In Vitro and in Vivo Assessment of the Effect of Impurities and Chirality on Methamidophos-Induced Neuropathy Target Esterase Aging

Thomas Kellner*,1, James Sanborn{dagger} and Barry Wilson{ddagger}

* Department of Pesticide Regulation, California Environmental Protection Agency, Sacramento, California 95814; {dagger} Department of Entomology, University of California, Davis, California; and {ddagger} Department of Animal Science, University of California, Davis, California

Received August 31, 1999; accepted November 18, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In vitro and in vivo studies evaluated neuropathy target esterase (NTE) inhibition and aging (i.e., loss of reactivation potential) by analytical and technical grade racemic and resolved L-(–) and D-(+) isomers of methamidophos (O,S-dimethyl phosphoramidothioate). For studies in vitro, microsomal protein from phenobarbital-induced livers was isolated from chick embryos and NTE inhibition assays were performed using chick embryo brain homogenate treated with 1 or 5 mM methamidophos (with and without metabolic enzymes); for studies in vivo, hens received 30 to 35 mg/kg methamidophos injected into the pectoral muscle. NTE aging in hens was assessed 24 h later or after 30 min to 1 h incubation in vitro using solutions of potassium fluoride (KF) reactivator. Technical methamidophos produced significantly higher levels of aged-inhibited NTE than analytical methamidophos or isolated optical isomers. In vivo, technical methamidophos produced 61% total NTE inhibition with 18% aged and 43% unaged NTE; hens receiving analytical grade averaged 6% aged, 52% unaged, and 58% total NTE inhibition. Results for 1 mM analytical methamidophos in vitro were 5% aged, 54% unaged, and 59% total inhibition; for 1 mM technical methamidophos, values averaged 11% aged, 50% unaged, and 60% total NTE inhibition. The degree of NTE aging obtained both in vivo and in vitro for the isolated D-(+) and L-(–) isomers never exceeded that obtained using analytical grade. These data indicate that impurities in methamidophos could contribute to OPIDN potential. The in vitro methodology described could be applied to first tier screening for detection of NTE inhibition and aging, thus reducing the need for whole-animal testing for OPIDN.

Key Words: methamidophos; delayed neurotoxicity; OPIDN; neuropathy target esterase; organophosphorus compound; stereoisomer; potentiation of toxicity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Some organophosphorus compounds (OPs) are capable of inducing a specific form of neurotoxicity known as organophosphate-induced delayed neuropathy (OPIDN), which is characterized by axonal degeneration and secondary demyelination of both ascending and descending nerve fibers (Cavanagh, 1964Go). A high degree of inhibition (70%) of a specific esterase fraction known as neuropathy target esterase (NTE) after single exposure to a compound correlates with delayed neurotoxicity and resulting ataxia 10–14 days later (Johnson, 1975aGo). Recent developments in NTE purification techniques have led to immunolocalization studies in neuronal cell bodies and axons and molecular cloning of NTE (Glynn et al., 1999Go).

The inhibition of NTE can result in either unaged NTE, with reactivation of enzymatic activity possible using nucleophilic reactivator potassium fluoride (KF), or aged NTE, which has undergone the loss of an alkyl group ("aging" reaction) and cannot be reactivated by KF. Structure-activity studies (Davis and Richardson, 1980Go; Johnson, 1975bGo; Johnson, 1988Go) and NTE inhibition/aging studies (Johnson, 1974Go; Johnson and Read, 1987Go; Johnson et al., 1988Go; Ohkawa, et al., 1980Go; Williams, 1983Go) have demonstrated a link between the production of aged-inhibited NTE and the subsequent appearance of OPIDN. Compounds such as diisopropyl fluorophosphonate (DFP), which inhibit 70–80% of NTE in vivo after single dosages and undergo rapid aging reactions, are considered full NTE agonists. In contrast, non-aging inhibitors such as phenylmethanesulfonyl fluoride (PMSF), which can protect from neuropathy when given before agonists or cause a mild neuropathy only after repeated dosing at almost complete NTE inhibition, are considered NTE antagonists (Lotti et al., 1995Go).

