Inhibition and aging of neuropathy target esterase by the stereoisomers of a phosphoramidate related to methamidophos

Toxicology Letters 93 (1997) 95 – 102

Inhibition and aging of neuropathy target esterase by the stereoisomers of a phosphoramidate related to methamidophos Miguel Angel Sogorb, Nuria Dı´az-Alejo, Marı´a C. Pellı´n, Eugenio Vilanova * Uni6ersidad Miguel Herna´ndez, Instituto de Neurociencias, Facultad de Medicina, Campus de San Juan, E-03550 San Juan de Alicante, Alicante, Spain Received 5 August 1996; received in revised form 1 August 1997; accepted 1 August 1997

Abstract Discrepancies in the aging reaction between neuropathy target esterase (NTE) inhibited in vitro and in vivo by racemic mixtures of O-alkyl O-2,5-dichlorophenyl phosphoramidates have been observed. It suggested the existence of differences in the interactions (inhibition and aging) between NTE and each stereoisomers of the above mentioned compounds. In order to verify this hypothesis, stereoisomers of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) were isolated by chiral column chromatography, followed by the evaluation of NTE inhibition and aging for each stereoisomers. The loss of reactivation capacity by KF was used as criterion of aging. The stereoisomer S-( − )-HDCP inhibited hen brain NTE with an I50 of 7.6 nM for 30 min of incubation, this being similar to the value obtained for the racemic mixture (I50 =6.2 nM), and much lower than that recorded for R-( + )-HDCP (I50 = 191 nM). NTE inhibited by HDCP racemic mixture and the stereoisomer S-( − )-HDCP was reactivated by KF after 20 h of incubation at 37°C. The NTE inhibited by R-(+ )-HDCP could not be fully reactivated after inhibition. © 1997 Elsevier Science Ireland Ltd. Keywords: Neuropathy target esterase; Phosphoramidate; Stereoisomer; Methamidophos; Organophosphorus compound

1. Introduction Certain organophosphorus compounds (OPs) cause a neurodegenerative syndrome known as organophosphorus induced delayed polyneuropa* Corresponding author. Tel.: + 34 6 5919477; fax: + 34 6 5919484; e-mail: [email protected]

thy (OPIDP) (Johnson, 1982). This disease is in turn related to the inhibition and posterior modification (aging) of an esterase known as neuropathy target esterase (NTE) (Clothier and Johnson, 1980). The phenomenon of aging is operatively defined as the loss of the reactivation capacity of the protein by nucleophilic agents (normally KF) (Clothier and Johnson, 1979).

0378-4274/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 3 7 8 - 4 2 7 4 ( 9 7 ) 0 0 0 8 4 - 2

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Methamidophos (O,S-dimethyl phosphorothioamidate) is an OP widely employed as insecticide. It is difficult to induce OPIDP in animal models with this compound due to its acute cholinergic effects (FAO, 1977), but some studies with the resolved stereoisomers have proved its neuropathic potential (Bertolazzi et al., 1991; Lotti et al., 1995). However, cases of the disease have been described in humans who ingested products containing this insecticide (Senanayake and Johnson, 1982). With the aim of characterizing both the mechanism by which this compound induces OPIDP and its interaction with NTE, different phosphoramidates (structural analogs of methamidophos in which NTE inhibiting power was enhanced) were developed (Vilanova et al., 1987). Hen brain NTE inhibited by racemic mixtures of the O-alkyl O-2,5-dichlorophenyl phosphoramidate family compounds was reactivated by KF, and was thus not affected by aging (Vilanova et al., 1987). However, when these compounds were administered to hens, they developed delayed neuropathy and NTE could no longer be reactivated by KF (Johnson et al., 1989). The following hypothesis of three points was proposed to account for these differences in behaviour in vitro and in vivo: (a) the two stereoisomers possess different NTE inhibiting capacities; (b) the more potent inhibitor produces NTE inhibition to a form that may be reactivated by KF, while the other yields a form that cannot be reactivated; and (c) the more potent inhibitor is degraded stereospecifically —the residual stereoisomer being responsible for the toxic effects observed with the racemic mixture. The present study investigates the existence of differences in NTE inhibition and aging between the stereoisomers of the O-alkyl O-2,5dichlorophenyl phosphoramidate family that may contribute to either support or reject the above hypothesis. To this effect, we employed a chiral HPLC technique to purify the stereoisomers of the compound O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP), and studied the inhibition and aging of hen brain NTE by both racemic mixture of HDCP and each of its pure stereoisomers. By comparing with the literature on stereoisomers of phosphoramidate analogs,

each HDCP stereoisomer was designated to a corresponding R– S configuration. 2. Material and methods

