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available for acetylcholinesterase inhibition. This study of the interaction of rat plasma and liver CarbE with dichlorvos, soman and sarin in vitro and in vivo was.
( Springer-Verlag 1996

Arch Toxicol (1996) 70: 444—450

OR I G I N AL I NV EST I G AT I ON

Milan Jokanovic´ · Melita Kosanovic´ · Matej Maksimovic´

Interaction of organophosphorus compounds with carboxylesterases in the rat

Received: 4 July 1995/Accepted: 26 September 1995

Abstract Carboxylesterases (CarbE) are involved in detoxication of organophosphorus compounds (OPC) through two mechanisms: hydrolysis of ester bonds in OPC which contain them and binding of OPC at the active site of CarbE which reduces the amount of OPC available for acetylcholinesterase inhibition. This study of the interaction of rat plasma and liver CarbE with dichlorvos, soman and sarin in vitro and in vivo was undertaken in order to contribute to better understanding of the role of CarbE in detoxication of OPC. The results obtained have shown that inhibitory potency (I ) of dichlorvos, sarin and soman towards rat 50 liver CarbE was 0.2 lM, 0.5 lM and 4.5 lM, respectively, for 20-min incubation at 25°C. Second-order rate constants (k ) for liver CarbE inhibition were ! 2.3]105 M~1 min~1, 6.9]104 M~1 min~1 and 1.1] 104 M~1 min~1 for dichlorvos, sarin and soman, respectively. The corresponding values for plasma CarbE could not be calculated because of dominant spontaneous reactivation of inhibited CarbE. CarbE inhibited with these OPC in vitro spontaneously reactivate with half-times of 18, 143 and 497 min for sarin, dichlorvos and soman in plasma and 111, 163 and 297 min for sarin, soman and dichlorvos in liver, respectively. These results were also confirmed in experiments in vivo in which rats were subcutaneously treated with 0.5 LD 50 of these agents. The half-times of spontaneous reactivation of rat plasma CarbE in vivo were 1.2, 2.0 and 2.7 h for dichlorvos, sarin and soman, respectively. These findings have changed current understanding of the mechanism of interaction of CarbE with OPC and involvement of the enzymes in detoxication of OPC, suggesting an active and important role of the enzymes M. Jokanovic´ ( ) 1 · M. Kosanovic´ · M. Maksimovic´ Faculty of Pharmacy, Department of Toxicology, Vojvode Stepe 450, YU-11000 Belgrade, Yugoslavia Present address: 1 Istituto di Medicina del Lavoro, Universita degli Studi de Padova, Via Facciolati 71, I-35127 Padova, Italy

in metabolic conversions of OPC to their less toxic metabolites. Key words Organophosphorus compounds · Carboxylesterases · Detoxication of organophosphates · Soman · Sarin · Dichlorvos

Introduction In recent years, several papers concerning the role of carboxylesterases (EC 3.1.1.1) (CarbE) in poisonings with organophosphorus compounds (OPC) have been published (Bos\ kovic´ 1979; Sterri et al. 1983; Bos\ kovic´ et al. 1984; Clement 1984; Maxwell et al. 1987; Jokanovic´ 1989; Maxwell 1992; Jokanovic´ et al. 1994). It was shown that CarbE are involved in detoxication of OPC through two mechanisms. First is the hydrolysis of the ester bonds in OPC which contain them such as malathion (World Health Organization 1986; Fukuto 1990). These compounds can be also degraded by OPC hydrolases which attack the bond between phosphorus and acyl radical. In addition, OPC can be bound at the active site of CarbE, after which their concentration in blood circulation decreases, causing less inhibition of acetylcholinesterase in target tissues such as respiratory muscles and brain (Clement 1984; Jokanovic´ 1989). This second mechanism appears to be more important, since it is potentially related to all OPC. In these papers the interaction of OPC with CarbE was not studied under in vitro conditions. The involvement of CarbE in detoxication of OPC is directly related to the chemical structure of these compounds. Since the ability of CarbE to bind OPC is limited with regard to the amount of active sites at which OP molecules can be bound, it was suggested that CarbE are more important in detoxication of highly toxic OPC such as soman, sarin, tabun and paraoxon (Maxwell 1992). Inhibition of CarbE, except for inhibition of neuropathy target esterase, which is

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involved in organophosphate-induced delayed polyneuropathy (Johnson 1982), does not cause any known deleterious effect. The purpose of this study was to examine the interaction of CarbE with soman, sarin and dichlorvos in vitro and in vivo. The results obtained will contribute to the better understanding of mechanisms of toxic action of OPC, particularly of the role of CarbE in detoxication of these compounds.