Various factors, including stereochemistry and presence of impurities, can modify OP toxicity. Although numerous studies have dealt with the differential inhibition of acetylcholinesterase (AChE) by the optical isomers of various compounds (Jianmongkol et al., 1999Go; Ohkawa et al., 1977Go; Ooms and Boter, 1965Go; Seiber and Tolkmith, 1969Go; Wustner and Fukuto, 1974Go), fewer studies of this type have focused on NTE inhibition. One such study by Johnson and Read (1987) demonstrated different responses of NTE to the L-(–) and D-(+) isomers of O-ethyl O-4-nitrophenyl phenylphosphonate (EPN oxon). More recently, Wu and Casida (1994) established that the resolved isomers of 2-substituted 4H-1,3,2-benzodioxaphosphorin 2-oxides differentially inhibited NTE. Another factor that can modify the OP toxicity is the presence of impurities. In the case of malathion, the isomalathion content and other contaminants such as trimethyl phosphorothioates were found to be important in increasing its toxicity (Aldridge et al., 1979Go).

Methamidophos (O,S-dimethyl phosphoramidothioate), sold as Monitor in the United States and Tamaron in other countries, has been linked to cases of organophosphate-induced delayed neuropathy (OPIDN) in Sri Lanka (Senanayake and Johnson, 1982Go) and China (Sun et al., 1998Go). The 10 patients in Sri Lanka had acute cholinergic symptoms immediately after accidental exposure or attempted suicide and neuropathy developed 2–3 weeks later, with at least six patients showing pyramidal-tract involvement. A bottle from one of the cases contained methamidophos and small amounts of impurities including O,O-dimethyl phosphoramidothioate and an N-methyl analogue of methamidophos. When this technical material was administered to hens at about twice the LD50 (hens received atropine protection), about 50% of brain NTE was inhibited.

Because NTE aging distinguishes inhibitors with high from those with low potency to induce OPIDN, an increased aging potential in the technical grade product compared to the analytical grade or the resolved isomers would indicate a contributing role of impurities in the subsequent formation of delayed neuropathy. The possibility that impurities in technical grade methamidophos could enhance neuropathic potential was evaluated by assessing NTE inhibition and aging by analytical and technical grade racemic and resolved L-(–) and D-(+) isomers of methamidophos both in vivo and in vitro.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Racemic methamidophos sources and optical isomer synthesis.
Both enantiomers of methamidophos [D-(+) and L-(–)] were synthesized by the method of Miyazaki with modifications (Miyazaki et al., 1988Go). Racemic methamidophos (analytical grade, 99.4% purity and technical grade, 73.1% purity) was obtained from Chevron, Inc. (structure shown in Fig. 1Go).



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FIG. 1. Structures of key chemicals tested for NTE inhibition (left column) and impurities detected in technical grade methamidophos by GC-MS (right column).

 
NTE inhibition/aging studies.
NTE aging assays were performed using brain homogenates from adult hens and 3-week-old chicks treated in vivo and from 18-day-old chick embryos treated in vitro; both experimental systems employed potassium fluoride (KF) as the nucleophilic reactivator. The in vitro assay, incorporating metabolic activation, NTE inhibition and measurement of aging, was used to determine the role metabolic activation in NTE inhibition and aging by various forms of methamidophos and to validate an in vitro alternative to whole-animal testing.

Hens 48–64 weeks of age were housed in individual cages. The animal rooms were kept in optimum conditions for maintaining hens in a laying condition (e.g., laying ration provided ad libitum with light/dark cycle of 16/8 h); in vivo experiments were designed to be short in duration in order to minimize discomfort to the animals.

Aging measurements in vivo.
For hens receiving methamidophos technical, a 70 mg/ml solution of the OP was prepared (in distilled water) and birds were dosed (im) in the pectoral muscle at a dose of 35 mg/kg (with 73.1% purity, actual methamidophos concentration of 25.6 mg/kg). Birds that received analytical methamidophos were dosed at 30 mg/kg (29.8 mg/kg with 99.4% purity; analysis by Chevron, Inc., verified by TLC and GC-MS). Although most of the experiments involved the use of adult hens, one experiment was performed with 3-week-old chicks to reduce use of limited supplies of optical isomers. Negative control birds received distilled water and positive controls received DFP (Sigma Chemical Co., St. Louis, MO) by im injection. This positive control rapidly forms aged-inhibited NTE (Clothier and Johnson, 1979Go; Williams and Johnson, 1981Go; Williams, 1983Go) and was used to verify if the assay could detect the loss of NTE reactivation capacity. The ability of the KF system to reactivate a reversible inhibitor was also tested, with some control birds receiving 10 mg/kg phenyl di-n-pentylphosphinate (PNPP), a compound that has been shown to inhibit NTE without aging (Johnson et al., 1988Go). All birds were dosed with 20 mg/kg atropine and 50 mg/kg 2-PAM (in the opposite pectoral muscle) before receiving the OP to reduce cholinergic symptoms.

