the specificity of carboxylesterase protection against the toxicity of organophosphorus compounds

TOXICOLOGY AND APPLIED PHARMACOLOGY 114, 306-312 (1992)

The Specificity of Carboxylesterase Protection against the Toxicity of Organophosphorus Compounds’

DONALD M. MAXWELL

U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010-5425

Received April 8, 1991; accepted February 18, 1992

The Specificity of Carboxylesterase Protection against the Toxicity of Organophosphorus Compounds. MAXWELL, D. M. (1992). Toxicol. Appl. Pharmacol. 114, 306-312.

The ability of endogenous carboxylesterase (CaE) to protect against the lethal effects of a variety of organophosphorus (OP) compounds was examined in rats. The in vivo protection provided by endogenous CaE was measured by the difference in the LDSO values of OP compounds in control rats and rats whose CaE activity had been inhibited by SC injection with 2 mg/kg of 2-(O- cresyl)4H-1,3,2-benzodioxaphosphorin-2-oxide. Endogenous CaE provided significant protection against the in viva toxicity of soman, sarin, tabun, and paraoxon, but not against dichlorvos, diisopropyl fluorophbsphate, or ethoxymethyl-S-[2-(diisopropylamino)ethyl] thiophosphonate (VX). The relationship between the in vivo CaE protection against OP compounds and their relative reactivities with CaE and acetylcholinesterase (AChE) was evaluated by measuring the in vitro bimolecular rate constants (ki) for inhibition of plasma CaE and brain AChE. Except for VX, ki values for CaE inhibition varied <IO-fold while ki values for AChE inhibition varied 105-fold. The degree of in vivo inhibition of CaE by equitoxic doses of the OP compounds increased as the CaE/AChE ki ratio increased. However, the protective ratio of the LDso values in control vs CaE-inhibited rats decreased as the CaE/AChE ki ratio increased. This inverse relationship between in vivo CaE protection and relative in vitro reactivity for CaE suggested that CaE detoxication is more important for highly toxic OP compounds (i.e., compounds with high AChE ki values and low LDso values) than for less toxic compounds. Q 1992 Academic PKSS, IN.

INTRODUCTION

The acute toxicity of organophosphorus (OP) compounds in mammals is usually attributed to their irreversible inhibition of acetylcholinesterase (AChE, EC 3.1.1.7), an enzyme that terminates the action of acetylcholine in the nervous

’ The opinions or assertions contained herein are the personal views of the author and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. In conducting the research described in this report, the investigator adhered to the Guidefor the Cure and Use ofLaboratory Animals, National Institutes of Health Publication 85-23.

system (Karczmar, 1970; Taylor, 1985). The increase of acetylcholine at cholinergic synapses resulting from the inhibition of AChE, particularly in brain and diaphragm, produces a variety of pharmacological effects that culminate in death by respiratory failure (Natoff, 197 1; Brimblecombe, 1977).

OP compounds also react irreversibly with two other esterases-carboxylesterase (CaE; EC 3.1.1.1) and butyrylcho- line esterase (BuChE; EC 3.1.1.8)-whose inhibition produces no toxicity (Koelle et al., 1974; Junge and Krisch, 197 5). The reactions of CaE and BuChE with OP compounds are important only as potential detoxication processes because they stoichiometrically reduce the amount of an OP compound available to inhibit AChE. The reaction of OP compounds with BuChE is probably not an important detoxication process inasmuch as in vivo inhibition of BuChE in plasma and liver does not potentiate OP toxicity in mice (Clement, 1984), and whole-body BuChE levels in rats are one-thousandth the levels of CaE (Maxwell et al., 1987b). In contrast to the observations with BuChE, in viva inhibition of CaE in plasma and liver potentiates the toxicity of many OP compounds in both mice (Boskovic, 1979; Boskovic et al., 1984; Clement, 1984) and rats (Lauwerys and Murphy, 1969; Sterri and Fonnum, 1984; Maxwell et al., 1987a), which suggests that the reaction of OP compounds with CaE may be a detoxication route of considerable significance in these species.