2.1. Reagents and biological material A racemic mixture (96% purity) of HDCP was synthesized by Drs J. Pardo and C. Na´jera, of the Departamento de Quı´mica Orga´nica (Universidad de Alicante, Alicante, Spain). Benzenesulphonyl fluoride was produced in our laboratory according to the method described by Johnson (1975). Mipafox (N,N%-di-isopropylphosphorodiamidic fluoride) and phenyl valerate were obtained from Lark Enterprises (Webster, MA). Paraoxon (O,O%-diethyl p-nitrophenyl phosphate) and the rest of the reagents and solvents employed were all of analytical grade and were purchased from Sigma Chemicals (Madrid, Spain). Particulate fraction of hen brain was used as the source of NTE. Adult red hens were housed in the installations of the Universidad Miguel Herna´ndez and sacrificed by decapitation; the brains were immediately removed and homogenized in cold 50 mM Tris/1 mM EDTA buffer pH 8.0. After that, the homogenate was centrifuged at 150 000× g over 60 min (4°C). Finally, the pellet was resuspended in the same buffer at 400 mg tissue/ml and was employed for NTE inhibitions and aging experiments.

2.2. HDCP stereoisomers The HDCP stereoisomers were obtained from the racemic mixture using a chiral chromatographic method initially developed in our laboratory for analytical purposes (Dı´az-Alejo and Vilanova, 1993). We applied this method but injecting the maximum amount possible of HDCP racemic mixture accepted by the column without loss of resolution (162.5 mg). An automatic fractions collector was placed at the base of the detector to collect the eluted compounds. The material from several programmed injections was collected automatically, dried and finally dissolved in acetone for the inhibition and aging assays.

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2.3. Determination of NTE acti6ity Pairs of samples of 40 mM paraoxon (sample B), or 40 mM paraoxon plus 250 mM mipafox (sample C) containing both 5 mg of hen brain particulate fraction were incubated for 30 min at 37°C. A 1 ml volume of phenyl valerate 2.7 mM was then added, and the reaction stopped after 30 min of incubation at 37°C. Finally, the phenol produced by the reaction was assayed in both samples, using the method described by Johnson (1977). NTE activity was calculated from the differences between samples B and C.

2.4. NTE acti6ity reacti6ation and aging assays 2.4.1. Protocol A: remo6ing inhibitor The tissue samples (400 mg/ml) were inhibited with 1.2 mM of HDCP racemic mixture or (−)HDCP, or 200 mM of ( +)-HDCP stereoisomers for 60 min at 37°C and; after that, samples were diluted 1/20 in cold 50 mM Tris/1 mM EDTA pH 8.0. Then, the inhibitor was washed by centrifugation (150 000×g, 20 min, three times). The final pellet was resuspended in cold 10 mM Tris/1 mM EDTA buffer pH 8.0 buffer. Two aliquots were prepared, one of which was immediately subjected to reactivation treatment, while the other was incubated at 37°C for 22 h to allow NTE aging. The reactivation treatment was based in a previously described procedure (Clothier and Johnson, 1979) with slight modifications. A 1 ml volume of sample (30 mg of tissue) was incubated for 60 min at 37°C with 1 ml of 300 mM KF (reactivating agent) or 300 mM KCl (ion strength control) both contained in 50 mM Tris/50 mM citrate/1 mM EDTA buffer pH 5.2. The chloride and fluoride were eliminated by centrifugation (150 000× g, 20 min, once), the final pellet was resuspended in 50 mM Tris/1 mM EDTA pH 8.0. 2.4.2. Protocol B: with short inhibition time and diluting the inhibitor The tissue samples (400 mg tissue/ml) were inhibited with 4 mM of ( −)-HDCP or 400 mM of ( +)-HDCP for 10 min at 37°C. Reactivation treatment was applied after inhibition by direct dilution of the samples with 200 ml of 50 mM