Materials and methods Animals and dosing The experiments were performed in male Wistar rats weighing 180—220 g. All animals had free access to food and water. The animals were killed by decapitation. Blood was collected into heparinized test tubes and centrifuged for 10 min at 3000 rpm for separation of plasma. Liver homogenate (1 : 10) was prepared in physiological saline. Plasma and liver homogenates were kept at #4°C and CarbE activity was assayed as soon as possible but always within few hours. In in vivo experiments the rats were subcutaneously dosed with 0.5 LD of soman, sarin and dichlorvos. Their solutions in distilled 50 water (1 ml/kg) were prepared immediately before use and given at total volume of 0.1% body weight.

activity of CarbE in the same samples without inhibitors served as control.

Spontaneous reactivation of CarbE activity in plasma Aliquots of 2 ml plasma were incubated for 5 min at 25°C with the inhibitor of appropriate concentration which was added in total volume of 1%. After 5 min, 0.3 ml plasma was taken out and diluted with 1.3 ml ice-cold physiological saline. The next samples were taken out at time intervals which are indicated in corresponding Figs 4—6. Before the start of titration all the samples were kept at #4°C. For the titration, 1.5 ml diluted plasma was used, which was added to 8.5 ml prepared substrate. CarbE activity in diluted plasma without inhibitor was used as control.

Spontaneous reactivation of CarbE activity in liver Liver homogenate 2 ml was incubated for 5 min at 25°C with inhibitor at an appropriate concentration (to give about 80% inhibition) which was added in total volume of 1%. At the same time, a control sample was incubated with inhibitor solvent (acetone diluted in physiological saline). After 5 min, 8 ml ice-cold saline was added to both samples and the tubes were centrifuged at 27 000 g for 20 min at 4°C. The inhibition of CarbE was stopped by cooling and dilution of inhibitor. The pellet was resuspended in 2 ml ice-cold saline and the control sample (0.1 ml) was taken out after 5, 10, 20, 30, 40, 50 and 60 min. All samples were kept at #4°C until enzyme assay.

Chemicals Soman (O-1,2,2-trimethylpropyl methylphosphonofluoridate) and sarin (O-isopropyl methylphosphonofluoridate) (purity'98%) were obtained from the Military Technical Institute in Belgrade. Dichlorvos (2,2-dichlorvinyl dimethylphosphate) (purity 98.5%) was obtained from Zorka, S[ abac, Yugoslavia. Tributyrin (glycerol tributyrate) was purchased from Fluka, Buchs, Switzerland. Tributyrin (6.5 mM) was used as the substrate as 0.2% emulsion in physiological saline. The quality of the emulsion was improved with the addition of arabic gum (1 g/l).

Enzyme assays CarbE activity was assayed by the titrimetric pH-stat method (Clement 1982). The butyric acid liberated during hydrolysis of substrate was titrated with a standard solution of sodium hydroxide using a Radiometer titrigraph. The appropriate amount of substrate emulsion (8.5—10 ml), from which CO was earlier removed, was 2 titrated with 0.01 N NaOH at 25°C and pH 7.60. After addition of 0.2 ml plasma or 0.1 ml liver homogenate the mixutre was titrated further for 5—10 min. The amount of NaOH used during the first 5 min, when the hydrolysis of the substrate was linear, indirectly enabled the calculation of CarbE activity.

Time-course of progressive inhibition of CarbE with soman, sarin and dichlorvos Aliquots of 2 ml plasma or 3 ml liver homogenate, prewarmed at 25°C, were incubated with OP inhibitors of appropriate concentration which were added in total volume of 1%. The mixture was incubated at 25°C and in certain time intervals (0, 5, 10, 15, 20 and 25 min) the samples were taken out for determination of CarbE activity. Before the assays, these samples were kept at #4°C. The

Results According to Clothier et al. (1981), the reaction of an esterase with OP inhibitors has the following characteristics: a) the lines are straight, b) the slopes of the lines are proportional to the inhibitor concentration used, and c) the lines intersect the ordinate at 2. On these graphs a progressive increase of the enzyme inhibition as a function of time was always present. In Figs 1—3 the time-course of inhibition of rat plasma and liver CarbE is presented, with at least three different concentrations of soman, sarin and dichlorvos. From parts of these graphs representing inhibition of plasma CarbE because of dominant spontaneous reactivation, we could not calculate kinetic parameters of inhibition such as second-order rate constant (k!) and concentration inhibiting 50% of CarbE activity (I50). This process of spontaneous reactivation of CarbE in liver occurred at a much slower rate and it was possible to calculate these parameters, which are presented in Table 1. Figures 4—6, in which the ordinate represents log% CarbE inhibition and the abscissa, time of inhibition, show spontaneous reactivation of rat plasma and liver CarbE inhibited with these OPC. The extent of CarbE inhibition was proportional to the concentration of inhibitor used. This process of spontaneous reactivation followed the first-order kinetics and the lines at the graphs representing the effect of particular concentration of OP inhibitor were parallel. From the slopes of