Twenty-four hours after dosing, the birds were sacrificed by exsanguination and whole brain was homogenized by teflon glass (10 strokes, 1200 rpm) and sonication (twice for 3 s, setting 4) in ice-cold 50 mM Tris-HCl, 0.2 mM EDTA, pH 8 buffer (NTE assay buffer). A fresh solution of potassium fluoride (KF) reactivation buffer and potassium chloride (KCl) was prepared (250 mM in 50 mM Tris-Citrate, 0.2 mM EDTA, pH 5.2) and reactivation was initiated by adding 1 ml brain homogenate to 10 ml KF reactivation buffer. Another 1-ml aliquot was added to 10 ml KCl-containing buffer in plastic test tubes (buffer prewarmed to 37°C in shaking water bath). After 30 min incubation, tubes were cooled on ice and 25 ml ice-cold distilled water was added to each to slow the reaction. In some experiments the reactivation volumes were reduced for convenience, with 0.5 ml of brain homogenate added to 3 ml reactivation buffer and reaction slowed with 8.5 ml ice-cold distilled water.

The cooled tubes were centrifuged at 27,000 x g for 60 min., the supernatant was discarded, and the pellet was resuspended in 2 ml NTE assay buffer. NTE activity (reported as nmoles/min/mg protein) was determined (using 15 or 20 µl of homogenate) using benzenesulphonyl fluoride (25 µl of 7.5 mM solution in acetone) instead of paraoxon (Johnson and Read, 1987Go). Total volume of the assay was 750 µl, as modified by Thomas et. al. (1989). At the end of the assay, three 300-µl samples from the final assay volume of 1.5 ml were transferred to wells of a 96-well microplate, and absorbances were read at 490 nm. Specific activity of NTE was determined after protein measurements (Lowry et al., 1951Go).

Aging measurements in vitro (with and without metabolic activation).
Microsomal protein from phenobarbital-induced livers was isolated from chick embryos, and NTE inhibition assays using chick embryo brain (with and without metabolic enzymes) were performed according to the method of Chow et al. (1986). Incubation time of test compound with embryo brain was 1 h [1 mM technical and analytical grade or 5 mM D-(+) and L-(–) isomers] or 30 min (positive controls). After incubation, 2.5 ml ice-cold 10 mM Tris-HCl buffer (pH 7.4) and 50 µl of CaCl2 (0.8 M) were added to each tube, followed by centrifugation at 24,000 x g (max) for 30 min; the supernatant was discarded. Pellets were resuspended in 1 ml 10 mM Tris-HCl buffer by short (3 s) bursts of sonication while in an ice bath. Reactivation of inhibited NTE using fresh solutions of potassium fluoride and measurement of enzyme activity proceeded as described previously. Leptophos (O-[4-bromo-2,5-dichlorophenyl] O-methyl phenylphosphonothioate) with metabolic activation and leptophos oxon without activation were incubated at 37°C for 30 min. at 0.1 mM concentrations and served as positive metabolic activation and positive aging controls, respectively. Leptophos requires metabolic activation to become a potent NTE inhibitor (Abou-Donia, 1983Go; Sanborn et al., 1977Go) and was used to verify the effectiveness of the in vitro metabolic activation system. Leptophos oxon has been shown to form aged-inhibited NTE (Clothier and Johnson, 1980Go).

Chemical analysis.
Analytical and technical methamidophos was analyzed by thin layer chromatography (TLC) using 8 x 3.5 cm (1-mm thickness) silica gel 1B plates, developed with iodine vapor (running solvent EtOH:ethyl acetate, 10:90). One TLC preparation of technical methamidophos included a high-resolution GC-MS (VG Masslab instrument with electron impact ionization (E.I.) detector, HP 5890 gas chromatograph, DB-1 column, 50°C (2 min) to 250°C (10 min) at 10°C/min). The TLC plate was loaded with a concentrated spot of technical methamidophos and the plate was run in the solvent mentioned above. Two spots, one corresponding to methamidophos (analytical was run side by side and developed with iodine) and another of unknown composition were seen. The unknown spot was cut out of the plate and dissolved in methanol and analyzed by high-resolution GC-MS.

