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Correlation of Binding Sites for Diisopropyl Phosphorofluoridate with Cholinesterase and Neuropathy Target Esterase in Membrane and Cytosol Preparations from Hen
NeuroToxicology 22 (2001) 203±214
Correlation of Binding Sites for Diisopropyl Phosphoro¯uoridate with Cholinesterase and Neuropathy Target Esterase in Membrane and Cytosol Preparations from Hen Ryo Kamata1,3,*, Shin-ya Saito2, Tadahiko Suzuki1, Tadashi Takewaki3, Haruo Kobayashi1 1
Department of Veterinary Pharmacology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Japan 2 Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan 3 United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan Received 23 February 2000; accepted 19 October 2000
Abstract To ®nd new putative target(s) for organophosphorus induced delayed neurotoxicity (OPIDN), we investigated the biochemical and pharmacological characteristics of [3H]diisopropyl phosphoro¯uoridate (DFP) binding to membrane and cytosol preparations from the brain and spinal cord of hens. Speci®c [3H]DFP binding was determined by subtracting non-speci®c binding from total binding. The binding sites of [3H]DFP, an organophosphate that induces OPIDN, were found not only on membrane but also in cytosol. Reduction of subsequent ex vivo speci®c [3H]DFP binding by in vivo pretreatment with unlabeled DFP was found in cytosol, not membrane. The reduced binding lasted to the onset of OPIDN, especially in spinal cord. These results suggest that the speci®c DFP binding sites in cytosol, rather than on membrane, are the most important with regard to the initiation of OPIDN. Inhibitors of cholinesterase (ChE) and neuropathy target esterase (NTE) other than DFP did not affect speci®c [3H]DFP binding to either membranes or cytosol in vivo. Additionally, inhibition of the activities of these esterases by these compounds was not consistent with either the degree of inhibition of the [3H]DFP binding or a time-dependent manner of OPIDN. These results suggest that DFP binding site(s) involved in the initiation of OPIDN may be different from the active sites of ChE and NTE. # 2001 Elsevier Science Inc. All rights reserved.
Keywords: Organophosphorus induced delayed neurotoxicity (OPIDN); Diisopropyl phosphoro¯uoridate (DFP); Cytosolic protein; Hen
INTRODUCTION A number of organophosphorus esters produce delayed neuropathy (OPIDN) in humans and other sensitive avian and mammalian species (AbouDonia, 1981). Inhibition and subsequent modi®cation (``aging'') of an enzyme known as neuropathy target
* Corresponding author. Tel.: 81-19-621-6215; Fax: 81-19-621-6215. E-mail address: [email protected] (R. Kamata).
esterase (NTE) has been proposed as the initial effect of compounds that induce OPIDN (Johnson, 1970, 1982). However, although numerous studies have attempted to associate NTE with OPIDN, the inhibition of NTE activity does not correspond with the onset of morphological/clinical signs of OPIDN (Abou-Donia and Lapadula, 1990) and the structure and physiological function(s) of NTE are still unknown. In the 1990's, many studies described ultrastructural changes at the cellular level during OPIDN. These changes include enhanced aggregation and phosphorylation of cytoskeletal proteins (i.e. microtubules and
0161-813X/01/$ ± see front matter # 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 1 - 8 1 3 X ( 0 0 ) 0 0 0 1 2 - 7
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neuro®laments), and the alteration of axonal transport (Abou-Donia and Lapadula, 1990; Gupta and AbouDonia, 1994). These effects on axoplasm have been proposed to result from alterations in Ca2/calmodulin dependent protein kinase activity (Gupta and AbouDonia, 1995; Gupta et al., 1997). These observations suggest that the initiation of neuropathy in OPIDN involves intracellular component(s) other than neuronal cell membrane. Diisopropyl phosphoro¯uoridate (DFP) is known to inhibit the activity of NTE and produce OPIDN. There have been many investigations of the DFP labeling sites (proteins) on membrane of hen brain. These have been mostly aimed at characterizing NTE or phenyl valerate esterases, because of the hypothesis that membrane bound esterases such as acetylcholinesterase (AChE) and NTE might be involved (Williams and Johnson, 1981; Pope and Padilla, 1989; Meredith and Johnson, 1989). However, it is our hypothesis that there may be DFP binding sites other than AChE and NTE in neural tissue with physiological functions in the pathogenesis of OPIDN. Carrington and Abou-Donia (1985) have reported that there are several DFP binding sites in both membrane and soluble fractions separated by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis from hen brain and the number of DFP binding sites does not necessarily correspond to phenyl valerate, an arti®cial substrate of NTE, hydrolysing activity in membrane. We also previously performed binding assays in membrane and cytosol preparations from the brain and spinal cord of chickens in vitro and con®rmed the presence of speci®c DFP binding sites in cytosol in addition to the expected binding sites in membrane. The binding sites in cytosol had a lower af®nity for DFP than membrane and were obviously different from cholinesterase (ChE) and NTE. The role of these cytosol DFP binding sites in the production of OPIDN appeared to warrant further investigation. The purpose of the present study was, therefore, to clarify the pharmacological and biochemical functions of the new cytosol DFP binding site(s) in production of OPIDN. Hens were pretreated, in vivo, with various inhibitors of ChE and NTE, and subsequent, ex vivo, speci®c binding of [3H]DFP in membrane and cytosol preparations from the brain and spinal cord was assayed. Additionally, the effects of AChE and NTE inhibitors on enzyme activity were examined in each preparation, and compared with the results of binding assay. The results suggest that speci®c DFP binding sites on membrane may be the active sites of ChE but the binding sites in cytosol seem to be unrelated to ChE
or NTE. It appears that the speci®c DFP binding sites in cytosol, especially in spinal cord, may have a role in the initiation of OPIDN. Furthermore, the initiation of OPIDN seems to be unrelated to inhibition of ChE or NTE. MATERIALS AND METHODS Chemicals DFP, diethyl p-nitrophenyl phosphate (paraoxon), phenylmethylsulfonyl ¯uoride (PMSF), O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate (chlorpyrifos), and N,N0 -diisopropyl phosphorodiamidic ¯uoridate (mipafox) were purchased from Wako Pure Chemical Industries (Osaka, Japan), Aldrich Chemical Co., Inc. (Milwaukee, WI), Nacalai Tesque Co., Ltd. (Kyoto, Japan), Kanto Chemical Co., Inc. (Tokyo, Japan), and Oriza Laboratories, Inc. (Newburyport, MA), respectively. Phenyl valerate was kindly synthesized and puri®ed by Dr. M. Ota and Dr. H. Kofujita (Department of Wood Science and Technology, Faculty of Agriculture, Iwate University, Japan) by the method of Johnson (1977). [3H]DFP (111 GBq/ mmol) was purchased from Dupont/New England Nuclear (Boston, MA). Animals and Treatment Adult white Leghorn laying hens (Gallus gallus domesticus, body weight 1.6±2.2 kg) were used. In dosing experiments in vivo, DFP (2 mg/ml), paraoxon (0.3 mg/ml), PMSF (30 mg/ml), and chlorpyrifos (30 mg/ml) were dissolved in corn oil, and injected subcutaneously in the posterior cervical region at a volume of 1 ml/kg of body weight. To reduce the acute cholinergic toxicity of organophosphorus compounds, atropine sulfate (20 mg/kg) dissolved in saline, was injected subcutaneously 20 min before the treatment with organophosphorus compounds. Control hens were treated with corn oil as vehicle at 1 ml/kg. The birds were sacri®ced 24 h or 8 days after the treatment, and brain and spinal cord were collected and assayed for speci®c DFP binding. Tissue Preparations Tissue preparations were made using a modi®ed method of Konno et al. (1994). The medulla and cerebellum, regions with neuropathological changes in OPIDN, were removed from the brain to compare
