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 21010-5425 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 TMB4, produced poor reactivation of OP-inhibited CaE. The best uncharged reactivator was 2,3butanedione 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 OPinhibited 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 catalyzingits 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 OPinhibited 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-Ccarboxamide)(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. Abbreviations: OP, organophosphorus; AChE, acetylcholinesterase; PAM, pyridine-2-aldoxime; TMB-4, N,N’-trimethylenebis(pyridine-4aldoxime); HI-6, N,N’-(oxydimethylene)(pyridine-4-carboxamide)(pyridine-2-aldoxime);BuChE, 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. @
1
dependent process in which an OP-inhibited enzyme becomes progressivelymore 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). Oximeinduced 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-inducedreactivation 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. Methyldichlorophospheoxide (I), 0-ethyl S42(diisopropylamino)ethyl]methylthiophosphonate (VI, VX) ,iso-
This article not subject to US. Copyright. Published 1994 by the American Chemical Society
Chem. Res. Toxicol., Vol. 7,No. 3, 1994 429
Reactivation of OP-Inhibited Carboxylesterase Table 1. Structures of Organophosphorus Compounds 0 II
A,-
P-X I
A2 ~~
organophosphorus compds instant aging
I I1 phosphinates I11
IV V
A1
Me Ph
C1 C1
c1 c1
Me Me Ph
Me Ph Ph
OPh-4-NOz OPh-4-NOz OPh-4-NOz
Me Me Me Me Me Me Me Me
OEt OPri OPin OHexC OPh OPh-4-OMe OPh-4-NOz OPh-4-CN
SC2HIN(Pri)2 F F F c1 C1 OPh-4-NOz OPh-4-CN
OMe OEt OPri OPh
OMe OEt OPri OPh
OCH=CC12 OPh-4-NO; F c1
phosphonates
VI (vx) VI1 (sarin)
VI11 (soman) IX (GF)
X XI XI1 XI11
phosphates XIV (dichlorvos) XV(paraoxon) XVI (DFP)
XVII
x
Az
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 methylphenylphosphinate (IV),and p-nitrophenyl diphenylphosphinate (V)were synthesized as previously described by Lieske et al. (13). Phenyl methylphosphonochloridate (XI, p-methoxyphenyl 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 characterized by 1H-NMRand 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(pyridine4-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’-(Oxydimethylene)(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 spectrophotometric method of Ecobichon and Comeau (16) with 1-naphthyl acetate as substrate.
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 microliters 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 correspondingcontrol 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-pLvolume) was reactivated at 25 OC by addition of 650 r L of 103M 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-inducedreactivation (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. Statistical Analysis. Significant differences < 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).
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 organophosphorus compds instant aging Ib IIb phosphinates I11 IV V phosphonates VI (VX)b VI1 (sarin) VI11 (soman) IX (GF) Xb XIb XI1 XI11 phosphates XIV (dichlorvos) XV (paraoxon) XVI (DFP) XVIIb
MINA
DAM 0 0
0 0
INAP
0 0
0 0
69 3 66 i 4 66 3
99 3 99*2 96*4
*3
5*3
86*5
6*3
90*4
96 f 4 85 6 68 5 74 f 4 40 4 23 f 3 19 f 4 0
98 2 99i3 97 4 96*4 45 f 5 55 2 9*2 15 3
45 15 0 0 0 0 0 0
75 f 7 64 i 5 71 f 4 4*1
98 f 3 97 5 5 i 1
20 0 0 0
* *
*
2-PAM
* 95 * 5
22
* 64
*6
cationic oxime TMB-4
0 0
HI-6 0 0
2*2
5 f 3 17 2 10* 1
*
0 10+3 9*4
6*4 0 0 0 0 0 0 0
9f3 0 0 0 0 0 0 0
5*3 0 0 0 0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
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 OPinhibited 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 abilityto reactivate OP-inhibited CaE decreased in the order DAM > MINA > INAP. For studies of oxime-inducedreactivation 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