Analysis of the technical grade methamidophos was also performed before in vivo or in vitro experiments using a Hewlett-Packard GC-MS with a MSD detector, 20m DB-17 column, injector temp. 250°C, column 50°C to 250°C at 7°C/min. This analysis was performed in two different solvents (methanol and ethyl acetate) to ensure that new products were not being formed by reaction with the solvent. About 1 year later, this same analysis was run on this sample of technical methamidophos (stored continuously at –20°C) and similar results were obtained. This later analysis also included high-resolution GC-MS using a VG Masslab instrument with an electron impact ionization (E.I.) detector (HP 5890 GC), conc. 2 mg/ml, DB-1 column, 50°C (2 min.) to 250°C (10 min.) at 10°C/min. Methamidophos optical isomers (and organic synthesis intermediates) were analyzed by nuclear magnetic resonance (NMR), 60, 360, and 500 Hz, after being dissolved in deuterated chloroform.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Synthesis of Methamidophos Optical Isomers
Results from specific rotation analyses are shown in Table 1Go. The specific rotation of the D-(+) isomer (+49.6°), slightly higher in magnitude than that of the L-(–) isomer (–40.2°), was similar to published values of +55.0° and –53.9°, respectively. Optical purity of the of D-(+) and L-(–) isomers was estimated by evaluating the precursor diastereomers of S-methyl N-prolinyl benzyl phosphorodiamidate by NMR (CDCl3), 500 MHz. Assessment of peaks centered at {delta} 4.39 and {delta} 4.20 (quartet, OCH2Ar) indicated an optical purity of > 99% and 97.5% for the forms corresponding to the D-(+) and L-(–) enantiomers, respectively. These results, together with NMR and GC-MS analyses of the final products, indicated that the isomers were suitable for NTE inhibition studies.


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TABLE 1 Specific Rotation and Purity of Resolved Isomers of Methamidophos
 
Chemical Analysis
TLC analysis showed one spot for the analytical sample, with an Rf value of 0.61. The two technical sample runs showed this spot with the same Rf values, plus another spot that migrated farther with the solvent phase Rf value of 0.89.

GC-MS analysis of technical grade methamidophos showed a principal peak of O,S-dimethyl phosphoramidothioate (M.W. 141, methamidophos) accounting for approximately 80% of the total based on total ion current values (Fig. 2Go). Three other major components included O,O-dimethyl phosphoramidothioate (also M.W. 141; about 10% of the total), O,O,N-trimethyl phosphoramidothioate (M.W. 155; 2% of total) and O,O,S-trimethyl phosphorothioate (M.W. 156; less than 1%).



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FIG. 2. Chromatogram from high-resolution GC-MS analysis (DB-1 column, E.I. detector) of technical methamidophos in methanol. Peak at position 696: O,S-dimethyl phosphoramidothioate (methamidophos); 516: O,O-dimethyl phosphoramidothioate; 546: O,O,N-trimethyl phosphoramidothioate; 537: O,O,S-trimethyl phosphorothioate.

 
NTE Inhibition/Aging in Vivo and in Vitro
In a series of four experiments, racemic analytical methamidophos and technical methamidophos were tested for their ability to produce aged-inhibited NTE in vivo 24 h after chemical exposure (Table 2Go). A 35 mg/kg dose of technical methamidophos produced an average of 61% total NTE inhibition at the end of the KF reactivation procedure, with 18% aged and 43% unaged NTE. Table 2Go also shows the results of birds that received analytical methamidophos. Although the actual dose of analytical methamidophos was slightly higher than the technical, the amount of aged-inhibited enzyme was significantly less (p < 0.05, Wilcoxon rank-sum test) with 6% aged-inhibited and 52% unaged-inhibited NTE. It should be noted the ratio of the unaged-inhibited NTE for technical and analytical methamidophos (Table 2Go: 43/52 = 0.83) reflected the difference in actual concentration of pure methamidophos administered to the birds (25.6 mg/kg/29.8 mg/kg = 0.86). The positive control birds given 200 µg/kg DFP showed virtually all inhibited NTE was of the aged type (48%). As expected, the opposite result was seen in birds administered 10 mg/kg PNPP, with all of inhibited NTE unaged.