Inhibition of CaE in plasma is particularly important for potentiation of the lethality of highly toxic OP compounds, such as soman. A dosage of CaE inhibitor that produces complete CaE inhibition in plasma and lung markedly potentiates the lethality of soman while inhibition of CaE in additional tissues (e.g., liver and kidney) by larger doses of inhibitor produces only a small increase in the potentiation of soman toxicity (Maxwell et al., 1987a). There is also a linear correlation between soman toxicity and changes in plasma CaE levels as rats develop (Sterri et al., 1985a) and age (Maxwell et al., 1988). The importance of plasma CaE for soman detoxication or potentiation appears to be primarily the result of the kinetics of OP absorption and dis- tribution whereby the tissue CaE that first encounters an OP

0041-008X/92 $5.00 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.

306

SPECIFICITY OF CARBOXYLESTERASE 307

compound (i.e., plasma CaE) is the CaE that preferentially detoxifies it (Maxwell et al., 1987b).

Although the in vitro reactivity of CaE with a variety of OP compounds has been studied (Ooms and Breebaart- Hansen, 1965; Hassan and Dauterman, 1968; Ecobichon and Comeau, 1973; Lee et al., 1978; Mentlein et al., 1984; Brestkin et al., 1986; Maxwell et al., 1988), the relationship between the in vitro reactivity of OP compounds for CaE and the in vivo protection of endogenous CaE against OP lethality has not been examined. The purpose of this paper is (1) to measure the in vitro reactivities of plasma CaE with a group of OP compounds that exhibit a range of in vivo toxicity values, (2) to compare the in vitro reactivities of these OP compounds for CaE and AChE, and (3) to inves- tigate the relationship of in vitro CaE reactivity and the in vivo protection provided by endogenous CaE against the toxicity of OP compounds.

METHODS AND MATERIALS

Materials. Pinacoloxymethyl fluorophosphonate (soman), isopropox- ymethyl fluorophosphonate (sarin), ethoxy-jV-dimethyl cyanophosphor- amidate (tabun). and ethoxymethyl-9[2-(diisopropylamino)ethyl] thiophosphonate (VX) were obtained from the Chemical Research, Development and Engineering Center (Aberdeen Proving Ground, MD). Diisopropyl fluorophosphate (DFP), dimethyl-2,2-dichlorovinyl phosphate (dichlorvos), and diethyl-4-nitrophenyl phosphate (paraoxon) were purchased from Ald- rich Chemical Co., Milwaukee, WI. 2-(0Cresyl)4H- 1,3,2benzodioxa- phosphorin-2-oxide (CBDP) was synthesized by Starks Associates (Buffalo, NY). [‘4C]Acetyl-~-methylchohne was synthesized by Amersham (Arlington Heights, IL). Triton X- 100, 1 -naphthyl acetate, physostigmine salicylate, and sucrose were purchased from Sigma Chemical Co. (St. Louis, MO). Sephadex G-200 was obtained from Pharmacia (Piscataway, NJ).

Enzyme preparation. Rat brain synaptosomal AChE was prepared for in vitro inhibition studies by differential centrifugation on sucrose density gradients as described by Maxwell et al. (1988). Synaptosomal AChE was solubilized with 0.5% Triton X-100 and centrifuged at 100,OOOg for 30 min to yield supernatants containing soluble AChE. In vitro inhibition of plasma CaE was studied with CaE that was purified from rat plasma by gel filtration on Sephadex G-200 by the method of Hashinotsume et al. (1978). Lung and liver tissues were prepared for measurement of in vivo inhibition of CaE by homogenization in 9 vol of saline containing 1% Triton X-100 by using a Potter-Elvehjem tissue grinder. Measurement of in vivo inhibition of plasma CaE was performed on heparinized blood samples that were obtained via cardiac puncture.