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Tris/50 mM citrate/1 mM EDTA buffer pH 5.2 containing 300 mM KF (reactivating agent) or 300 mM KCl (ion strength control). After inhibition, reactivation and removal of chloride and fluoride the residual NTE activity was hen assayed using benzenesulphonyl fluoride at a concentration of 100 mM in place of paraoxon, as the latter in the presence of fluoride ion traces is converted to a potent NTE inhibiting derivative (Clothier and Johnson, 1979). The results were expressed in percentages with respect to a control subjected to the same process but without initial inhibition by HDCP. 3. Results

3.1. Purification of HDCP stereoisomers After concentration of the purified stereoisomers, purity was estimated by injecting the samples with the same chromatographic protocol. The samples of each stereoisomer showed no detectable levels of the other isomer, absolute purification degree was higher than 99.5%. The sign of polarized light rotation allowed us to designate as (+ )-HDCP and (−)-HDCP those stereoisomers eluted in first and second place from the HPLC column.

3.2. Hen brain NTE inhibiting power of the HDCP stereoisomers Hen brain samples (5 mg tissue/ml) were simultaneously incubated with paraoxon (sample B) or paraoxon plus mipafox (sample C) and different concentrations of HDCP racemic mixture or each of its stereoisomers. The substrate was then added and residual NTE activity assayed according to standard protocol. The results were expressed as percentage activity with respect to a control preincubated without HDCP, and were plotted against inhibitor concentration (Fig. 1). For 30 min of incubation, (− )-HDCP yielded an I50 of 7.6 nM, and was a much more potent NTE inhibitor than (+)-HDCP, with an I50 of 191 nM (Table 1). The value recorded for the racemic mixture (I50 = 6.2 nM) was fundamentally attributable to the effect of (− )-HDCP (Table 1).

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3.3. Aging of hen brain NTE acti6ity inhibited by HDCP racemic mixture, (+) -HDCP and ( − ) -HDCP The concentrated hen brain samples (400 mg tissue/ml) were inhibited with HDCP, as men-

Table 1 I50 values deduced from the NTE inhibition by the HDCP racemic mixture and stereoisomers Compound

I a50 (nM)

Racemic mixture R-(+)-HDCP S-(−)-HDCP

6.2 (2) 191 (3) 7.6 (2)

Mean for (n) independent experiments measured in triplicate is presented. a The I50 values refered to 30 min incubation and are graphically deduced from the plots of % NTE activity versus inhibitor concentration (see Fig. 1).

Fig. 1. Hen brain NTE activity inhibition curves by the HDCP racemic mixture (panel A), R-(+)-HDCP (panel B), and S-( −)-HDCP (panel C). The 100% activity controls exhibited activities of 19389540 mU/g tissue (mean9 S.D., n= 6). The results of one experiment measured in triplicate is illustrated. Several experiments were carried out which exhibited similar results. The mean of all I50 calculated is presented in Table 1.

tioned in Section 2.4.1. The employed concentrations were 1.2 mM of racemic mixture and (− )-HDCP and 200 mM of ( + )-HDCP. The higher inhibitor concentrations than in the I50 determinations were needed because partial degradation of the compound occurred at high tissue concentration. The existence of phosphotriesterase activity in hen brain capable of hydrolysing HDCP was previously demonstrated (Diaz-Alejo et al., 1990; Dı´az-Alejo et al., 1994; Reiner et al., 1993). The presence of this phosphotriesterase explains the necessity of the higher HDCP concentrations employed in aging experiments, since inhibition it is performed in homogenates of 400 mg tissue/ml while I50 values are calculated for homogenates of 5 mg tissue/ml. So that, the degradation effect was more marked in aging studies than in inhibition experiments and, therefore, we needed high inhibitor concentrations to get high percentage of inhibition. After incubating the tissue with the inhibitors, these were washed thoughtfully from the medium by three centrifugations and resuspension in cold buffer. Finally, reactivation with F − (or Cl − as control) was carried out in one aliquot immediately after removing the inhibitors, and in other after 22 h of incubation at 37°C. Table 2 shows the percentage of NTE activity corresponding to each sample assayed. NTE phosphorylised by the racemic mixture and by the stereoisomer (− )HDCP was reactivated even after 22 h of incubation at 37°C. Therefore, (− )-HDCP inhibits NTE but modifies it to a form that may be reactivated with KF, i.e. aging is not induced. In contrast, the

