosphe oxide (I), 0-ethyl S42-

(diisopropylamino)ethyl]methylthiophosphonate (VI, VX) , iso-

This article not subject to US. Copyright. Published 1994 by the American Chemical Society

Reactivation of OP-Inhibited Carboxylesterase

Table 1. Structures of Organophosphorus Compounds 0 I I

I A,- P - X

A 2 ~~

organophosphorus compds A1 Az x instant aging

phosphinates

I Me C1 c1 I1 Ph C1 c1

I11 Me Me OPh-4-NOz IV Me Ph OPh-4-NOz V Ph Ph OPh-4-NOz

VI (vx) Me OEt SC2HIN(Pri)2 VI1 (sarin) Me OPri F VI11 (soman) Me OPin F IX (GF) Me OHexC F X Me OPh c1 XI Me OPh-4-OMe C1 XI1 Me OPh-4-NOz OPh-4-NOz XI11 Me OPh-4-CN OPh-4-CN

phosphonates

phosphates XIV (dichlorvos) OMe OMe OCH=CC12 XV(paraoxon) OEt OEt OPh-4-NO; XVI (DFP) OPri OPri F XVII OPh OPh c1

propyl methylphosphonofluoridate (VII, sarin), pinacolyl methylphosphonofluoridate (VIII, soman), and cyclohexyl methylphosphonofluoridate (IX, GF) were obtained from the Chemical Research, Development and Engineering Center (Aberdeen Proving Ground, MD). Their purities were >99% as determined by GC and *lP-NMR analysis. Phenyldichlorophosphine oxide (11) and diphenyl phosphorochloridate (XVII) were purchased from Pfaltz and Bauer (Waterbury, CT) and were used as supplied at >98% purity. 2,2-Dichlorovinyl dimethyl phosphate (XIV, dichlorvos), p-nitrophenyl diethyl phosphate (XV, paraoxon), and diisopropyl phosphorofluoridate [XVI, DFP (diisopropyl fluorophosphate)] were purchased from Aldrich Chemical Co. (Milwaukee, WI) and were used as supplied at >98% purity. p-Nitrophenyl dimethylphosphinate (III), p-nitrophenyl me- thylphenylphosphinate (IV), and p-nitrophenyl diphenylphos- phinate (V) were synthesized as previously described by Lieske et al. (13). Phenyl methylphosphonochloridate (XI, p-methoxy- phenyl methylphosphonochloridate (XI), bise-nitrophenyl) methylphosphonate (XII), and bise-cyanophenyl) methylphosphonate (XIII) were synthesized as described by Hovanec and Lieske (14). These phosphinates and phosphonates were char- acterized by 1H-NMR and elemental analyses, and their purities (>99%) were determined by GC and 31P-NMR. Caution: All of the above-mentioned O P compounds are hazardous and should be handled carefully (1). Monoisonitrosoacetone (MINA), 2,3-butanedione monoxime (DAM; diacetylmonoxime), isonitrosoacetophenone (INAP), and N,N’-trimethylenebis(pyridine- 4-aldoxime) (TMB-4) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Pyridine-2-aldoxime (PAM) was purchased from Ayerst Laboratories (New York, NY). N,N’-(Oxydimeth- ylene) (pyridine-4-carboxamide) (pyridine-2-aldoxime) (HI-6) was obtained from the Defense Research Establishment (Suffield, Canada). The purities of these oximes were >97 % as determined by HPLC. Sephadex G-200 was obtained from Pharmacia (Piscataway, NY).

Enzyme Preparation. CaE was enriched from rat plasma (Pel-Freez, Rogers, AR) by gel filtration on Sephadex G-200 by the method of Hashinotaume et al. (15). The CaE preparation eluted as a single peak (0.14 unit/mg of protein) that was devoid of arylesterase (EC 3.1.1.2) or butyrylcholinesterase (EC 3.1.1.8) contamination.

Enzyme Analysis. CaE was assayed by the spectrophoto- metric method of Ecobichon and Comeau (16) with 1-naphthyl acetate as substrate.