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with the spinal cord, a susceptible tissue. The remaining brain tissue and spinal cords were cleaned free of blood vessels and meninges, and homogenized on ice in 10 vol (w/v) of ice-cold HEPES buffer (50 mM, pH 7.4) using a Te¯on-glass homogenizer. These homogenates were centrifuged at 1000 g for 10 min at 48C, and the supernatants were centrifuged again at 50,000 g for 10 min at 48C. The resulting supernatants were used as cytosol preparation. Furthermore, the pellets from the last 50,000 g centrifugation were resuspended in HEPES buffer, and recentrifuged at 50,000 g for an additional 10 min at 48C and the supernatants were discarded. HEPES buffer was then added to these washed pellets, which were then resuspended using Polytron homogenizer, so the concentration of these preparations were about 50 mg tissue/ml. The resulting suspensions were used as the membrane preparation. Assay of [3H]diisopropyl Phosphorofluoridate Binding To assay the speci®c binding of [3H]DFP to the membrane and cytosol preparations, the membrane or cytosol preparations were diluted to 5 or 10 vol, respectively, with 10 mM sodium phosphate buffer (pH 7.4). Diluted preparations of 100 ml each and 100 ml of 10 mM sodium phosphate buffer or 100 ml of 25 mM unlabeled DFP (®nal concentration of 10 mM) were incubated with 50 ml of [3H]DFP at 378C for 60 min. For experiments examining concentration dependent (saturation) binding of [3H]DFP, or in dosing experiments in vivo, the solution of [3H]DFP was adjusted to 2±128 nM (®nal concentrations of 0.4±25.6 nM), or 4, 16 or 64 nM (0.8, 3.2 or 12.8 nM), respectively. The reaction was terminated by ®ltration through a GF/B glass ®lter (24 mm in diameter, Whatman International Ltd., Maidston, UK). The ®lter was washed twice with 5 ml of ice-cold Tris±HCl buffer (10 mM Tris, 140 mM NaCl, pH 7.4). The ®lters were soaked in 0.3% polyethylenimine for 1±24 h before use and were placed on the ®lter apparatus without washing according to the method of Bruns et al. (1983). After ®lters had been soaked overnight in 5 ml of scintillation cocktail (ACS-II; Amersham, Bucks, UK), the radioactivity attributable to tritium was counted (LSC-5100, Aloka Co. Ltd., Tokyo). Speci®c binding of [3H]DFP was de®ned as difference between the total binding and non-speci®c binding measured in the absence and presence of unlabeled DFP, respectively.
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Determination of NTE and AChE Activities and Protein Activity of NTE was determined as described by Johnson (1977). The activity was de®ned as the difference between the colorimetric determinations of the phenol liberated from the paraoxon-resistant (40 mM) and mipafox-sensitive (50 mM) hydrolysis of phenyl valerate, and expressed as nanomoles of phenyl valerate hydrolyzed per minute per milligram of protein. Activity of AChE in each preparation was assayed by the method of Ellman et al. (1961). Concentration of protein was measured by the method of Lowry et al. (1951) with bovine serum albumin as the standard. Data Analysis Saturation binding data were converted to Scatchard plots, from which af®nity constants Kd and Bmax values were determined as ±1/slope and the x-axis intercept, respectively. Data from [3H]DFP binding in vivo and the activities of AChE and NTE are presented as means S:E. Comparisons between each groups were performed by using a one-way analysis of variance (ANOVA). Post hoc analysis was performed with Dunnett's test. The activity of NTE under 30% of control value was taken as a threshold level, because it has been proposed that OPIDN is caused by 70±80% inhibition of NTE (Johnson and Glynn, 1995). RESULTS Saturation Binding of [3H]DFP The characteristics of [3H]DFP binding to membrane and cytosol preparations from brain and spinal cord of untreated hens was analyzed by saturation curves of the speci®c binding which reached a plateau. The Kd and Bmax values of [3H]DFP binding in each preparation were determined using Scatchard analysis. Similar results were obtained by non-linear regression (not shown). Kd values (nM) of membrane preparations were 1.40 for spinal cord and 3.36 for brain. Those of cytosol preparations were 4.03 for spinal cord and 7.41 for brain. These results show that the Kd values were consistently lower in membrane than in cytosol. The Bmax values (pmol/mg protein) of membrane preparations were 1.71 for spinal cord and 1.49 for brain, and those of cytosol preparations were 0.57 for spinal cord and 0.41 for brain. These results show that the
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Bmax values were higher in membrane than in cytosol. Overall, the minimum Kd and maximum Bmax values were found in membrane preparations from spinal cord. Concentrations of [3H]DFP from 0.8 to 12.4 nM were used to study binding to preparations from hens pretreated with organophosphates or PMSF. Effects of Pretreatment with Organophosphates and PMSF on [3H]DFP Binding Ex Vivo The effects of pretreatments with DFP, chlorpyrifos, paraoxon and PMSF on subsequent speci®c [3H]DFP binding to membrane and cytosol preparations from brain and spinal cord of hens are shown in Figs. 1 and 2, respectively. In membrane preparations, the speci®c [3H]DFP binding to brain and spinal cord from
hens 24 h after an exposure to DFP was signi®cantly decreased at each concentration of [3H]DFP (Fig. 1A and B). However, DFP did not signi®cantly decrease the binding to either tissue 8 days after exposure except for the lowest concentration of [3H]DFP (®nal, 0.8 nM) (Fig. 1C and D). Paraoxon, which does not induce OPIDN, PMSF, an antagonist of NTE, and chlorpyrifos, which is proposed as a delayed neurotoxic compound, hardly decreased the binding to either tissue 24 h and 8 days after exposure. In cytosol preparations, speci®c [3H]DFP binding to brain and spinal cord 24 h after pretreatment with DFP was signi®cantly decreased at ®nal concentrations of 3.2 and 12.8 nM [3H]DFP (Fig. 2A and B). In addition, DFP continued to signi®cantly decrease the speci®c binding to spinal cord even 8 days after pretreatment (Fig. 2D). Paraoxon, PMSF and chlorpyrifos hardly
Fig. 1. Effects of organophosphates and PMSF on specific [3H]FP binding to membrane preparations from brain (A) and spinal cord (B) 24 h after treatment, and brain (C) and spinal cord (D) 8 days after treatment. Membrane preparations were incubated in the presence of 0.8, 3.2 or 12.8 nM [3H]DFP. Data are shown as means with standard error bars for four experiments. Asterisks indicate experimental values that are significantly different from the respective control (P < 0:05, P < 0:01).
