* Dipartimento di Medicina Ambientale e Sanità Pubblica, Università degli Studi di Padova, Padova, Italy;
Laboratoire de Neuropathologie R Escoulle, INSERM U306, Paris, France; and
Zeneca Central Toxicology Laboratory, Alderley Park, Cheshire, United Kingdom
Received February 5, 2001; accepted April 4, 2001
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ABSTRACT |
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Key Words: OP neuropathy; protection; promotion; molinate; organophosphate; esterase; pathology; inhibition.
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INTRODUCTION |
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The thiocarbamate herbicide molinate is an inhibitor of esterases in vivo (Jewell et al., 1998) and its testicular toxicity in rats is thought to be related to inhibition of Leydig cell neutral cholesterol ester hydrolase activity (Ellis et al., 1998
; Wickramaratne et al., 1998
). Esterase inhibition is due to molinate sulfoxide and molinate sulfone, the oxidation metabolites of molinate known to be formed by NADPH-dependent liver microsomal enzymes (Jewell and Miller, 1998
). We report here in vitro studies with molinate oxides on NTE and M200 activities from hen, rat, and human nervous tissues and the effects of molinate in both rats and hens when tested for protection from and promotion of neuropathy induced by di-n-butyl dichlorovinyl phosphate (DBDCVP).
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MATERIALS AND METHODS |
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Animals and dosing.
Adult hens (1.72.5 kg bw) were purchased from a local breeder. Male Wistar rats (1-month-old, 0.10.2 kg bw and 6-month-old, 0.50.7 kg bw) were purchased from Charles River Italia, (Calcio, LC, Italy). Animals were allowed food and water ad libitum. Molinate and DBDCVP were dissolved in glycerol formal immediately before use and administered sc in the anterothoracic region (hens) or in the nape of the neck (rats) at volumes of 0.2 ml/kg (hens) or 0.5 ml/kg (rats). Controls were injected with a corresponding volume of vehicle. Experiments in hens were conducted with either technical molinate or the commercial formulation whereas rats were treated with commercial formulation only, because the purpose of the latter experiment was only confirmatory. Hens and rats were observed for clinical signs of peripheral neuropathy every other day from day 7 through 21 after treatment. Assessment of hens was based on a 0 to 8 point scale (Lotti et al., 1991) and assessment of rats was based on 0 to 3 point scales for walking and rod test (Moretto et al., 1992b
). Scores observed on day 21 are reported in the results.
Tissues.
Brains and sciatic nerves of hens and rats were excised from animals after decapitation and stored at 80°C until assay. Samples of human nucleus caudatus and cerebral cortex were obtained during routine postmortem examinations performed within 36 h of death from the bodies of 2 male subjects who died of non-neurological diseases.
Biochemistry.
NTE activity was determined according to Johnson (1977) and Moretto et al. (1989). AChE activity in whole brain (hens and rats) or nucleus caudatus (humans) was determined according to Ellman et al. (1961), with modifications of volumes and tissue concentrations to obtain adequate absorbances while maintaining linearity of color development. M200 is defined as the activity resistant to paraoxon (40 µM) plus mipafox (50 µM) and sensitive to mipafox (1 mM) (Lotti and Moretto, 1999). It was determined using the same procedure of NTE assay with slight modifications of tissue concentration (13.3 and 80 mg/kg for brain and peripheral nerve, respectively) and time of hydrolysis (35 min) in order to obtain adequate absorbances. Nerves of rats were pooled (6 nerves from 3 animals) due to the small amount of tissue. Rates of reappearance of NTE and M200 activities in hen brain and peripheral nerve after treatment with molinate (180 mg/kg, sc) were calculated from the semilog plot of days after dosing vs. log % recovery. The recovery was calculated as percentage of inhibition found on day 1 after dosing. The I50 (concentration that inhibits 50% of enzyme activity) for both NTE and M200 in hen, rat, and human (mixture of cerebral cortex and nucleus caudatus) homogenates for molinate sulfone and sulfoxide were derived as follows: Inhibitors were dissolved in acetone and mixtures were incubated for 20 min at 37°C before addition of PV. I50 values were calculated by regression analysis of the semilog plots of percent remaining activity vs. inhibitor concentrations (710 concentrations up to 510 x I50). Time courses (up to 180 minutes) of inhibition were also performed with inhibitor concentrations corresponding to 0.52 x I50 at 20 min.
The reversibility of brain NTE inhibition was examined as follows: Homogenates of hen, rat, and human brain (6.6 mg/ml) were incubated with each molinate metabolite (at concentrations corresponding to 24 x I50) for 20 min. Inhibition was halted by adding 30 volumes of ice-cold Tris buffer followed by centrifugation at 30,000 x g for 30 min. Pellets were resuspended in the initial volume and NTE activity was determined immediately or after 120 min incubation at 37°C. Because M200 activity is in the soluble fraction, a dilution procedure to assess reversibility was applied. Thus, homogenates (100 mg/ml) were incubated with molinate sulfone or sulfoxide (at concentrations corresponding to 0.5 x I50 for 20 min) for 60 min at 37°C. Then 15 volumes of Tris were added and M200 activity measured immediately or after 120 min incubation. In a concurrent control, part of the sample was diluted with Tris containing the same concentrations of molinate sulfone or sulfoxide. In addition, the reversibility of NTE activity was also measured in these conditions.
Pathology.