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TABLE 2 Derived Values for Unaged and Aged Inhibited NTE after OP Treatment in Vivo
 
The results for 1 mM analytical methamidophos (30 min to 1 h incubation with metabolic activation) obtained in vitro were almost identical to those obtained in vivo, with 5% aged, 54% unaged, and 59% total NTE inhibition (Table 3Go). The in vitro data for 1 mM technical methamidophos indicated 11% aged, 50% unaged, and 60% total NTE inhibition. The concentrations of technical methamidophos were formulated to compensate for the 73.1% purity, so that the brain homogenate was exposed to 1 mM pure methamidophos in addition to impurities. This was reflected in the nearly equal unaged-inhibited NTE noted for technical and analytical, which was 50 and 54%, respectively. Administration of 0.1 mM leptophos oxon (without metabolic activation) resulted in derived values of 25% aged, 74% unaged, and 99% total NTE inhibition. Leptophos was also tested (with and without metabolic activation) and as expected, leptophos without activation exhibited low total NTE inhibition (37%) and with activation showed 84% inhibition. The degree of aging was less than leptophos oxon (12% vs 25%, respectively).


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TABLE 3 Derived Values for Unaged and Aged Inhibited NTE in Hen Brain Treated in Vitro with OPs with and without Metabolic Activation
 
Because of the limited amount of material synthesized, most of the experiments involving optical isomers were performed in vitro. In the lone in vivo experiment using 3-week-old chicks, two out of six birds receiving 25 mg/kg D-(+) isomer died less than 1 h after dosing (Table 4Go). The brain of one of these birds was immediately removed and frozen (–65°C) and was assayed with the rest of the samples taken 24 h later. The remaining D-(+) dosed birds required additional doses of atropine to relieve cholinergic signs. Only one bird received L-(–) isomer and showed no cholinergic signs. The bird receiving 200 mg/kg DFP showed the expected high level of aged-inhibited NTE (63%) and no unaged NTE. The L-(–) dosed bird had 14% aged NTE, but the total NTE inhibition was low (almost all of the inhibited enzyme of the aged type). The D-(+) isomer results showed 10% aged, 29% unaged, and 39% total NTE inhibition. It is interesting to note that the degree of aging for the D-(+) isomer was lower than the L-(–) isomer (14%), but the total NTE inhibition by D-(+) was higher (added inhibition in the form of unaged NTE). These results were mirrored in the in vitro data obtained using the metabolic activation/aging assay (5 mM concentrations of each isomer, 1 h incubations) (Table 5Go). Again, the total NTE inhibition by L-(–) isomer (33%) was less than half that of the D-(+) (73%) and the degree of aging of the L-(–) was slightly higher than D-(+) (6% and 4%, respectively).


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TABLE 4 Derived Values for Unaged and Aged Inhibited NTE in Chick Brain after Treatment with Resolved OP Isomers in Vivo
 

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TABLE 5 Derived Values for Unaged and Aged Inhibited NTE in Hen Brain Treated in Vitro with Resolved OP Isomers with and without Metabolic Activation
 
Although the absolute value of aged NTE was lower for both isomers using the in vitro assay (compared to values obtained in vivo), the degree of difference between values obtained for the two isomers was consistent using either assay. This phenomenon was also noted in the comparison of technical versus analytical methamidophos, in which the absolute aged NTE values were higher using the in vivo assay. This probably reflected the longer period of chemical exposure in vivo (24 h vs 1 h for the in vitro method), thus allowing chemicals that have a long half-life of aging to have more effect. It is interesting to note that the D-(+)-dosed bird that was found dead (less than 1 h after dosing) had aged NTE levels equal to the in vitro assay (4%), illustrative of the time dependence of degree of aging (Table 5Go).

One in vitro assay (1 mM conc., 1 h incubation) was performed to compare NTE inhibitions by the isomers with and without metabolic activation. No difference in the extent of NTE inhibition or aging was seen between these treatments, either with or without activation (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Technical methamidophos produced a slightly higher level of aged NTE than analytical methamidophos or isolated optical isomers. The amount of unaged-inhibited NTE seen probably reflected the actual dosage of methamidophos administered; the extra aged-inhibited enzyme observed with technical grade may have been due to one of the impurities mentioned. Senanayake and Johnson (1982) reported that a methamidophos precursor (namely, O,O-dimethyl phosphoramidothioate) and an N-methyl analogue of methamidophos (presumably, O,O,N-trimethyl phosphoramidothioate) were present in samples obtained from the Sri Lanka poisonings. These impurities were present in the technical material used in the current studies. Other studies involving the comparison of the reactivation of AChE inhibited by methamidophos with that of crufomate (N-methyl analogue of methamidophos) and tabun have shown that reactivation decreased with increased alkylation of the P-NH2 group (de Jong et al., 1982Go).