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Fig. 2. Effects of organophosphates and PMSF on specific [3H]DFP binding to cytosol preparations from brain (A) and spinal cord (B) 24 h after treatment, and brain (C) and spinal cord (D) 8 days after treatment. Cytosol preparations were incubated in the presence of 0.8, 3.2 or 12.8 nM [3H]DFP. Data are shown as means with standard error bars for four experiments. Asterisks indicate experimental values that are significantly different from the respective control (P < 0:05, P < 0:01).
decreased the binding to either tissue 24 h and 8 days after an exposure, except for the binding at ®nal concentrations of 3.2 nM [3H]DFP 24 h after exposure to paraoxon. Acetylcholinesterase Activities The results of in vivo pretreatments with organophosphates and PMSF on AChE activity in membrane and cytosol preparations are shown in Figs. 3 and 4, respectively. As expected, the AChE activity in cytosol was far lower than in membrane from both brain and spinal cord of untreated hens (Table 1). In membrane preparations, the most potent compounds in inhibiting the AChE activity in brain and spinal cord 24 h after pretreatment were DFP and paraoxon, and the activities were signi®cantly inhibited (Fig. 3A and B). PMSF and chlorpyrifos did not signi®cantly inhibit
the activity in membranes from either tissue. The activities in both brain and spinal cord membranes were recovered and were not signi®cantly different Table 1 Acetylcholinesterase (AChE) and neuropathy target esterase (NTE) activities in membrane and cytosol preparations from brain and spinal cord of untreated hensa Tissue
Preparation
AChE
NTE
Brain
Membrane Cytosol
442.33 19.40 45.11 6.88**
143.27 19.27 26.88 3.93**
Spinal cord
Membrane Cytosol
190.38 13.97 21.89 0.16**
88.34 2.61 12.71 3.41**
a Activity of AChE is expressed as mmol acetylthiocholine hydrolyzed/ min/g protein, mean S.E.M. of four expremenents for each preparation. Activity of NTE is expressed as nmol phenyl valerate hydrolyzed/min/mg protein, mean S.E.M. of four experiments for each preparation. ** Asterisks indicate experimental values significantly different from the values of respective membrane preparation (P < 0:01).
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Fig. 3. Effects of organophosphates and PMSF on AChE activity in membrane preparations from brain (A) and spinal cord (B) 24 h after treatment, and brain (C) and spinal cord (D) 8 days after treatment. Data are shown as means with standard error bars for four experiments. Asterisks indicate experimental values that are significantly different from the respective control (P < 0:05, P < 0:01).
from those of control at 8 days, regardless of pretreatment compound (Fig. 3C and D). In cytosol preparations, the most potent AChE inhibitors in both tissues 24 h after pretreatment were, like membrane, DFP and paraoxon, which produced signi®cant inhibition (Fig. 4A and B). PMSF produced no signi®cant inhibition in cytosol from either brain or spinal cord but chlorpyrifos produced slight but statistically signi®cant inhibition in spinal cord. The AChE activities in cytosol from both brain and spinal cord were recovered and were not signi®cantly different from those of control at 8 days, regardless of pretreatment compound (Fig. 4C and D). Neuropathy Target Esterase Activities The results of in vivo pretreatments with organophosphates and PMSF on NTE activity in membrane
and cytosol preparations are shown in Figs. 5 and 6, respectively. The NTE activity in cytosol was far lower than in membrane from both brain and spinal cord of untreated hens (Table 1). In membrane preparations, the most potent inhibition of NTE activity in brain and spinal cord 24 h after pretreatment was produced by DFP and PMSF. In these cases, the activities were very low, under 30% of control value, the proposed threshold level for OPIDN (Fig. 5A and B). However, the NTE activity in both brain and spinal cord membranes recovered to far above this proposed threshold by 8 days (Fig. 5C and D), even though slight but signi®cant inhibition remained in spinal cord. Paraoxon and chlorpyrifos did not signi®cantly inhibit NTE activity in membranes in either brain or spinal cord either 24 h or 8 days after pretreatment. In cytosol preparations, the most potent inhibitors of NTE activity in both brain and spinal cord 24 h after
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Fig. 4. Effects of organophosphates and PMSF on AChE activity in cytosol preparations from brain (A) and spinal cord (B) 24 h after treatment, and brain (C) and spinal cord (D) 8 days after treatment. Data are shown as means with standard error bars for four experiments. Asterisks indicate experimental values that are significantly different from the respective control (P < 0:05, P < 0:01).
the pretreatment were, like membrane, DFP and PMSF. Again, the NTE activity in cytosol after pretreatment with DFP and PMSF were very low, under 30% of control (Fig. 6A and B). However, the activities in both brain and spinal cord cytosol 8 days after the exposure to each compound had recovered far above the proposed threshold for OPIDN (30% of control) (Fig. 6C and D), even though signi®cant inhibition remained in brain pretreated with both DFP and PMSF, and in spinal cord pretreated with DFP, respectively. Paraoxon and chlorpyrifos did not signi®cantly inhibit NTE activity in either brain or spinal cord either 24 hr or 8 days after pretreatment. DISCUSSION To ®nd and clarify the biochemical and pharmacological characteristics of [3H]DFP binding site(s) other than membrane components, we used cytosol
preparations from brain and spinal cord of hens and compared the results with the membrane preparations. In the present study, [3H]DFP bound not only to the membrane preparation but also to the cytosol preparation of brain and spinal cord. The existence of the DFP binding sites in cytosol may support the existence of new target site(s) of OPIDN. However, both the number of speci®c [3H]DFP binding sites and the af®nity of the binding to cytosol were lower than those of membrane, even though the cytosol contains a large number of proteins. This indicates that there is weak interaction between cytosolic structures and DFP. On the other hand, the af®nities in spinal cord were higher than those in brain, and the number of binding sites were similar in brain and spinal cord. The higher af®nity in spinal cord is consistent with the distribution of neuropathologic lesions on OPIDN, which are not seen in higher brain but in spinal cord and peripheral nerves (Carrington et al., 1988).