EnWme analysis. AChE activity was measured by the method of Siakotos et al. (1969) with [14C]acetyl-~-methylcholine as substrate. CaE was assayed by the spectrophotometric method of Ecobichon (1970) with I-naphthyl acetate as substrate. CaE activity in tissue samples was measured in the presence of lo-’ M physostigmine to inhibit naphthyl acetate hydrolysis due to the presence of BuChE activity. Physostigmine was not required for measurement of purified plasma CaE activity.

In vitro envyme inhibition. The reactivity of OP compounds with CaE or AChE was evaluated by following the in vitro time course of enzyme inhibition at pH 7.4 and 37°C. Bimolecular rate constants (k,) were determined by the method of Aldridge and Reiner (1972) with at least three concentrations of each OP compound and four incubation times. Selected concentrations of each OP compound contained in 20 pl were incubated with 380 pl of AChE or CaE diluted in 0.1 M phosphate (pH 7.4). The concentration of enzyme used for these inhibition measurements was 0.3

pM except when OP compounds had ki values > 20’ M-’ min-‘, which necessitated reducing the enzyme concentration IO-fold. Concentrations of OP compounds that produced ~90% inhibition within 2 to 30 min were used. The time course of enzyme inhibition was determined from measurements of enzyme activity in 30-~1 samples of the enzyme-inhibitor incubation mixture. Enzyme inhibition was stopped by the 100-fold dilution of the 30- ~1 samples in 3 ml of substrate when enzyme activity was measured. When inhibitor concentration (r) is much greater than enzyme concentration (E) and I approaches the dissociation constant (&) of the enzyme-inhibitor complex, the bimolecular reaction of enzyme and inhibitor follows pseudo- first-order kinetics. If graphs of log percentage enzyme activity vs time were linear (i.e., kinetics were pseudo-first-order), k, was calculated from the first- order kinetics equation k, = ( l/ZOt)In(&/E,), where 1, is the initial concentration of OP inhibitor, t is the incubation time of inhibitor and enzyme before substrate is added, E. is the initial enzyme concentration, and E, is the enzyme concentration after t minutes of incubation with inhibitor. When graphs of log percentage enzyme activity vs time were not linear (i.e., ex- perimental conditions for enzyme inhibition did not satisfy the pseudo-fimt- order requirements that Z$E and I = &), k, was calculated from the second- order kinetics equation k, = (l/(& - ZO)t)ln(lO(& - E,)/E&, - E,)).

In viro toxicity. Male Sprague-Dawley rats (200-250 g) were obtained from Charles River Laboratories (Wilmington, MA) and housed in tem- perature-controlled animal quarters that were maintained on a 12-hr aher- nating light/dark cycle. Animals were allowed free access to food and water before and after OP compound administration. LDSo values in rats were calculated by probit analysis (Finney. 197 1) of deaths occurring within 24 hr following SC injection of OP compounds at five different doses with six animals per dose. LD,, values were determined in control animals and animals whose CaE had been inhibited with 2 mg/kg CBDP, a specific in vivo inhibitor of CaE (Maxwell et al., 1987a). Soman, sarin, tabun, dichlorvos, VX, paraoxon, and DFP were administered as solutions in isotonic saline. CBDP was administered SC as a solution in propylene glycol containing 5% ethanol. Animals receiving multiple injections were injected in alternate hind limbs.

Data analysis. Significant differences between pairs of LDSo values were identified by the Newman-Keuls test (Hicks, 1982). Enzyme activities were compared by Student’s t test. Differences were considered significant if p < 0.05. Rate constants (ki) were calculated from the time course of enzyme inhibition by linear regression of ln(E,,/&) vs t for first-order kinetic data and In(l,,(& - E,)/E&, - E,)) vs t for second-order kinetic data.