Chem. Res. Toxicol., Vol. 7, No. 3, 1994 429

CaE Inhibition and Reactivation. Inhibition of CaE was performed in 0.05 M phosphate buffer (pH 7.4) at 25 OC with solutions of each OP compound in acetonitrile. Twenty micro- liters of inhibitor solution was added to 1.00 mL of phosphate buffer containing 3 nmol of CaE. The CaE activity in the reaction mixture was measured after a 10-min incubation to confirm that >95% inhibition of CaE had occurred. The OP-inhibited CaE was then separated from excess inhibitor by HPLC by injection of 100 pL of the reaction mixture onto a TSK-GEL 2OOOSW column (300 mm X 7.5 mm; Thomson Instruments Co., Spring field, VA) equilibrated with phosphate buffer. With a column flow rate of 1.3 mL/min, OP-inhibited CaE eluted in the column fractions collected between 9 and 10 mL after sample injection, where control CaE samples were previously found to elute. The activities of control CaE samples were unaffected by passage through the HPLC column. The activity of each OP-inhibited CaE sample was compared to a corresponding control CaE sample that had received identical incubation and chromatography treatment except for the presence of OP inhibitor. After chromatographic separation the sample of OP-inhibited CaE (1300-pL volume) was reactivated at 25 OC by addition of 650 r L of 103 M oxime in 0.05 M phosphate (pH 7.4). Samples of the oxime reaction mixture (300 pL) were assayed sequentially for CaE activity at time intervals up to 2 h.

The observed rate constant (hob) for oxime reactivation of OP-inhibited CaE was calculated from the velocity of CaE activity (v) observed at sequential times ( t ) after the addition of oxime using the equation

where V,, was the maximal recovery of CaE activity. Inasmuch as the oxime-induced reactivation and aging of OP-inhibited enzymes are parallel first-order reactions, the true rate constants for oxime-induced reactivation (k,) and aging (k,) were calculated similarly to the method of Hovanec and Lieske (14) using the equations

and

k i k a = ?6 reactivation/(lOO - 7% reactivation)

where % reactivation is the maximal extent of oxime-induced reactivation of OP-inhibited CaE. This analysis assumed that oxime-induced reactivation was much greater than spontaneous reactivation (i.e., reactivation in the absence of oxime) under the conditions of our experiments. When spontaneous reactivation of OP-inhibited CaE was >1% of oxime-induced reactivation, k, was corrected for spontaneous reactivation.

< 0.05) were identified by the Newman-Keuls test (17). Regression analysis was performed by the use of RS/1 software (BBN Software Products, Cambridge, MA).

Statistical Analysis. Significant differences

Results Inhibition of CaE was achieved under the conditions

described in the Materials and Methods with concentrations of lo4 M for all OP compounds except I, 11, VI, X, XI, and XVII, where concentrations of M had to be used (Table 2). The poor reactivity of VI has been previously documented (18) and is probably related to the poor reactivity of cationic OP compounds for CaE, inasmuch as this compound is charged at physiological pH. The high concentration of the other compounds required for CaE inhibition is most likely due to their rapid degradation inasmuch as compounds 1,11, X, XI, and XVII are all phosphoryl chlorides and are readily hydrolyzed into noninhibitory OP compounds (14, 19).

430 Chem. Res. Toxicol., VoE. 7, No. 3, 1994 Maxwell et al.

Table 2. Oxime-Induced Reactivation of Carboxylesterase Inhibited by Organophoaphorue Compounds. % reactivation

uncharged oxime cationic oxime organophosphorus compds MINA DAM INAP 2-PAM TMB-4 HI-6

Ib 0 0 0 0 0 0 IIb 0 0 0 0 0 0

I11 69 3 99 * 3 22 * 3 5 * 3 5 f 3 0 IV 66 i 4 9 9 * 2 86*5 6*3 17 * 2 1 0 + 3 V 66 3 9 6 * 4 9 0 * 4 2 * 2 10* 1 9 * 4

VI (VX)b 96 f 4 98 2 45 * 6 6 * 4 9 f 3 5 * 3 VI1 (sarin) 85 * 6 9 9 i 3 15 4 0 0 0 VI11 (soman) 68 5 97 4 0 0 0 0 IX (GF) 74 f 4 9 6 * 4 0 0 0 0 Xb 40 * 4 45 f 5 0 0 0 0 XIb 23 f 3 55 2 0 0 0 0 XI1 19 f 4 9 * 2 0 0 0 0 XI11 0 15 * 3 0 0 0 0

XIV (dichlorvos) 75 f 7 95 * 5 20 * 6 0 0 0 XV (paraoxon) 64 i 5 98 f 3 0 0 0 0 XVI (DFP) 71 f 4 97 5 0 0 0 0 XVIIb 4 * 1 5 i 1 0 0 0 0

instant aging

phosphinates

phosphonates

phosphates

0 Two-hour reactivation with 0.3 mM oxime at 25 "C (pH 7.4). Oxime reactivation corrected for spontaneous reactivation and e x p r d as means i SD (n = 3). b Required 109 M concentration for inhibition of CaE, while all other OP compounds required 1 V M.