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Fig. 5. Effects of organophosphates and PMSF on NTE activity in membrane preparations from brain (A) and spinal cord (B) 24 h after treatment, and brain (C) and spinal cord (D) 8 days after treatment. Data are shown as means with standard error bars for four experiments. Sharps indicate experimental values that are under 30% of respective control value, as a threshold level, because it has been proposed that OPIDN is caused at 70±80% inhibition of NTE. Asterisks indicate experimental values that are significantly different from the respective control (P < 0:05, P < 0:01).
DFP produced severe reduction of speci®c [3H]DFP binding to both membrane and cytosol preparations 24 h after pretreatment in vivo. However, the effects of pretreatment with DFP on speci®c [3H]DFP binding in cytosol even 8 days after treatment were still marked, especially in spinal cord. In contrast to cytosol, the effects of DFP pretreatment on membrane at 8 days were slight or not signi®cant. The latent period of OPIDN after a single dose of organophosphates is approximately 1±3 weeks (Lotti, 1992). In the present study, hens pretreated with DFP also exhibited the early clinical signs of OPIDN 8 days later (data not shown). These observations indicate that the continuous binding sites of DFP are present in cytosol rather than on membrane, whose region inhibited continuously (spinal cord) is consistent with the neuropathology of OPIDN. Moreover, the effect of DFP on cytosol
continues to the onset of OPIDN or longer. These data suggest, therefore, target site(s) for the initiation of DFP induced delayed toxicity may be in cytosol rather than on membrane, especially in spinal cord. Although paraoxon produced strong reductions on speci®c [3H]DFP binding to membrane in vitro (Konno et al., 1994; Kamata et al., 2001), there was hardly any effect from in vivo pretreatment in either membrane or cytosol in the present study. Pretreatment in vivo with chlorpyrifos and PMSF also produced no reduction in speci®c [3H]DFP binding to either membrane or cytosol. Paraoxon, chlorpyrifos and PMSF, and their reactive metabolites are expected to have no effect on speci®c [3H]DFP binding after in vivo pretreatment in either brain or spinal cord, because of detoxication mechanisms such as carboxylesterase and cytochromes P450 (Atterberry et al., 1997). It is interesting to note,
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Fig. 6. Effects of organophosphates and PMSF on NTE activity in cytosol preparations from brain (A) and spinal cord (B) 24 h after treatment, and brain (C) and spinal cord (D) 8 days after treatment. Data are shown as means with standard error bars for four experiments. Sharps indicate experimental values that are under 30% of respective control value, as a threshold level, because it has been proposed that OPIDN is caused at 70±80% inhibition of NTE. Asterisks indicate experimental values that are significantly different from the respective control (P < 0:05, P < 0:01).
however, that these compounds still produced inhibition of AChE and NTE activity in the present study. Our results also show that the activities of AChE and NTE in cytosol preparation are far lower than those in membrane, in agreement with a previous study (Sogorb et al., 1994). The results are consistent with the idea that AChE and NTE are membrane-bound proteins. DFP and paraoxon produced marked inhibition of AChE activity in both membranes and cytosol 24 h after pretreatment in vivo. Pretreatment with DFP and PMSF in vivo also produced strong inhibition of NTE activity as measured at 24 h. If the initiation and progression of OPIDN results from inhibition of AChE and/or NTE activity, it is expected that some moderate inhibition of these esterases would continue at least until the onset of OPIDN or thereafter. However, AChE
activity exhibited good recovery in both membrane and cytosol by 8 days after in vivo pretreatment. This suggests that AChE inhibition by DFP or paraoxon is acute and does not correlate with the progression of OPIDN. Similarly, NTE activity also exhibited good recovery in both membrane and cytosol by 8 days after in vivo pretreatment, even though some inhibition (above the proposed threshold) remained. The half-life of recovery of NTE in hen brain after treatment with neuropathic compounds has been reported to be between 4 and 6 days (Johnson, 1974), which is similar to data from spinal cord and sciatic nerve (Caroldi and Lotti, 1982). And then, NTE activity gradually recovers. However, the clinical symptoms of OPIDN progress more and more in a time-dependent manner and are not necessarily consistent with NTE inhibition (Kamata et al., 1999).
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NTE has been reported to account for only a small proportion of total phenyl valerate hydrolyzing activity in hen brains (Abou-Donia and Lapadula, 1990; Lotti, 1992). Therefore, NTE is only a portion of many carboxylesterases which hydrolyze phenyl valerate as an arti®cial substrate. It is important to note that PMSF can irreversibly inhibit carboxylesterase, including NTE, without inducing neuropathy (CeÂspedes et al., 1997). Moreover, neural tissue, including glial and satellite cells, has a high level ChEs such as AChE (Lefkkowitz et al., 1996). Paraoxon, as an organophosphate, inhibits both carboxylesterases and ChE (Maxwell, 1992). However, because NTE is de®ned as phenyl valerate esterase activity resistant to paraoxon and sensitive to mipafox (Johnson, 1977), paraoxon does not, by de®nition, inhibit NTE. In this study, the inhibition of ChE and NTE does not account for the reduction of speci®c [3H]DFP binding produced by in vivo pretreatment with these ChE and NTE inhibitors. Reductions in speci®c [3H]DFP binding does not correlate to ChE and NTE inhibition either with regard to the degree of inhibition or the course, even though paraoxon and PMSF are ChE and NTE inhibitors, respectively. This suggests that the target site(s) for DFP are different from the active sites of ChE and NTE (and carboxylesterase). Chlorpyrifos has been reported to cause OPIDN in man (Lotti and Moretto, 1986) and in hens (Capodicasa et al., 1991). Capodicasa and coworkers (1991) concluded that the minimal neuropathic dose of chlorpyrifos was 60±90 mg/kg p.o., corresponding