RESULTS

The in vitro reactivities of OP compounds for reaction with CaE and AChE were compared by in vitro measurements of the rate constants (kJ for inhibition of rat plasma CaE and rat brain AChE. The ki values that were <lo6 M-’ min-’ were calculated from linear regression analysis of the pseudo-first-order kinetics of enzyme inhibition (see Fig. 1 for a typical graph). For inhibitors with ki > lo6 M-’ min-‘, ki values were calculated from linear regression of the second- order kinetics of enzyme inhibition (see Fig. 2), since the inhibition of enzyme did not follow first-order kinetics at the concentrations of OP compounds used for inhibition (see Fig. 2, insert). The variation among the OP compounds in their ki values for inhibition of CaE was less than IO-fold, except for the ki value for VX. VX was 500-fold less reactive with CaE than the other OP compounds, which had k; values in a narrow range from 7.1 X lo5 to 5.9 X lo6 M-’ mini’ (Table 1). In contrast, the OP compounds exhibited a 105-

308 DONALD M. MAXWELL

‘V 8 10 12 14 16

Time (min)

FIG. 1. First-order kinetics of AChE inhibition by DFP at pH 7.4 and 37°C. Initial concentrations of DFP were 3.2 X 10e5 M (O), 1.0 X low5 M

(m), and 3.2 X 10e6 M (A). Initial AChE concentration was 3.0 x lo-’ M.

fold variation in ki values for inhibition of AChE. The ki values for AChE inhibition ranged from 1.4 X lo8 M-i min-’ for VX to 1.1 X lo3 M-’ min-’ for CBDP. The variation in the CaE/AChE ratio of ki values (Table 1) was primarily the result of differences in ki values for inhibition of AChE. VX was the most specific inhibitor of AChE, with a CaE/AChE ki ratio < 10p4. CBDP was the most specific inhibitor of CaE with a CaE/AChE ki ratio > 103.

The relationship between the variation in the CaE/AChE ki ratio and the in viva reaction with CaE was examined with pharmacologically equivalent doses of each OP compound (Table 2). When 0.9 LDSO of each OP compound was administered to rats, VX produced no inhibition of CaE in plasma, lung, or liver. For the other OP compounds, plasma CaE was nearly completely inhibited and the in vivo inhibition of CaE in lung and liver was found to increase as the CaE/AChE ki ratio of the OP compounds increased. Lung

012345678

Time (min)

FIG. 2. Second-order kinetics of CaE inhibition by DFP at pH 7.4 and 37°C. The insert shows CaE inhibition by DFP on a first-order kinetic plot. Initial concentrations of DFP were 6.0 X IO-’ M (O), 4.5 X lo-’ M (m), and 3.1 X lo-’ M (A). Initial CaE concentration was 3.0 X lo-’ M.

TABLE 1 In Vitro Inhibition of Acetylcholinesterase and

Carboxylesterase by Organophosphorus Compounds

Inhibition rate constant (k, )

Compound AChE”

(M-’ min-‘) CaE”

(M-’ min-‘) k, ratio

(CaE/AChE)

vx 1.4 (20.3) x 108 b 1.5 (+o.l) x IO3 l/93.300 Soman 3.6 (20.5) x 10’ b 5.1 (r+_O.9) x lo6 b l/7 Sarin 1.2 (kO.3) x 10’ b 3.0 (kO.3) x lo6 b l/4 Tabun 4.5 (kO.7) x 106 * 2.4 (kO.5) x lo6 b 112 Paraoxon 9.0 (kl.1) x IO5 4.6 (kO.7) x IO6 b 5/l DFP 2.9 (kO.3) x lo4 3.6 (kO.3) x lo6 b 124/t Dichlorvos 1.4 (kO.2) x lo3 7.1 (kO.6) x 10’ 507/l CBDP 1.1 (kO.2) x IO3 5.9 (k0.9) x IO6 b 5360/l

’ Brain acetylcholinesterase (AChE) and plasma carboxylesterase (CaE) inhibited at pH 7.4 and 37°C. k, values are means i SD (n = 3).

h k, calculated from the second-order kinetic equation because inhibition was not first order at the concentrations of organophosphorus compound used for inhibition.