The ability of a range of commonly used oximes to reactivate OP-inhibited CaE is shown in Table 2. Cationic oximes were uniformly poor in their reactivation of OP- inhibited CaE, regardless of the structures of the OP compound. Uncharged oximes were much better reactivators of OP-inhibited CaE than cationic oximes. Among the uncharged oximes the ability to reactivate OP-inhibited CaE decreased in the order DAM > MINA > INAP. For studies of oxime-induced reactivation the only substituents of interest are A1 and A2 since X is cleaved from the OP compound when it inhibits CaE. The best reactivator, DAM, was able to reactivate completely all OP compounds except for those containing a chloro substituent in the A2 position (I and 111, which exhibited no reactivation, and compounds containing a phenoxy group (X-XIII, XVII), which exhibited <55 % reactivation. The compounds containing a chloro substituent in position A2 (I, 11) were used as negative controls for oxime-induced reactivation since P-C1 bonds are known to hydrolyze readily, leaving a P-O- inhibiting moiety which is not reactivated (i.e., instant aging) (19). The phosphinate inhibitors (III-V) were used as positive controls for reactivation since they containA1 and A2 substituents that have P-C bonds, which are not subject to hydrolysis (i.e., aging) and are, therefore, capable of complete oxime-induced reactivation (1 3).

Compounds VI-XI11 are organophosphonates that contain one P-0-C bond that is susceptible to aging. Compounds XIV-XVII are phosphates which contain two P-0-C bonds that are also susceptible to aging. The influence of the structure of OP moieties on the oxime reactivation of OP-inhibited CaE was evaluated by measuring the observed rate constants of oxime-induced reactivation (koa) by DAM, the best reactivator of OP- inhibited CaE. The observed rates of oxime-induced reactivation were separated into their constituent rate constants of reactivation (k,) and aging (k,) which are shown in Table 3. As expected, compounds I and 11, which have structures consistent with instant aging, have no observable oxime-induced reactivation; and compounds III-V, which have structures that cannot age, have complete oxime-induced reactivation with DAM. The rate

constants for reactivation of different phosphinate- inhibited CaE were not statistically different (1.3-1.4 h-1).

CaE that was inhibited by phosphonates had rate constants of oxime reactivation that varied in a narrow range from 1.3 to 1.5 h-l, which was similar to k, for phosphinates. The alkoxy-containing phosphonates (VI- IX) exhibited no aging while the aryloxy-containing phosphonates (X-XIII) exhibited a wide variation in rate constants of aging, ranging from 1.6 to 15.2 h-l. The aging rate for the aryloxy phosphonates appears to be related to Hammett's electronic parameter (a), which is an estimate of the ability of a substituent on an aryl ring to withdraw (+a) or donate (-a) electrons to the ring. Figure 1 shows the linear relationship between a and the rate constants of aging for the H, OMe, CN, and NO2 Substituents of aryloxy-containing phosphonate inhibitors of CaE.

CaE that was inhibited by phosphates had rate constants of oxime reactivation that varied in a narrow range from 0.7 to 1.0 h-l, which was about two-thirds of the values of k, for phosphinates and phosphonates. The alkoxy- containing phosphates (XIV-XVI) exhibited no aging, while the sole aryloxy-containing phosphate (XVII) had a rate constant of aging of 17.1 h-l, which was 20 times larger than its reactivation rate constant.

Discussion Oxime-induced reactivation and aging of OP-inhibited

CaE have several characteristics that distinguish them from the more extensively studied oxime-induced reactivation and aging of OP-inhibited AChE. First, the oximes that are effective reactivators of OP-inhibited CaE-MINA, INAP,andDAM-areallunchargedoximes, while the most ineffective oximes-PAM, TMB-4, and HI-6-are all cationic oximes (Table 2). This pattern of oxime effectiveness is quite different from the pattern of oxime reactivation for OP-inhibited AChE, where cationic oximes are far superior to uncharged oximes (6,101. This phenomenon is probably related to the differences in the substrate and inhibitor specificities of these two enzymes.