to four to six times the estimated LD50, even though pralidoxime (2PAM) in conjunction with atropine was necessary to reverse AChE inhibition and cholinergic toxicity. This single oral dose represented an anomaly in which the inhibition of NTE reached threshold (greater than 70%) within 5±6 days but the high inhibition of AChE (greater than 90%) was measured within hours after dosing. However, in contrast, with continuous dosing of hens (either orally or dermally), Francis et al. (1985) reported that chlorpyrifos did not produce typical OPIDN. The syndrome produced differed in the delay of onset after dosing and in the apparent reversibility of the ataxia. Chlorpyrifos may be a dif®cult compound to experimentally detect evident symptoms of OPIDN. Moreover, the acute toxicity of chlorpyrifos results from its reactive metabolite, termed `oxon-form' (Sultatos et al., 1984), therefore, chlorpyrifos may exhibit the inhibitory effects in later periods. Although chlorpyrifos was used at a comparatively low dose in the present study, it hardly had any effect on either the [3H]DFP binding or the activities of AChE and NTE at
either 24 h or 8 days, except for the reduced activity of AChE in spinal cord cytosol 24 h after pretreatment. Therefore, it appears that chlorpyrifos may have delayed neurotoxicity at a very high dose without the interaction with ChE or NTE. In this decade, Abou-Donia and coworkers have reported ultrastructural changes at cellular level during OPIDN. These were characterized by enhanced phosphorylation of various cytoskeletal proteins and autophosphorylation of Ca2/calmodulin dependent protein kinase II (CaM kinase II) (Abou-Donia and Lapadula, 1990; Gupta and Abou-Donia, 1994). OPs decreased Ca2 pump activity and would result in increased total nerve intracellular calcium level (Sharma and Bhattacharya, 1995). CaM kinase II has been proposed to correlate with increased Ca2 in axoplasm and to result in enhanced Ca2-activated proteolysis of cytoskeletal proteins and a later degeneration of the axon (AbouDonia and Lapadula, 1990). In deed, recently, enhanced CaM kinase II activity in DFP-treated hen brain supernatant has been reported to cause increased phosphorylation of cytoskeletal proteins, such as neuro®lament and tau, and to be consistent with the observed anomalous aggregations (Gupta and AbouDonia, 1995, 1998, 1999). Moreover, DFP treatment induced transient increases in mRNA expression of CaM kinase IIa subunit and neuro®lament subunits in central nervous system of hen (Gupta et al., 1998, 1999). Therefore, the interaction between cytoskeletal proteins and enzymes that regulate the phosphorylation of these proteins, such as CaM kinase, may play a signi®cant role in the induction of OPIDN. How these observations relate to our results is unknown. However, the binding sites of DFP also existed in cytosol, and both the properties of those binding sites in the present study and the reported alterations of cytoskeletal proteins were consistent with a time-dependent manner of OPIDN. Therefore, the target site(s) for DFP toxicity, which is proposed to exist in cytosol in the present study, may not be membrane-bound structures like Ca2 pump and ChE, but intracellular functional protein(s). In summary, we demonstrated the existence of the speci®c binding sites of [3H]DFP not only in membrane but also in cytosol preparations from brain and spinal cord. The assays of speci®c [3H]DFP binding indicated that the DFP binding lasts longer in cytosol, up to at least 8 days. Similar long-lasting DFP binding on membrane was not observed. Furthermore, we attempted to clarify a correlation between the binding sites and ChE and NTE, and found that the binding sites were different from the active sites of ChE and
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carboxylesterase, including NTE. Therefore, the target site(s) for the initiation of DFP induced delayed toxicity may be present in cytosol rather than on membrane, especially in spinal cord. And the speci®c binding sites of DFP in cytosol, which were found in the present study, may play a signi®cant role in the initiation of OPIDN, and may be unrelated to the inhibition of ChE and/or NTE. ACKNOWLEDGEMENTS The authors wish to thank Dr. Michikazu Ota and Dr. Hisayoshi Kofujita for the supply of synthesized phenyl valerate, Dr. Donald E. Moss for reviewing the manuscript and valuable advice, and Ms. Kaori Yotsuya for useful advice on statistical analysis. REFERENCES Abou-Donia MB. Organophosphorus ester-induced delayed neurotoxicity. Annu Rev Pharmacol Toxicol 1981;21:511±48. Abou-Donia MB, Lapadula DM. Mechanisms of organophosphorus ester-induced delayed neurotoxicity: type I and type II. Annu Rev Pharmacol Toxicol 1990;30:405±40. Atterberry TT, Burnett WT, Chambers JE. Age-related differences in parathion and chlorpyrifos toxicity in male rats: target and nontarget esterase sensitivity and cytochrome P450-mediated metabolism. Toxicol Appl Pharmacol 1997;147:411±8. Bruns RF, Lawson-Wendling K, Pugsley TA. A rapid filtration assay for soluble receptors using polyethylenimine-treated filters. Anal Biochem 1983;132:74±81. Capodicasa E, Scapellato ML, Moretto A, Caroldi S, Lotti M. Chlorpyrifos-induced delayed polyneuropathy. Arch Toxicol 1991;65:150±5. Caroldi S, Lotti M. Neurotoxic esterase in peripheral nerve: assay, inhibition, and rate of resynthesis. Toxicol Appl Pharmacol 1982;62:498±501. Carrington CD, Abou-Donia MB. Characterization of [3 H]diisopropyl phosphorofluoridate-binding proteins in hen brain. Rates of phosphorylation and sensitivity to neurotoxic and nonneurotoxic organophosphorus compounds. Biochem J 1985;228:537±44. Carrington CD, Brown HR, Abou-Donia MB. Histopathological assessment of triphenyl phosphite neurotoxicity in the hen. NeuroToxicology 1988;9:223±34. CeÂspedes MV, Escudero MA, Barril J, Sogorb MA, Vicedo JL, Vilanova E. Discrimination of carboxylesterases of chicken neural tissue by inhibition with a neuropathic, non-neuropathic organophosphorus compounds and neuropathy promoter. Chem Biol Interac 1997;106:191±200. Ellman GL, Courtney KD, Andres Jr. V, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88±95. Francis BM, Metcalf RL, Hansen LG. Toxicity of organophosphorus esters to laying hens after oral and dermal administration. J Environ Sci Health 1985;B20:73±95.