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Table 2 Reactivation of NTE inhibited by HDCP mixture or its pure stereoisomers assayed removing inhibitors NTE activity (%)a

Inhibitor compound

After removing the inhibitor

Racemic mixture R-(+)-HDCP S-(−)-HDCP

After 22 h for aging

Cl− (control)

F−

Cl− (control)

F−

29/6 26/15 2/2

87/75 8/5 91/100

22/9 7/6 1/2

76/85 16/14 95/80

Reactivation was assayed according to protocol A described in Section 2.4. The results of two independent experiments measured each one by duplicate are shown. a The 100% activity control exhibited an activity of 21569 174 mU/g tissue (mean9S.D.). There was no significant differences in controls not incubated and incubated for 22 h.

stereoisomer (+)-HDCP could not be reactivated even after the inhibition and removal of the inhibitor. In order to check if ( +)-HDCP can be reactivated immediately after inhibition, experiments were performed according to protocol B described in Section 2.4.2. Applying this protocol, inhibitor concentration was increased and inhibition time was reduced to 10 min. In these experiments, the inhibitor was not washed out but the solution (0.525 ml) was diluted with 200 ml of buffer containing Cl − or F − . Reactivation time was 60 min. This type of assay had the inconvenience that the inhibitor is not removed but presented the possibility of reducing the elapsed time for aging after the inhibition. Results are showed in Table 3 Reactivation of NTE inhibited by HDCP mixture or its pure stereoisomers assayed without removing inhibitors Inhibitor compound

R-(+)-HDCP S-(−)-HDCP

NTE activity (%)a with: Cl− (control)

F−

5/1 11/33

33/30 90/97

Reactivation was assayed according to protocol B described in Section 2.4.2. Inhibition was carried out by incubating the tissue at 37°C for 10 min. Immediately after, samples were diluted with buffer containing Cl− or F− for reactivation. The results of two independent experiments, each one measured in duplicate are shown. a The 100% activity control exhibited an activity of 1953 mU/g tissue.

Table 3. We recorded that samples treated with(+ )-HDCP and Cl − exhibited an activity about 1–5% of control, while, activity reached 30–33% of control in samples reactivated with F − indicating that some practical but significant reactivation can be observed using this protocol that reduces as long as possible the aging time. NTE activity of samples inhibited with (− )HDCP and reactivated in the same conditions was recovered almost totally (from 11–33% with Cl − to 90–97% with F − ). 4. Discussion The present study shows that the two stereoisomers of a phosphoramidate analog of the commercial insecticide methamidophos exhibit different properties upon interacting with NTE from hen brain. The stereoisomer (+)-HDCP was found to be a less potent NTE inhibitor than (−)-HDCP; consequently, the latter was the main responsible for the NTE inhibition observed with the racemic mixture. In addition, both the racemic mixture and (− )-HDCP inhibited NTE with modification to a fluoride-reactivatable form, which indicates that aging did not occur. However, NTE inhibited by (+)-HDCP could not be totally reactivated with fluoride. Therefore, the results obtained confirm the existence of different inhibiting capacities between the stereoisomers, and a different modification of the NTE inhibited by them.

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Fig. 2. Inhibition of NTE by compound of the families of methamidophos and HDCP. As the leaving groups are methanothiol and 2,5-dichlorophenol both family compounds yield the same form of inhibited NTE. (R = alkyl group; E-OH =NTE).

HDCP racemic mixture exhibited an I50 of 6.21 nM for 30 min of incubation of hen brain NTE (Table 1). This value is comparable to the value reported by Vilanova et al. (1987) (4.4 nM) for 20 min of incubation and by Reiner et al. (1993) for the same time (4.9 nM). The fact that the value presented in this study is slightly higher can be explained, as it is obtained for samples with a tissue concentration of 5 mg/ml, while Reiner et al. reported the value for homogenates of 2.5 mg/ml. It is known that, as it has been previously explained, due to the enzymes which degradate HDCP, higher concentrations of inhibitor are required for inhibition in more concentrated homogenates, as reported by HDCP (Diaz-Alejo et al., 1990; Reiner et al., 1993; Dı´az-Alejo et al., 1994) and its family compound (Vilanova et al., 1987; Diaz-Alejo et al., 1990). This can explain why in the aging experiments using high tissue concentrations (400 mg/ml) a much higher HDCP concentration was needed for high NTE inhibition than expected from I50 deduced from diluted tissues homogenates (5 mg/ml). Moreover, the longer incubations times employed also can account for a more complete HDCP degradation, and therefore, higher HDCP concentrations are required to reach the same inhibition. NTE inhibited by (+ )-HDCP was not reactivated neither after removing the inhibitor nor 22 h later (Table 2). Two possible explanations arise from this result: (1) ( + )-HDCP inhibits NTE in a stable form that can not be reactivated under any