Reactivation of OP-Inhibi ted Carboxylesterase Chem. Res. Toxicol., Vol. 7, No. 3, 1994 431

Table 3. Aging and Reactivation by 2,a-Butanedione Monoxime (DAM) of Carboxylesterase Inhibited by Organophosphorus Compounds 0 I I

A,- 7 -0 -CaE

inhibited species rate constant (h-l)a

organophosphorus compds Ai Az k o b kr k , instant aging

phosphinates

I Me I1 Ph

I11 Me IV Me V Ph

VI (vx) Me VI1 (sarin) Me VI11 (soman) Me IX (GF) Me X Me XI Me XI1 Me XI11 Me

XIV (dichlorvos) OMe XV (paraoxon) OEt XVI (DFP) OPri XVII OPh

phosphonates

phosphates

a Reactivation with 0.3 mM DAM at 25 "C (pH 7.4). phosphinates and phosphonates.

1.5

c1 0 c1 0

Me 1.4 f 0.1 1.4 f 0.1 0 Ph 1.3 f 0.2 1.3 f 0.2 0 Ph 1.4 f 0.3 1.4 f 0.2 0

OEt 1.3 f 0.2 1.3 f 0.2 0 OPri 1.4 f 0.2 1.4 f 0.2 0 OPin 1.3 f 0.1 1.3 f 0.1 0 OHexC 1.5 f 0.3 1.5 f 0.3 0 OPh 2.9 f 0.2 1.3 f 0.1 1.6 f 0.1 OPh-4-OMe 2.5 f 0.2 1.4 f 0.1 1.1 f 0.1 OPh-4-NOz 16.7 f 2.2 1.5 f 0.2 15.2 f 2.0 OPh-4-CN 9.3 f 1.1 1.4 f 0.2 7.9 f 0.9

OMe 0.8 f 0.1 0.8 f O.lb 0 OEt 1.0 f 0.2 1.0 f 0.2b 0 OPri 0.7 f 0.1 0.7 f O . l b 0 OPh 26.0 f 2.3 0.9 f O.lb 17.1 f 2.2

Rate constants are expressed as means f SD (n = 3). b Significant difference from

Electronic Effect ((3-) of Substituent

Figure 1. Effect of electronic parameter (u-) of aryloxy substituent on aging rate (k.) of CaE inhibited by organophosphorus compounds. Substituents are presented in order of increasing U- for inhibited species Me(OPhX)P(O)-CaE, where X = H, OMe, CN, or NOz. Data for u- from Hansch and Leo (24).

AChE has such a strong specificity for cationic substrates (e.g., acetylcholine) and inhibitors (e.g., VX) that it has been suggested that it contains an anionic center in its active site ( I ) . In contrast, CaE does not react well with acetylcholine, VX, or a variety of other charged substrates and inhibitors, but it has a broad specificity for uncharged substrates and inhibitors (18). Second, although the aging processes of OP-inhibited CaE and AChE share some similarities, the aging process in these enzymes appears to occur by different mechanisms. Both CaE and AChE that have been inhibited by instant aging compounds, such as I and 11, are not susceptible to oxime reactivation, and both CaE and AChE that have been inhibited by phosphinates are completely reactivated by effective oximes (13; Table 2). However, the structure-activity patterns of aging for CaE and AChE that have been inhibited by phosphonates and phosphates are quite different. The most rapidly aging OP compounds for AChE contain highly branched alkoxy substituents, such as isopropoxy (e.g., sarin, DFP) or pinacolyloxy (e.g., soman), which can

stabilize the formation of a carbonium ion that is believed to be produced during aging that involves a cationic dealkylation mechanism (8, 10). OP compounds that contain these highly branched alkoxy substituents do not age after their inhibition of CaE (Table 3). Therefore, the mechanism of aging of OP-inhibited CaE probably does not proceed through a cationic dealkylation mechanism.