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Gupta RP, Abdel-Rahman A, Wilmarth KW, Abou-Donia MB. Alteration in neurofilament axonal transport in the sciatic nerve of the diisopropyl phosphorofluoridate (DFP)-treated hen. Biochem Pharmacol 1997;53:1799±806. Gupta RP, Abou-Donia MB. In vivo and in vitro effects of diisopropyl phosphorofluoridate (DFP) on the rate of hen brain tubulin polymerization. Neurochem Res 1994;19:435±44. Gupta RP, Abou-Donia MB. Neurofilament phosphorylation and [125 I]calmodulin binding by Ca2/calmodulin-dependent protein kinase in the brain subcellular fractions of diisopropyl phosphorofluoridate (DFP)-treated hen. Neurochem Res 1995;20:1095±105. Gupta RP, Abou-Donia MB. Tau proteins-enhanced Ca2/ calmodulin (CaM)-dependent phosphorylation by the brain supernatant of diisopropyl phosphorofluoridate (DFP)-treated hen: tau mutants indicate phosphorylation of more amino acids in tau by CaM kinase II. Brain Res 1998;813:32±43. Gupta RP, Abou-Donia MB. Tau phosphorylation by diisopropyl phosphorofluoridate (DFP)-treated hen brain supernatant inhibits its binding with microtubules: role of Ca2/calmodulindependent protein kinase II in tau phosphorylation. Arch Biochem Biophys 1999;365:268±78. Gupta RP, Bing G, Hong J, Abou-Donia MB. cDNA cloning and sequencing of Ca2/calmodulin-dependent protein kinase IIa subunit and its mRNA expression in diisopropyl phosphorofluoridate (DFP)-treated hen central nervous system. Mol Cell Biochem 1998;181:29±39. Gupta RP, Lin WW, Abou-Donia MB. Enhanced mRNA expression of neurofilament subunits in the brain and spinal cord of diisopropyl phosphorofluoridate-treated hens. Biochem Pharmacol 1999;57:1245±51. Johnson MK. Organophosphorus and other inhibitors of brain ``neurotoxic esterase'' and the development of delayed neurotoxicity in hens. Biochem J 1970;120:523±31. Johnson MK. The primary biochemical lesion leading to the delayed neurotoxic effects of some organophosphorus esters. J Neurochem 1974;23:785±9. Johnson MK. Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential. Arch Toxicol 1977;37:113±5. Johnson MK. The target for initiation of delayed neurotoxicity by organophosphorus esters: biochemical studies and toxicological applications. Rev Biochem Toxicol 1982;21:141±212. Johnson MK, Glynn P. Neuropathy target esterase (NTE) and organophosphorus-induced delayed polyneuropathy (OPIDP): recent advances. Toxicol Lett 1995;82/83:459±63. Kamata R, Suzuki T, Saito S, Kofujita H, Ota M, Kobayashi H. Lack of correlation of organophosphorus-induced delayed neuropathy with neuropathy target esterase in hens and Japanese quails. J Health Sci 1999;45:209±16. Kamata R, Saito S-Y, Suzuki T, Takewaki T, Kofujita H, Ota M, Kobayashi H. A comparative study of binding sites for diisopropyl phosphorofluoridate in membrane and cytosol preparations from spinal cord and brain of hens. NeuroToxicology 2001;22:191±202. Konno N, Suzuki N, Horiguchi H, Fukushima M. Characterization of high-affinity binding sites for diisopropylfluorophosphate (DFP) from chicken spinal cord membranes. Biochem Pharmacol 1994;48:2073±9. Lefkkowitz RJ, Hoffmen BB, Taylor P. Neurotransmission: the autonomic and somatic motor nervous systems. In: Hardman
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JG, Limbrid LE, Gilman AG, editors, Goodman and Gilman's the pharmacological basis of therapeutics, 9th ed. New York: McGraw-Hill, 1996. p. 106±39. Lotti M. The pathogenesis of organophosphate polyneuropathy. Toxicology 1992;21:455±87. Lotti M, Moretto A. Inhibition of lymphocyte neuropathy target esterase predicts the development of organophosphate polyneuropathy in man. Hum Toxicol 1986;5:114. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265±75. Maxwell DM. The specificity of carboxylesterase protection against the toxicity of organophosphorus compounds. Toxicol Appl Pharmacol 1992;114:306±12. Meredith C, Johnson MK. Species distribution of paraoxonresistant brain polypeptides radiolabelled with diisopropyl phosphorofluoridate ([3 H]DiPF): electrophoretic assay for the aged polypeptide of [3 H]DiPF-labelled neuropathy target esterase. J Neurochem 1989;52:1248±52.
Pope CN, Padilla SS. Chromatographic characterization of neurotoxic esterase. Biochem Pharmacol 1989;38:181±8. Sharma SK, Bhattacharya BK. Altered glycine transport by cerebral tissue and decreased Na and Ca pump activities during organophosphorus-ester-induced delayed neurotoxicity development period. J Biochem Toxicol 1995;10: 233±8. Sogorb MA, Viniegra S, Reig JA, Vilanova E. Partial characterization of neuropathy target esterase and related phenyl valerate esterases from bovine adrenal medulla. J Biochem Toxicol 1994;9:145±52. Sultatos LG, Shao M, Murphy SD. The role of hepatic biotransformation in mediating the acute toxicity of the phosphorothionate insecticide chlorpyrifos. Toxicol Appl Pharmacol 1984;73:60±8. Williams DG, Johnson MK. Gel-electrophoretic identification of hen brain neurotoxic esterase, labelled with tritiated di-isopropyl phosphorofluoridate. Biochem J 1981;199: 323±33.
Correlation of Binding Sites for Diisopropyl Phosphoro¯uoridate with Cholinesterase and Neuropathy Target Esterase in Membrane and Cytosol Preparations from Hen Ryo Kamata1,3,*, Shin-ya Saito2, Tadahiko Suzuki1, Tadashi Takewaki3, Haruo Kobayashi1 1
Department of Veterinary Pharmacology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Japan 2 Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan 3 United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan Received 23 February 2000; accepted 19 October 2000
Abstract To ®nd new putative target(s) for organophosphorus induced delayed neurotoxicity (OPIDN), we investigated the biochemical and pharmacological characteristics of [3H]diisopropyl phosphoro¯uoridate (DFP) binding to membrane and cytosol preparations from the brain and spinal cord of hens. Speci®c [3H]DFP binding was determined by subtracting non-speci®c binding from total binding. The binding sites of [3H]DFP, an organophosphate that induces OPIDN, were found not only on membrane but also in cytosol. Reduction of subsequent ex vivo speci®c [3H]DFP binding by in vivo pretreatment with unlabeled DFP was found in cytosol, not membrane. The reduced binding lasted to the onset of OPIDN, especially in spinal cord. These results suggest that the speci®c DFP binding sites in cytosol, rather than on membrane, are the most important with regard to the initiation of OPIDN. Inhibitors of cholinesterase (ChE) and neuropathy target esterase (NTE) other than DFP did not affect speci®c [3H]DFP binding to either membranes or cytosol in vivo. Additionally, inhibition of the activities of these esterases by these compounds was not consistent with either the degree of inhibition of the [3H]DFP binding or a time-dependent manner of OPIDN. These results suggest that DFP binding site(s) involved in the initiation of OPIDN may be different from the active sites of ChE and NTE. # 2001 Elsevier Science Inc. All rights reserved.