CaE was less inhibited by soman or satin (CaE/AChE k, ratio G l/4) than by DFP or dichlorvos (CaE/AChE ki ratio >, 124). In a similar fashion liver CaE was not significantly inhibited by soman or sarin (CaE/AChE k; ratio =G l/4), but was moderately inhibited (25-35%) by tabun and paraoxon (CaE/AChE ki ratios of l/2 and 5, respectively) and nearly completely inhibited (86-87%) by DFP and dichlorvos (CaE/ AChE ki ratio >, 124).

The protection against the in vivo toxicity of OP compounds that was provided by endogenous CaE was estimated by the difference in LDjO values for control rats and rats whose CaE had been inhibited by administration with 2 mg/

TABLE 2 In Vivo Inhibition of Carboxylesterase

by Organophosphorus Compounds

% CaE inhibition’ k, ratiod Dose (CaE/

Compound (rmol/kg)” Plasma Lung Liver AChE)

vx 0.027 0 0 0 l/93,300 Soman 0.30 93 ?I 46 65 -t 56 0 l/7 Satin 0.5 I 94 2 5b 71 *4b 0 l/4 Tabun 0.97 95 + 6’ 78 k 7b 25 + 3b 112 Paraoxon 1.64 93 + 56 81 +6” 35 +4b 5/l DFP 8.78 96 f 3b 84 +- 7b 86 + 8’ 124/I Dichlorvos 88.56 95 * 5* 85 + 6b 87 + 6’ 507/l

a 0.9 LDSo from Table 1. b Significant difference from CaE activity in control rats (p < 0.05). c Carboxylesterase (measured I hr after 0.9 LDSo of organophosphorus

compound in rats. Values are means + SD (n = 6). d From Table 1.


TABLE 3 Protective Effect of Endogenous Carboxylesterase against

In Viva Toxicity of Organophosphorus Compounds

LDm (wmllkg)

Compound Control CaE

inhibitedb Protective Protective

ratiod difference’

vx 0.03 0.03 1.0 - (0.03-0.04) (0.02-0.04)

Soman 0.33 0.04’ 8.3 0.29 (0.25-0.36) (0.03-0.06)

Sarin 0.57 0.07’ 8.1 0.50 (0.43-0.78) (0.05-O. 10)

Tabun 1.08 0.24’ 4.5 0.84 (0.85-1.35) (0.19-0.31)

Paraoxon 1.82 0.92’ 2.0 0.90 (1.37-2.42) (0.75-1.21)

DFP 9.75 9.78 1.0 - (7.84-12.52) (7.95-12.48)

Dichlorvos 98.4 100.2 1.0 - (77.4-130.5) (78.1-133.4)

CBDP 1080.0 (821-1370) - - -

a In viva toxicity (LD,,) of organophosphorus (OP) compounds administered sc with 95% confidence limits in parentheses. LDW values represent only toxic (P-) isomers for racemic OP compounds.

b LD,, value of OP compound in rats receiving OP 1 hr after sc injection with 2 mg/kg CBDP.

c Significant difference between LDSo values in control rats and CaE- inhibited rats (p < 0.05).

d Ratio between LDSo values in control rats and CaE-inhibited rats. e Difference between LD5,, values in control rats and CaE-inhibited

rats.

kg CBDP. This dose of CBDP has been previously demonstrated to completely inhibit plasma and lung CaE in vivo without inhibiting AChE in brain or diaphragm (Maxwell et al., 1987a). A comparison of the in viva toxicity of OP compounds in control and CaE-inhibited rats is shown in Table 3. No CaE protection was observed for VX, DFP, or dichlorvos. Excluding VX, for which CaE detoxication does not appear to be important (Table 2), the protective ratio of the OP compounds decreased approximately 8-fold as the CaE/AChE ratio of ki values increased 3500-fold from soman to dichlorvos (Fig. 3).