Many technical products of OP insecticides contain appreciable amounts of reactive phosphorylating impurities; a frequently cited example is the isomalathion and trimethyl phosphorothioate content in malathion (Aldridge et al., 1979Go). One such component, O,O,S-trimethyl phosphorothioate, is very toxic to rats (LD50 60 mg/kg compared with 10,700 mg/kg for malathion). Animals dosed with approximately 3 x LD50 gradually deteriorated in condition and were found dead 3–4 days later. After removing these compounds from technical malathion, the oral toxicity of malathion was reduced from an LD50 of 1580 mg/kg to 8000 mg/kg (Pellegrini and Santi, 1972Go). Trimethyl phosphorothioates have also been found in technical acephate and are believed to contribute to its toxicity (Umetsu et al., 1977Go).

Methamidophos was shown to be a relatively weak inhibitor of NTE in this system, compared to a potent NTE inhibitor such as DFP. Although the NTE aging potential of methamidophos was enhanced by the presence of impurities, the effect was not robust and probably does not explain fully how humans could have developed OPIDN after exposure to methamidophos. The question of whether methamidophos or impurities were interacting with another target site (e.g., a promotion site) was not investigated (Lotti et al., 1995Go).

The proposed action of the resolved D-(+) and L-(–) isomers of methamidophos at the NTE catalytic center and at the promotion site has been examined by combination studies involving a non-aging NTE inhibitor (PMSF) and a rapidly aging NTE inhibitor, dibutyl dichlorovinyl phosphate (DBDCVP) (Lotti et al., 1995Go). The D-(+) isomer was considered a weak NTE antagonist because a very marginal neuropathic response was correlated with about 80% NTE inhibition, which is less than the almost 100% required by classical NTE antagonists such as PMSF. L-(–) methamidophos was classified an NTE agonist because it did not protect from DBDCVP neuropathy (protection is related to pretreatment with non-aging NTE inhibitor such as PMSF). The mild OPIDN caused by L-(–) methamidophos alone was considered self-promoted because the neuropathy it initiates was only slightly promoted by PMSF (i.e., when L-(–) form initiates neuropathy, it will also occupy the promotion site, which will not be available to PMSF). The putative OPIDN promotion site (called M200) has been further characterized in recent studies (Gardiman et al., 1999Go).

In the current study, the D-(+) isomer showed more acute toxicity (all chicks required extra doses of atropine protection); two out of six chicks died when dosed with D-(+), whereas the L-(–) bird showed no cholinergic effects. This is in agreement with published rate constant (Ka) data for AChE inhibition by isomers of methamidophos that indicated higher acute toxicity for the D-(+) isomer (Bertolazzi et al., 1991Go). The D-(+) isomer also showed greater total NTE inhibition [39% vs 14% for L-(–)], but slightly less aged-inhibited enzyme [10% vs 14% for L-(–) isomer]. This was in agreement with published NTE inhibition data, in which a greater degree of NTE reactivation was achieved in the case of inhibition by the D-(+) isomer (Johnson et al., 1991Go).

Because of the small yield of L-(–) isomer in the current study, the in vivo study for this isomer consisted of only one bird. In the case of the L-(–) isomer, principal consideration should be placed on data obtained in vitro. Because only a small amount of test material is needed for the in vitro assay, reliable NTE inhibition data could still be obtained for this isomer. These results mirrored the data obtained in vivo, with the L-(–) showing lower total NTE inhibition, but slightly higher aged-inhibited NTE. Other investigators have noted similar patterns of NTE inhibition and aging after treatment with the stereoisomers of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP), a phosphoramidate related to methamidophos (Sogorb et al., 1997Go). The more potent NTE inhibitor [the S-(–) HDCP isomer with an IC50 of 7.6 nM], did not age [i.e., comparable to the D-(+) isomer of methamidophos]. The R-(+)-HDCP isomer was a less potent NTE inhibitor (IC50 191 nM), but did exhibit aging akin to the L-(–) isomer.