Sampling was performed 2122 days after treatment. Hens were anesthetized with ketamine and halothane, and perfused through the heart with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Brain and medulla, cervical, thoracic and lumbosacral spinal cord, and sciatic, peroneal, and tibial nerves from both limbs were collected. Paraffin sections were prepared from 6 transverse sections of the brain and medulla, and stained with hematoxylin and eosin. Spinal cord samples were sectioned transversely and peripheral nerves were sectioned both transversely and longitudinally. Tissues were then embedded in methacrylate resin, sectioned, and stained with toluidine blue. Axonal degeneration was found in the spinal cord and peripheral nerve and was scored as: 0 = none, 1 = minimal, 2 = slight, 3 = moderate, 4 = marked degeneration.
Statistical analysis.
Comparison of in vivo inhibitions, clinical and morphological scores were performed by either the U-test or the Kruskal-Wallis test and significance set at p < 0.05. Differences between I50s and in vivo recovery of activities were tested by comparing regression coefficients and significance was set at p < 0.05.
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RESULTS |
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DISCUSSION |
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Protection from DBDCVP neuropathy given to both hens and rats by the pretreatment with molinate was correlated with NTE inhibition, probably through carbamylation of the active site that was shown to occur when a rat testicular esterase (hydrolase A) is inhibited by molinate (Jewell and Miller, 1998). Carbamylated NTE is known not to undergo the molecular rearrangements thought to be necessary to trigger OPIDP (Johnson, 1990
; Lotti et al., 1993
). Therefore, when NTE is inhibited by molinate (> 75%), as shown in Table 2
, no neuropathy develops and these animals are protected from challenging doses of the neuropathic DBDCVP (Tables 4 and 6
).
Promotion of DBDCVP neuropathy by molinate was associated with M200 inhibition higher than 50%, in both hens and rats, probably by carbamylation as well (Tables 2, 4 and 6). All compounds tested so far indicate that promotion is associated with inhibition of M200, whereas neuropathic OPs have negligible effect when given at doses causing high inhibition of NTE (> 70%) and OPIDP (Lotti and Moretto, 1999
).
The recovery half-life of M200 activity after inhibition by a single dose of molinate was found to be about 5 days (Table 3). Within this time frame, another promoter (the phosphorothioic acid-O-(2-chloro-2,3,-trifluoro-cyclobutyl)-O-ethyl S-propyl ester), not inhibiting NTE, was found to be effective when given before the initiating dose of a neuropathic OP (Moretto et al., 1994
). However, it is not known whether M200 inhibition lasting for a few days is required for promotion to occur or if a short-duration, higher inhibition is needed, similar to that of NTE for OPIDP development (Lotti and Johnson, 1980
).
We previously mentioned that M200, as currently measured, is not the activity of a single enzyme and studies are underway to characterize the relevant protein. This protein is likely located in the soluble fraction of hen peripheral nerve where a PV esterase was found, approximately 60 kDa molecular mass (Escudero et al., 1997; Escudero and Vilanova, 1997
), which shares some biochemical characteristics with M200.
It is interesting that molinate causes testicular toxicity in rats through inhibition of Leydig cell hydrolase A, whose activity is sometimes determined as nonspecific esterase (NSE) using either -naphtyl acetate or p-nitrophenyl acetate as substrates (Ellis et al., 1998
; Jewell and Miller, 1998
). This is a key enzyme in the synthesis of testosterone. It has a molecular mass of about 60 kDa and is also inhibited by PMSF, the prototype promoter, which causes testicular lesions in rats as well (Jewell and Miller, 1998
). Tri-ortho-cresyl phosphate (TOCP) is also a known testicular toxicant in rats (Somkuti et al., 1987a
) and this effect is probably mediated by NSE inhibition in testes (Chapin et al., 1990
, 1991
; Somkuti et al., 1987b
). Moreover, both hydrolase A and M200 are inhibited in vitro by the 2 oxide metabolites of molinate, sulfone and sulfoxide.
Exaggeration of DBDCVP-induced histopathological changes was observed after molinate treatment causing more than 78% M200 inhibition, whereas protection from DBDCVP induced histopathological changes was observed with molinate pretreatment causing more than 80% (almost complete protection) or more than 33% (partial protection) NTE inhibition (Tables 2 and 5). The comparison of morphological lesions in spinal cord and peripheral nerves of hens displaying DBDCVP neuropathy, or DBDCVP neuropathy promoted by molinate confirmed that promotion causes an exacerbation of axonal degeneration, without other types of lesions or involvement of parts of the nervous system that are usually not affected by OPIDP (Harp et al., 1997
; Lotti et al., 1991
; Pope et al., 1992
; Randall et al., 1997
).
The data presented here also confirm that older rats do express clinical OPIDP (Moretto et al., 1992b). Neuropathy can either be protected or promoted when molinate or other non-neuropathic NTE inhibitors are given before or after the neuropathic OP. Resistance of younger rats to OPIDP can not likely be ascribed to a difference in the disposal of compounds because the amount of inhibition of NTE was not significantly different across ages. Data in Table 2
confirm that inhibition of NTE and M200 is not different when molinate was given to either 1- or 6-month-old rats.
In conclusion, since human NTE and M200 have a sensitivity to the active metabolites of molinate comparable to that of hens and rats (Table 1), it is conceivable that protection from and promotion of OPIDP by molinate might occur in humans. However, since sulfur oxidation represents about 30% and 1% of molinate metabolism in rats and humans, respectively (Wickramaratne et al., 1998
), high exposures to molinate should be envisaged for such effects to occur in humans.
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ACKNOWLEDGMENTS |
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NOTES |
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Part of this work was presented at the 38th annual meeting of the Society of Toxicology, March 1999, New Orleans, LA, and at the 39th annual meeting of the Society of Toxicology, March 2000, Philadelphia, PA.
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