The LDso values in CaE-inhibited rats were significantly reduced from the LD50 values in control rats for soman, sarin, tabun, and paraoxon (Table 3), indicating that endogenous CaE provided protection against lethality for these compounds. For those compounds where CaE provided sta- tistically significant protection, the protective difference increased as the CaE/AChE ki ratio increased from l/7 for soman to l/2 for tabun (Fig. 4). The protective difference remained constant as the CaE/AChE ki ratio increased lo- fold from l/2 for tabun to 5 for paraoxon. A maximal pro-

loT

2- I\ l ---.-. Op 0.1 1 10 100 1000

ki ratio (CoE/AChE)

FIG. 3. Relationship between CaE/AChE ratio of in vitro reactivities (k,) and in vivo CaE protection against OP compounds expressed as protective ratios. CaE/AChE k, ratios were taken from Table 1. Protective ratios were taken from Table 2.

tective difference of approximately 0.9 pmol/kg was achieved where the CaE/AChE k, ratio 3 l/2.

DISCUSSION

In general, the ki values for in vitro inhibition of CaE by OP compounds that were obtained in this study were com- parable to the ki values for other OP compounds in recent studies by Brestkin et al. (1986) and greater than those obtained in earlier investigations by Ooms and Breebaart-Han- sen (1965). These differences may result from enzyme iso- lation procedures, since the same techniques were employed in all three studies for determination of ki values, but the preparation of CaE used by Ooms and Breebaart-Hansen

ki ratio (CoE/AChE)

FIG. 4. Relationship between CaE/AChE ratio of in vitro reactivities (k,) and in vivo CJaE protection against OP compounds expressed as protective differences. CaE/AChE k, ratios were taken from Table 1. Protective differences were taken from Table 2. Protective differences were not presented for VX, DFP, and dichlorvos because there was no significant difference between LD,, values in control and CaE-inhibited rats for these OP compounds.


was isolated from an acetone powder and subjected to heat denaturation and lyophilization during purification, whereas the CaE used in the present study and the work by Brestkin and co-workers was prepared using much milder purification conditions. The ki values for OP inhibition of AChE that were obtained in the present study were in good agreement with the results of Andersen et al. (1977), where a similar rat brain synaptosomal preparation was used.

The variation in ki values for inhibition of CaE was less than 1 O-fold, except for the ki value for VX, which was 500- fold less than any other Ici value (Table 1). This generally broad specificity of CaE for OP inhibitors is consistent with its well-documented wide specificity for uncharged substrates (Junge and Krisch, 1975). The lower reactivity of VX for CaE has been attributed by Ooms ( 196 1) to the partial pro- tonation of VX at physiological pH, inasmuch as CaE has such poor reactivity for cationic substrates, such as acetylcholine or butyrylchohne, that they have been routinely used to differentiate CaE from AChE and BuChE (Augustinsson, 1958).

The variation in the CaE/AChE ki ratio (Table 1) was primarily the result of changes in ki values for inhibition of AChE. VX was the most specific in vitro inhibitor of AChE, which correlated with the observations that endogenous CaE was not inhibited by VX (Table 2) and that prior CaE inhibition by CBDP in rats did not increase the in vivo lethality of VX (Table 3). CBDP was the most specific in vitro inhibitor of CaE, in accord with its previously reported in vivo ability to inhibit plasma and lung CaE > 95% without significantly inhibiting AChE (Maxwell et al., 1987a).

The in vivo tissue inhibition of CaE by equitoxic doses of OP compounds, an indicator of the relative level of detoxication of OP compounds by CaE, increased as the CaE/AChE ratio of in vitro reactivities increased (Table 2). The tissue most sensitive to in vivo CaE inhibition was plasma, which was inhibited >93% by all OP compounds with CaE/AChE ki ratios > l/7. Lung was nearly as sensitive with 65% inhibition at a CaE/AChE ratio of l/7 increasing to 85% inhibition at a ki ratio of 507. Liver was the least sensitive tissue to CaE inhibition, exhibiting significant inhibition only for OP compounds with CaE/AChE ki ratios > l/2 and increasing to 85% inhibition at a ki ratio of 507. Although there are differences in isoelectric point, substrate specificity, and oxime reactivation in the CaE enzymes found in different tissues, these different types of CaE have similar in vitro sen- sitivities to OP inhibition (Sterri et al., 1985b; Sterri and Fonnum, 1987). Therefore, the variation in tissue sensitivity to CaE inhibition by OP compounds was probably related to either (1) the differences in the blood flow to tissues, where plasma and lung receive 100% of the cardiac output and liver receives only 25% of the cardiac output, or (2) the amount of each OP compound that was administered as a 0.9-LD5,, dosage, which increased from 0.30 to 88.56 pmol/ kg as the CaE/AChE ratio increased from l/7 to 507 (Table