The structural characteristic of OP compounds that was most related to the aging of OP-inhibited CaE was the PKa of the acid formed by the hydrolysis of P-0-C bond. The OP compounds that aged contained aryloxy groups while the compounds that did not age contained alkoxy groups (Table 3). The pKavalues of the alkoxy groups are >14 while the pKa values of the aryloxy groups are <10.2 (20). The lower PKa of the aryloxy groups suggests that the P-0-C bonds of OP compounds containing this group are more labile than the corresponding bonds for alkoxy groups. This argument could be extended to substituents on phenoxy groups as well as differences between alkoxy and aryloxy groups. Electron-withdrawing substituents, such as CN or NOz, on phenoxy-containing phosphonates increased the aging rate constant in comparison to unsubstituted phenoxy phosphonates, while electron- donating substituents, such as OMe, decreased the aging rate constant (Figure 1). This type of oxime-induced aging has previously been described for OP-inhibited chymotrypsin that ages by a hydrolytic mechanism rather than a cationic dealkylation mechanism (21-23).

The most significant practical finding of this research was that CaE that has been inhibited by alkoxy-containing OP compounds, which constitute most of the commercial and military OP compounds, can be rapidly and completely reactivated. The only OP compounds that undergo aging after reaction with CaE are the aryloxy-containing OP compounds. Thus, endogenous CaE, which is an important stoichiometric detoxication enzyme for OP compounds

432 Chem. Res. Toxicol., Vol. 7, No. 3, 1994 Maxwell et al.

polymer to increase its molecular weight and reduce its penetration into the central nervous system.

(18), when in the presence of an uncharged oxime, becomes even more effective because it is easily reactivated for further stoichiometric removal of most OP compounds. To put the magnitude of these rates of CaE reactivation into perspective, the rates of DAM reactivation of VX-, sarin-, or soman-inhibited CaE were approximately equal to the rates of TMB-4 reactivation of AChE and about one-fifth the rates of HI-6 reactivation of AChE that has been inhibited by these same OP compounds (6,25, 26). Therefore, oxime-induced reactivation of OP-inhibited CaE for detoxication occurs at approximately the same rate as oxime-induced reactivation of OP-inhibited AChE for pharmacological intervention.

A comprehensive evaluation of oxime-induced reactivation of OP-inhibited CaE as a means of detoxication of OP compounds must include an analysis of the stereospecificity of inhibition, reactivation, and aging of OP- inhibited CaE. Many OP compounds, such as IV and VI-XI in our study, exist as enantiomers which may exhibit large differences in their reactivities with esterases, as has been observed for inhibition (27), reactivation (26), and aging of OP-inhibited AChE (28). Although the stereospecificity of aging and reactivation of OP-inhibited CaE have not been investigated, the stereospecificity of OP inhibition of CaE has been examined with several compounds. These studies revealed that the P(-) enantiomers of all the chiral OP compounds that have been examined are much more reactive with CaE than the P(+) enantiomers (29). This suggests that the OP-inhibited CaE conjugates whose reactivation and aging rates were measured in our study were probably inhibited by the P(-) enantiomers in our racemic mixtures. An unequivocal demonstration of this point in future work is important for development of oxime-induced reactivation of OP- inhibited CaE as a detoxication mechanism inasmuch as the P(-) enantiomers of chiral OP compounds are the most toxic (27).

Exploitation of oxime reactivation of CaE to increase its efficiency as an OP scavenger requires some consid- eration of the plasma concentration, biological half-life, and pharmacological effects of potential oximes. Inasmuch as DAM was the most effective oxime for CaE reactivation in our research (Table 2), its pharmacokinetic and pharmacological properties are informative. DAM has been reported to produce plasma concentrations of 1.5,2, and 0.25 mM and biological half-lives of 137,270, and 430 min in rats, dogs, and humans, respectively (30). These pharmacokinetic properties, which are superior to charged oximes, suggest that an uncharged oxime, such as DAM, can achieve adequate plasma concentrations for sufficient time to reactivate OP-inhibited CaE under conditions such as those described in our study (0.3 mM DAM). Although lack of charge confers pharmacokinetic advantages on oximes, such as DAM, it also produces pharmacological disadvantages. Cationic oximes, such as PAM or TMB-4, produce minimal pharmacological effects because their charges retard their penetration into the nervous system, while uncharged oximes, such as DAM, distribute easily into the nervous system, producing greater pharmacological effects, such as tachycardia, changes in blood pressure, postural hypotension, and disruption of electroencepha- logram activity (31). To reduce these detrimental pharmacological effects, it may be necessary to develop an uncharged oxime reactivator of CaE that is restricted to the plasma compartment by coupling the oxime to a

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