446 Fig. 1 Time-course of inhibition of rat plasma and liver carboxylesterases with sarin

Fig. 2 Time-course of inhibition of rat plasma and liver carboxylesterases with soman

the lines, the rate constants of spontaneous reactivation of plasma and liver CarbE (k`3) and half-times of spontaneous reactivation (t1@2) have been calculated, and the values are given in Table 1. The results of our experiments in vitro were directly confirmed in experiments in vivo in which the rats were subcutaneously treated with 0.5 LD50 of soman, sarin and dichlorvos (Fig. 7). After obtaining the highest possible inhibition of plasma CarbE 15—30 min after poisoning, spontaneous reactivation of the enzyme appeared and it occurred in two different phases. The first phase of reactivation lasted until about 50% of total CarbE activity was achieved and then the spontaneous reactivation was continued at

a slower rate. Four hours after poisoning with sarin and dichlorvos, plasma CarbE activity was completely restored, but after soman treatment it reached about 70% of control activity. The half-times of spontaneous reactivation of plasma CarbE treated with 0.5 LD50 of dichlorvos, sarin and soman were 1.2, 2.0 and 2.7 h, respectively.

Discussion Acute toxic effects in poisoning with OPC are the consequence of inhibition of acetylcholinesterase

447 Fig. 3 Time-course of inhibition of rat plasma and liver carboxylesterases with dichlorvos

Table 1. Kinetic parameters of interaction of rat plasma and liver carboxylesterases with sarin, soman and dichlorvos in vitro. Control carboxylesterase activity in rat tissues was 20.5$3.7 lmol/min per g liver and 0.43$0.16 lmol/min per ml plasma (n"62). The values represent the mean$SD for 3—9 separate determinations with different inhibitor concentrations at 25°C. k second-order rate constant, a I concentration of inhibitor that inhibits 50% of enzyme activity, k first order rate constant of spontaneous reactivation, t half-time of 50 `3 1@2 spontaneous reactivation Inhibitor

Tissue

k ! (M~1min~1)

I 50 (lM/20 min)

k ]10~3 `3 (min~1)

t 1@2 (min)

Sarin

Liver Plasma Liver Plasma Liver Plasma

6.9$1.3]104 a 1.1$0.8]104 a 2.3$0.5]105 a

0.5$0.1 a 4.5$2.0 a 0.2$0.04 a

6.3$0.7 39.6$11.1 4.4$1.0 1.5$0.6 2.4$0.2 4.9$0.6

111$14 18$4 163$43 497$214 297$28 143$20

Soman Dichlorvos

!cannot be calculated because of dominant spontaneous reactivation

Fig. 4 Spontaneous reactivation of rat plasma and liver carboxylesterases inhibited with sarin

448 Fig. 5 Spontaneous reactivation of rat plasma and liver carboxylesterases inhibited with soman

Fig. 6 Spontaneous reactivation of rat plasma and liver carboxylesterases inhibited with dichlorvos

(AChE) and accumulation of endogenous acetylcholine at the sites of cholinergic transmission. The process of detoxication of OPC occurs at the same time as AChE inhibition and it represents a defensive mechanism of the organism in these poisonings. Detoxication of OPC is accomplished through several different reactions, including spontaneous hydrolysis and hydrolysis catalysed by OPC hydrolases, reactions with proteins (among them CarbE are of particular importance), deposition of OPC to tissue depots (such as lipid tissue) from which these compounds can be mobilized in their original form, which is capable of inhibiting AChE. Metabolic transformations of OPC through reactions with mixed function oxidases and glutathione-depen-

dent mechanisms also occur. Which of these processes of detoxication will be dominant depends mainly on the chemical structure of OPC and the affinity of enzyme systems to react with organophosphates. In Table 1, kinetic parameters of the reaction of CarbE with soman, sarin and dichlorvos are shown. In the literature there are various values of k and I ! 50 for this reaction because of different purity and origin of OPC, different methods, tissue fractions and substrates for the assessment of CarbE activity. Sterri and Fonnum (1987) found values of I of 2—5 lM (30 min 50 incubation at 30°C and pH 7.80) for inhibition of some isoenzymes of guinea-pig liver CarbE with soman, which is similar to our value of 4.5 lM for 20-min