2). With respect to the influence of blood flow, a close correlation between tissue variation in enzyme inhibition and differences in tissue blood flow has been previously observed for the in vivo inhibition by OP compounds of other esterases, such as AChE (Maxwell et al., 1987b). With respect to the effect of variation in the administered dosages of OP compounds, the expectation that CaE in tissues would be more inhibited by OP compounds with larger LDsO values was demonstrated in Table 2, where lung and liver were pro- gressively more inhibited by OP compounds as the LDso values increased 300-fold from soman to dichlorvos.

Previous investigations have revealed that CBDP at the 2 mg/kg dose used in the present experiments inhibits plasma and lung by >95% and liver by 20% without inhibiting AChE in brain or diaphragm (Maxwell et al., 1987a). The impact of this pattern of CaE inhibition on CBDP’s ability to potentiate OP lethality varied among the OP compounds examined in the present study. For OP compounds, such as soman and sarin, where OP detoxication by CaE (i.e., CaE inhibition) was observed only in plasma and lung (see Table 2), prior CaE inhibition by CBDP potentiated the lethality of the OP compounds 8-fold (Table 3). For tabun and paraoxon, liver CaE was inhibited about one-third as much as CaE in plasma and lung (Table 2), but the number of CaE binding sites in liver has been estimated to be seven times larger than that in plasma and lung (Maxwell et al., 1988) which suggests that CaE detoxication of these compounds in liver may be of equal or greater importance than CaE detoxication in plasma and lung. In accord with the prop- osition that CaE detoxication in lung and plasma is relatively less important for these compounds than for soman or satin, the LD,o values of tabun and paraoxon were potentiated only 4.5- and 2-fold, respectively, which was much less than the potentiation of soman or sarin. For DFP and dichlorvos, where liver CaE was inhibited as much as plasma and lung (Table 2), the larger number of CaE binding sites in liver supports the conclusion that CaE detoxication in liver for these OP compounds is much more important than CaE detoxication in plasma and lung. Thus, CaE inhibition by CBDP, which is primarily restricted to plasma and lung, produced no significant potentiation of lethality for DFP or dichlorvos (Table 3). The general relationship between the pattern of in vivo CaE inhibition in tissues and the variation in the potentiation of lethality by CBDP suggests that CaE detoxication of highly toxic OP compounds (e.g., soman) is nearly completely accomplished by CaE in plasma and lung, whereas an LDso dosage of a low-toxicity OP compound (e.g., dichlorvos) exceeds the number of CaE binding sites available for stoichiometric OP detoxication in plasma and lung and is, therefore, primarily detoxified by CaE in liver.

The apparent paradoxes in the present study were (1) the inverse relationship between in vivo protective ratio for CaE and the relative in vitro reactivity for CaE vs AChE and (2) the absence of significant in vivo protection by CaE against