Keywords: Organophosphorus induced delayed neurotoxicity (OPIDN); Diisopropyl phosphoro¯uoridate (DFP); Cytosolic protein; Hen
INTRODUCTION A number of organophosphorus esters produce delayed neuropathy (OPIDN) in humans and other sensitive avian and mammalian species (AbouDonia, 1981). Inhibition and subsequent modi®cation (``aging'') of an enzyme known as neuropathy target
* Corresponding author. Tel.: 81-19-621-6215; Fax: 81-19-621-6215. E-mail address: [email protected] (R. Kamata).
esterase (NTE) has been proposed as the initial effect of compounds that induce OPIDN (Johnson, 1970, 1982). However, although numerous studies have attempted to associate NTE with OPIDN, the inhibition of NTE activity does not correspond with the onset of morphological/clinical signs of OPIDN (Abou-Donia and Lapadula, 1990) and the structure and physiological function(s) of NTE are still unknown. In the 1990's, many studies described ultrastructural changes at the cellular level during OPIDN. These changes include enhanced aggregation and phosphorylation of cytoskeletal proteins (i.e. microtubules and
0161-813X/01/$ ± see front matter # 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 1 - 8 1 3 X ( 0 0 ) 0 0 0 1 2 - 7
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neuro®laments), and the alteration of axonal transport (Abou-Donia and Lapadula, 1990; Gupta and AbouDonia, 1994). These effects on axoplasm have been proposed to result from alterations in Ca2/calmodulin dependent protein kinase activity (Gupta and AbouDonia, 1995; Gupta et al., 1997). These observations suggest that the initiation of neuropathy in OPIDN involves intracellular component(s) other than neuronal cell membrane. Diisopropyl phosphoro¯uoridate (DFP) is known to inhibit the activity of NTE and produce OPIDN. There have been many investigations of the DFP labeling sites (proteins) on membrane of hen brain. These have been mostly aimed at characterizing NTE or phenyl valerate esterases, because of the hypothesis that membrane bound esterases such as acetylcholinesterase (AChE) and NTE might be involved (Williams and Johnson, 1981; Pope and Padilla, 1989; Meredith and Johnson, 1989). However, it is our hypothesis that there may be DFP binding sites other than AChE and NTE in neural tissue with physiological functions in the pathogenesis of OPIDN. Carrington and Abou-Donia (1985) have reported that there are several DFP binding sites in both membrane and soluble fractions separated by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis from hen brain and the number of DFP binding sites does not necessarily correspond to phenyl valerate, an arti®cial substrate of NTE, hydrolysing activity in membrane. We also previously performed binding assays in membrane and cytosol preparations from the brain and spinal cord of chickens in vitro and con®rmed the presence of speci®c DFP binding sites in cytosol in addition to the expected binding sites in membrane. The binding sites in cytosol had a lower af®nity for DFP than membrane and were obviously different from cholinesterase (ChE) and NTE. The role of these cytosol DFP binding sites in the production of OPIDN appeared to warrant further investigation. The purpose of the present study was, therefore, to clarify the pharmacological and biochemical functions of the new cytosol DFP binding site(s) in production of OPIDN. Hens were pretreated, in vivo, with various inhibitors of ChE and NTE, and subsequent, ex vivo, speci®c binding of [3H]DFP in membrane and cytosol preparations from the brain and spinal cord was assayed. Additionally, the effects of AChE and NTE inhibitors on enzyme activity were examined in each preparation, and compared with the results of binding assay. The results suggest that speci®c DFP binding sites on membrane may be the active sites of ChE but the binding sites in cytosol seem to be unrelated to ChE
or NTE. It appears that the speci®c DFP binding sites in cytosol, especially in spinal cord, may have a role in the initiation of OPIDN. Furthermore, the initiation of OPIDN seems to be unrelated to inhibition of ChE or NTE. MATERIALS AND METHODS Chemicals DFP, diethyl p-nitrophenyl phosphate (paraoxon), phenylmethylsulfonyl ¯uoride (PMSF), O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate (chlorpyrifos), and N,N0 -diisopropyl phosphorodiamidic ¯uoridate (mipafox) were purchased from Wako Pure Chemical Industries (Osaka, Japan), Aldrich Chemical Co., Inc. (Milwaukee, WI), Nacalai Tesque Co., Ltd. (Kyoto, Japan), Kanto Chemical Co., Inc. (Tokyo, Japan), and Oriza Laboratories, Inc. (Newburyport, MA), respectively. Phenyl valerate was kindly synthesized and puri®ed by Dr. M. Ota and Dr. H. Kofujita (Department of Wood Science and Technology, Faculty of Agriculture, Iwate University, Japan) by the method of Johnson (1977). [3H]DFP (111 GBq/ mmol) was purchased from Dupont/New England Nuclear (Boston, MA). Animals and Treatment Adult white Leghorn laying hens (Gallus gallus domesticus, body weight 1.6±2.2 kg) were used. In dosing experiments in vivo, DFP (2 mg/ml), paraoxon (0.3 mg/ml), PMSF (30 mg/ml), and chlorpyrifos (30 mg/ml) were dissolved in corn oil, and injected subcutaneously in the posterior cervical region at a volume of 1 ml/kg of body weight. To reduce the acute cholinergic toxicity of organophosphorus compounds, atropine sulfate (20 mg/kg) dissolved in saline, was injected subcutaneously 20 min before the treatment with organophosphorus compounds. Control hens were treated with corn oil as vehicle at 1 ml/kg. The birds were sacri®ced 24 h or 8 days after the treatment, and brain and spinal cord were collected and assayed for speci®c DFP binding. Tissue Preparations Tissue preparations were made using a modi®ed method of Konno et al. (1994). The medulla and cerebellum, regions with neuropathological changes in OPIDN, were removed from the brain to compare
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with the spinal cord, a susceptible tissue. The remaining brain tissue and spinal cords were cleaned free of blood vessels and meninges, and homogenized on ice in 10 vol (w/v) of ice-cold HEPES buffer (50 mM, pH 7.4) using a Te¯on-glass homogenizer. These homogenates were centrifuged at 1000 g for 10 min at 48C, and the supernatants were centrifuged again at 50,000 g for 10 min at 48C. The resulting supernatants were used as cytosol preparation. Furthermore, the pellets from the last 50,000 g centrifugation were resuspended in HEPES buffer, and recentrifuged at 50,000 g for an additional 10 min at 48C and the supernatants were discarded. HEPES buffer was then added to these washed pellets, which were then resuspended using Polytron homogenizer, so the concentration of these preparations were about 50 mg tissue/ml. The resulting suspensions were used as the membrane preparation. Assay of [3H]diisopropyl Phosphorofluoridate Binding To assay the speci®c binding of [3H]DFP to the membrane and cytosol preparations, the membrane or cytosol preparations were diluted to 5 or 10 vol, respectively, with 10 mM sodium phosphate buffer (pH 7.4). Diluted preparations of 100 ml each and 100 ml of 10 mM sodium phosphate buffer or 100 ml of 25 mM unlabeled DFP (®nal concentration of 10 mM) were incubated with 50 ml of [3H]DFP at 378C for 60 min. For experiments examining concentration dependent (saturation) binding of [3H]DFP, or in dosing experiments in vivo, the solution of [3H]DFP was adjusted to 2±128 nM (®nal concentrations of 0.4±25.6 nM), or 4, 16 or 64 nM (0.8, 3.2 or 12.8 nM), respectively. The reaction was terminated by ®ltration through a GF/B glass ®lter (24 mm in diameter, Whatman International Ltd., Maidston, UK). The ®lter was washed twice with 5 ml of ice-cold Tris±HCl buffer (10 mM Tris, 140 mM NaCl, pH 7.4). The ®lters were soaked in 0.3% polyethylenimine for 1±24 h before use and were placed on the ®lter apparatus without washing according to the method of Bruns et al. (1983). After ®lters had been soaked overnight in 5 ml of scintillation cocktail (ACS-II; Amersham, Bucks, UK), the radioactivity attributable to tritium was counted (LSC-5100, Aloka Co. Ltd., Tokyo). Speci®c binding of [3H]DFP was de®ned as difference between the total binding and non-speci®c binding measured in the absence and presence of unlabeled DFP, respectively.