Validation of the in vitro methodology was augmented by the data obtained for leptophos and leptophos oxon (positive metabolic activation and positive aging controls, respectively). Clothier and Johnson (1980) tested the aging potential of leptophos oxon using a significantly different test protocol, demonstrating significant amounts of aged-inhibited enzyme, over 50% unaged NTE, and a total NTE inhibition of 97% (without metabolic activation). Despite the different methods used, similar results were obtained in the current study with derived values of 25% aged, 74% unaged, and 99% total NTE inhibition for 0.1 mM leptophos oxon. Leptophos itself was also tested; without activation it produced low total NTE inhibition (37%) and with activation showed greater (84%) inhibition. The level of aging was lower than leptophos oxon, probably reflecting the time required to convert leptophos to the oxon form before subsequent inhibition and aging.

The data obtained both in vivo and in vitro suggest that technical grade methamidophos is more characteristic of an NTE agonist than either analytical grade racemic methamidophos or the resolved isomers. Studies performed in vitro suggested that bioactivation was required for the enhanced aging potential of the technical material (probably via reactions with impurities). Metabolic activation had no discernible effect on the NTE inhibition/aging potential of analytical racemic methamidophos or the resolved isomers, in agreement with researchers that have called it a poor substrate for microsomal mixed-function oxidase systems and for glutathione-dependent transferases (Magee, 1982Go; Suksayretrup and Plapp, 1977Go). However, some recent studies have suggested that brain AChE inhibition and poisoning signs from methamidophos are greatly delayed in mice and houseflies pretreated with oxidase inhibitors (Mahajna and Casida, 1998Go).

Consideration of cumulative risk of multiple pesticide exposures mandated under the Food Quality Protection Act (FQPA) has made it critical to determine the additive effect of compounds that may have a common endpoint such as OPIDN, but may be contributing to this disorder by different mechanisms (e.g., NTE vs promotion site). This consideration could also apply to a single product such as technical grade methamidophos, which may also contain significant levels of phosphorylating impurities of unknown toxicity. In the area of regulatory toxicology, it has been recognized that registration of compounds used as economic poisons must involve standardized toxicity tests performed using the highly pure active ingredient (generally > 95% purity) as well as the formulation that is actually used in the field.

Further experimentation should be performed to gauge the relative contribution of impurities to the enhanced neurotoxic potential of methamidophos (e.g., possible binding to a promotion site could be determined in manner similar to that described for PMSF). By varying the level and order of addition (before and after methamidophos), the degree and possible site of action (NTE catalytic center or promotion site) may be further elucidated. Progress has been achieved in characterizing the catalytic center of NTE (serine active site) following NTE purification studies (Glynn et al., 1999Go). As advances are made in the purification and elucidation of the three-dimensional structure of NTE, the binding characteristics of inhibitors (and isolated chiral isomers) at the serine active site will be modeled, giving further insight into the initial stages of delayed neurotoxicity.

Other routes of investigation involving the in vitro methodology described in this paper could include screening large numbers of pesticide samples for their NTE aging potential, both in the presence and absence of metabolic activation. A large sampling of technical grade pesticides from various sources could be screened for NTE inhibition and aging, thus allowing detection of pesticides with impurities that require metabolic activation for enhanced neuropathic potential. The use of in vitro methodology for this first-tier screening would be imperative with respect to the varied pesticide manufacturing and storage practices that exist throughout the world and to the important goal of reducing whole-animal testing in toxicology.


    NOTES
 
Supported in part by NIH ES 00202 and Chevron.

1 To whom correspondence should be addressed at Dept. of Pesticide Regulation, California Environmental Protection Agency, 830 K St., Sacramento, CA 95814-3510. Fax: (916) 324-3506. E-mail: tkellner{at}cdpr.ca.gov. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abou-Donia, M. B. (1983). Toxicokinetics and metabolism of delayed neurotoxic organophosphorus esters. Neurotoxicology 4, 113–129.

Aldridge, W. N., Miles, J. W., Mount, D. L., and Verschoyle, R. D. (1979). The toxicological properties of impurities in malathion. Arch. Toxicol. 42, 95–106.[ISI][Medline]

Bertolazzi, M., Caroldi, S., Moretto, A., and Lotti, M. (1991). Interaction of methamidophos with hen and human acetylcholinesterase and neuropathy target esterase. Arch. Toxicol. 65, 580–585.[ISI][Medline]

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