OP compounds that exhibited the highest levels of in vivo detoxication by CaE. With respect to the first paradox, the protective ratio decreased from 8.3 to 1 as the CaE/AChE ki ratio increased from l/7 for soman to 507 for dichlorvos (Fig. 3). This observation contradicts the intuitive expectation that there would be more protection by CaE against OP compounds as they become more reactive with CaE, which produces a protective effect, in comparison to their reactivity with AChE, which produces a toxic effect. However, if CaE protection was measured as the protective difference instead of the protective ratio, the intuitive expectation that CaE protection would increase with the increase in the CaE/AChE ki ratio was observed. For OP compounds with significant differences between the LDsO values for control and CaE- inhibited rats (see Table 3), the protective differences increased as the CaE/AChE ki ratios increased to a maximal value (Fig. 4). With respect to the second paradox, CaE provided no significant protection, measured as either protective ratio or protective difference, against OP compounds with low toxicity, such as DFP and dichlorvos, even though these compounds exhibited the greatest degree of in vivo CaE detoxication (i.e., CaE inhibition) of the OP compounds tested (Table 2). This observation emphasized that CaE inhibition was particularly important in the modification of LDso values for highly toxic OP compounds, such as soman and satin, and much less important for low-toxicity OP compounds, such as DFP and dichlorvos. This was probably the result of the limitations of CBDP as a molecular probe for deter- mining the importance of CaE as a detoxication mechanism for OP compounds and also due to the relative importance of OP hydrolases vs CaE as detoxication mechanisms for OP compounds of differing toxicity.

Using a compound such as CBDP as a pharmacological probe to determine the importance of CaE as a detoxication mechanism for OP compounds requires careful analysis of its in vivo specificity for inhibition of CaE in comparison to AChE, whose inhibition is directly involved in the lethality of OP compounds. The 2 mg/kg dose of CBDP used in the present experiments was the largest dose that could be used without inhibiting AChE in brain or diaphragm (Maxwell et al., 1987a). which would have compromised the determination of the importance of CaE detoxication alone on OP lethality. At the 2 mg/kg dose of CBDP only lung and plasma CaE were completely inhibited; CaE in liver was only 20% inhibited and CaE in kidney was only 35% inhibited (Maxwell et al., 1987a). Therefore, the use of CBDP in the present study primarily resulted in the estimation of the effect on OP lethality of CaE detoxication in plasma and lung, and another probe would be necessary to evaluate the effect of CaE detoxication in liver and other tissues. Recent investigations have attempted to discover better CaE inhibitors (Chambers et al., 1991) but CBDP has remained the best inhibitor yet found.

In addition to the limitations of inhibitor specificity for potentiating OP lethality to evaluate the importance of CaE detoxication of OP compounds, it should be noted that the rates of detoxication of OP compounds in rats have varied between the SC and ip routes of OP administration (Steni, 198 1). Correspondingly, the degree of potentiation of OP lethality after CaE inhibition by tri-o-cresyl phosphate in rats has varied between SC and inhalation exposure routes for soman (Sterri, 1981; Aas et al., 1985). Boskovic (1979) has also observed differences in the degree of potentiation of OP lethality after CaE inhibition by CBDP in mice exposed to soman by SC and ip routes of administration. In view of this variation in the magnitude of the effect of CaE detoxication on OP lethality between the SC, ip, and inhalation routes of OP administration, it will probably not be possible to extrapolate the results of the present study to other routes of OP exposure.

Among the diverse biochemical reactions involved in detoxication of OP compounds, CaE appears to perform the role of a high-affinity/low-capacity detoxication process. Physiological concentrations of most OP compounds (i.e., 0.0 1 to 1 .O PM) will react rapidly with CaE inasmuch as their ki were found to be > lo6 M-’ min-‘, but they react by an irreversible 1: 1 stoichiometry with the active site of CaE (Junge and Krisch, 1975). Therefore, the capacity of CaE to detoxify OP compounds is quantitatively limited by the number of available CaE molecules. CaE detoxication con- trasts with other detoxication enzymes, such as OP hydrolases, that are low-affinity/high-capacity enzymes that can detoxify 1 03- 1 O6 OP molecules/min/active site, but have K,,, for OP compounds in the millimolar concentration range (De Jong et al., 1989). Consequently, OP hydrolases would be expected to be more important for low-toxicity OP compounds that achieve the high in vivo concentrations required for effective detoxication by a low-affinity/high-capacity enzyme, while CaE is primarily important for detoxication of highly toxic OP compounds that achieve lower in vivo concentrations, for which the high affinity of the detoxication enzyme is more important than its detoxication capacity.

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