circumstance; or (2) NTE inhibited by (+ )HDCP undergoes a fast aging reaction during the time of inhibition (60 min) and removal of the enzyme by centrifugation (longer than 60 min). Since, the chemical nature of the bond between NTE and the phosphorus atom of HDCP it is the same, and NTE inhited by ( − )-HDCP could be reactivated under all conditions assayed (Tables 2 and 3), it is unlikely the first hypothesis. Moreover we were able to get a partial reactivation of NTE inhibited by (+ )-HDCP when times of aging were reduced as technically possible using protocol B (Table 3). These considerations argue in favour that the NTE inhibited by this compound undergoes a quick aging and, only technical problems hindered the reactivation. Compounds of the family of methamidophos and O-n-hexyl S-methyl phosphorothioamidate (HSMet) and HDCP produces the same form of inhibited NTE (Fig. 2) (Vilanova et al., 1987; Johnson et al., 1989). It was been previously demonstrated that one of the stereoisomers of methamidophos (Johnson et al., 1991) and HSMet (Johnson and Safi, 1993; Johnson and Read, 1993) inhibited NTE to a reactivable form (as HDCP) while the other steroeisomers inhibited the enzyme to a form that ages. Therefore, the aging reaction is the only logical explanation to the results obtained for (+ )-HDCP (Tables 2 and 3), which produces the same inhibited NTE that the stereoisomer of HSMet configuration that ages.

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The data presented in the present study may be correlated to those published for the isomers of methamidophos, according to which the NTE inhibited by the R(+ ) isomer (Fig. 3) of this compound was reactivated, while that inhibited by the S(−) isomer was not (Johnson et al., 1991). The same was observed with the isomers of HSMet (Johnson and Safi, 1993; Johnson and Read, 1993). Thus, in comparison with these two studies, the stereoisomer (+)-HDCP which inhibits NTE and induces aging, exhibits the same properties upon interacting with NTE as the S( − ) isomers of methamidophos and HSMet; it may thus be concluded that, since both compounds produce the same form of inhibited NTE (Fig. 2), the ( +)-HDCP enantiomer possesses the same spatial configuration as these compounds. Consequently, on applying the RS or Cahn-IngoldPrelog nomenclature rules, it can be deduced that the stereoisomer (+ )-HDCP corresponds to R( + )-HDCP, and stereoisomer (−)-HDCP to S( −)-HDCP (Fig. 3). Preliminary studies carried out in our laboratory confirm the existence of stereospecific degradation in hen liver and brain (Dı´az-Alejo et al., 1994) and the same was demonstrated in rabbit serum for HSMet (Johnson and Read, 1993). In

Fig. 3. Configuration of the stereoisomers of methamidophos and HDCP, which inhibit NTE but do not not induce aging.

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vitro, the HDCP racemic mixture produces an inhibited NTE form that does not age. In this study we have demonstrated S-(− )-HDCP, the most potent NTE inhibitor, does not induce NTE aging and should be responsible of the in vitro properties of the racemic mixture. However, it has been stated that R-(+ )-HDCP is less potent NTE inhibitor and induces aging, as observed in vivo after dosification with racemic mixture. Therefore, R-(+ )-HDCP should be the enantiomer that remains in vivo at concentrations to reach the nervous system and inhibit NTE.

Acknowledgements This work was financed by SAF 96/0168 and by FISSS (95/0053). M.A.S. and N.D.A. received a grant from the Spanish Ministry of Education and Science. The authors thank Drs J.J. Ortiz and C. Na´jera (Departamento de Quı´mica Orga´nica of the Universidad de Alicante) for their technical assistance in determining the specific rotation of the HDCP stereoisomers.

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