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Fig. 7 Time-course of inhibition of plasma carboxylesterases in rats subcutaneously poisoned with 0.5 LD of soman, sarin and dichlor50 vos

incubation at 25°C and pH 7.60. Also, our value of 0.2 lM/20 min for inhibition of liver CarbE with dichlorvos is similar to the value of 0.085 lM/5 min at 25°C reported by Ecobichon et al. (1973). Our values of k for inhibition of liver CarbE with soman and sarin ! are different, but for dichlorvos are within the same order of magnitude as those reported by Maxwell (1989) (5.2]106 M~1 min~1 for soman, 3.1]106 M~1 min~1 for sarin and 7.1]105 M~1 min~1 for dichlorvos), which are related to CarbE in microsomal fraction of rat liver and determined towards a-naphtyl acetate as substrate. In Figs 4—6 the spontaneous reactivation of plasma and liver CarbE inhibited by soman, sarin and dichlorvos is shown under in vitro conditions. In the literature there are some data about this process but only for the specific CarbE called neuropathy target esterase (NTE) which is involved in organophosphate-induced delayed polyneuropathy and assayed towards phenyl valerate as substrate (Johnson 1982). After inhibition of hen brain NTE with some phosphoramidates there was about 90% spontaneous reactivation during 20 h at 37°C (Jokanovic´ and Johnson 1993). There was also some spontaneous reactivation of NTE inhibited with soman. Johnson et al. (1985) have shown that after inhibition of hen brain NTE with P(!) stereoisomers of soman, there appears at most 40% of spontaneous reactivation during 18 h incubation at 37°C. These results, which show much slower and incomplete spontaneous reactivation of CarbE inhibited by OPC than reported in Table 1, did not support this phenomenon.

Spontaneous reactivation of CarbE inhibited by OPC under in vivo conditions has been reported in a few papers but it occurred at a much slower rate. Bos\ kovic´ et al. (1984) have shown spontaneous reactivation of plasma CarbE in rats poisoned with 0.75 LD of soman, sarin and tabun. Gupta et al. (1987a) 50 have found 50% of spontaneous reactivation of plasma CarbE in rats treated with 100 lg/kg soman 24 h after poisoning. Gupta et al. (1987b) also reported 94% of reactivation of plasma CarbE in rats treated with 200 lg/kg tabun, but 7 days after poisoning. These results confirm the recent findings of Shih et al. (1994), who reported much faster elimination of sarin than soman in urine of rats in the form of alkylmethylphosphonic acid. This metabolite is also formed during spontaneous reactivation of CarbE by cleavage of the bond between phosphoryl residue and hydroxyl group at the active site of CarbE. If soman irreversibly binds to proteins, as stated by Shih et al. (1994), and having in mind that CarbE are more, while OPC hydrolases are less, important in detoxication of highly toxic organophosphates (Maxwell 1992), then on the basis of our results it appears that CarbE are one of the major factors involved in formation of this metabolite at least for soman.

Role and importance of CarbE in detoxication of OPC CarbE are involved in detoxication of OPC through two mechanisms — hydrolysis of ester bonds in OPC which contain them (World Health Organization 1986; Fukuto 1990) and binding reactions which reduce the amount of OPC available to react with acetylcholinesterase (Clement 1984; Jokanovic´ 1989). It was apparently shown that the reaction of OPC and CarbE is not irreversible as was thought previously (Junge and Krisch 1975; Maxwell 1992). The results of this study, especially those of rapid spontaneous reactivation of CarbE inhibited by sarin, soman and dichlorvos in vitro and in vivo, enabled us to propose a new role for CarbE in this process. The phosphorylation of CarbE occurs through binding of phosphorus from OPC to the serine hydroxyl group at the active site of CarbE. During spontaneous reactivation this phosphoryl residue is separated from CarbE, accepting a hydroxyl group as its new acyl radical. Recovered CarbE can be reinhibited by OP inhibitors and this newly formed OPC (i.e. organophosphorus acid) is much weaker esterase inhibitor which represents a non-toxic metabolite of the parent OP compound. We suggest an active role of CarbE in detoxication of OPC which occurs through metabolic transformations of OPC to their non-toxic and non-reactive metabolites. Acknowledgement This study was supported in part by the grant (Project 1810) from Serbian Ministry for Science and Technology.

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