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Determination of NTE and AChE Activities and Protein Activity of NTE was determined as described by Johnson (1977). The activity was de®ned as the difference between the colorimetric determinations of the phenol liberated from the paraoxon-resistant (40 mM) and mipafox-sensitive (50 mM) hydrolysis of phenyl valerate, and expressed as nanomoles of phenyl valerate hydrolyzed per minute per milligram of protein. Activity of AChE in each preparation was assayed by the method of Ellman et al. (1961). Concentration of protein was measured by the method of Lowry et al. (1951) with bovine serum albumin as the standard. Data Analysis Saturation binding data were converted to Scatchard plots, from which af®nity constants Kd and Bmax values were determined as ±1/slope and the x-axis intercept, respectively. Data from [3H]DFP binding in vivo and the activities of AChE and NTE are presented as means S:E. Comparisons between each groups were performed by using a one-way analysis of variance (ANOVA). Post hoc analysis was performed with Dunnett's test. The activity of NTE under 30% of control value was taken as a threshold level, because it has been proposed that OPIDN is caused by 70±80% inhibition of NTE (Johnson and Glynn, 1995). RESULTS Saturation Binding of [3H]DFP The characteristics of [3H]DFP binding to membrane and cytosol preparations from brain and spinal cord of untreated hens was analyzed by saturation curves of the speci®c binding which reached a plateau. The Kd and Bmax values of [3H]DFP binding in each preparation were determined using Scatchard analysis. Similar results were obtained by non-linear regression (not shown). Kd values (nM) of membrane preparations were 1.40 for spinal cord and 3.36 for brain. Those of cytosol preparations were 4.03 for spinal cord and 7.41 for brain. These results show that the Kd values were consistently lower in membrane than in cytosol. The Bmax values (pmol/mg protein) of membrane preparations were 1.71 for spinal cord and 1.49 for brain, and those of cytosol preparations were 0.57 for spinal cord and 0.41 for brain. These results show that the
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Bmax values were higher in membrane than in cytosol. Overall, the minimum Kd and maximum Bmax values were found in membrane preparations from spinal cord. Concentrations of [3H]DFP from 0.8 to 12.4 nM were used to study binding to preparations from hens pretreated with organophosphates or PMSF. Effects of Pretreatment with Organophosphates and PMSF on [3H]DFP Binding Ex Vivo The effects of pretreatments with DFP, chlorpyrifos, paraoxon and PMSF on subsequent speci®c [3H]DFP binding to membrane and cytosol preparations from brain and spinal cord of hens are shown in Figs. 1 and 2, respectively. In membrane preparations, the speci®c [3H]DFP binding to brain and spinal cord from
hens 24 h after an exposure to DFP was signi®cantly decreased at each concentration of [3H]DFP (Fig. 1A and B). However, DFP did not signi®cantly decrease the binding to either tissue 8 days after exposure except for the lowest concentration of [3H]DFP (®nal, 0.8 nM) (Fig. 1C and D). Paraoxon, which does not induce OPIDN, PMSF, an antagonist of NTE, and chlorpyrifos, which is proposed as a delayed neurotoxic compound, hardly decreased the binding to either tissue 24 h and 8 days after exposure. In cytosol preparations, speci®c [3H]DFP binding to brain and spinal cord 24 h after pretreatment with DFP was signi®cantly decreased at ®nal concentrations of 3.2 and 12.8 nM [3H]DFP (Fig. 2A and B). In addition, DFP continued to signi®cantly decrease the speci®c binding to spinal cord even 8 days after pretreatment (Fig. 2D). Paraoxon, PMSF and chlorpyrifos hardly
Fig. 1. Effects of organophosphates and PMSF on specific [3H]FP binding to membrane preparations from brain (A) and spinal cord (B) 24 h after treatment, and brain (C) and spinal cord (D) 8 days after treatment. Membrane preparations were incubated in the presence of 0.8, 3.2 or 12.8 nM [3H]DFP. Data are shown as means with standard error bars for four experiments. Asterisks indicate experimental values that are significantly different from the respective control (P < 0:05, P < 0:01).
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Fig. 2. Effects of organophosphates and PMSF on specific [3H]DFP binding to cytosol preparations from brain (A) and spinal cord (B) 24 h after treatment, and brain (C) and spinal cord (D) 8 days after treatment. Cytosol preparations were incubated in the presence of 0.8, 3.2 or 12.8 nM [3H]DFP. Data are shown as means with standard error bars for four experiments. Asterisks indicate experimental values that are significantly different from the respective control (P < 0:05, P < 0:01).
decreased the binding to either tissue 24 h and 8 days after an exposure, except for the binding at ®nal concentrations of 3.2 nM [3H]DFP 24 h after exposure to paraoxon. Acetylcholinesterase Activities The results of in vivo pretreatments with organophosphates and PMSF on AChE activity in membrane and cytosol preparations are shown in Figs. 3 and 4, respectively. As expected, the AChE activity in cytosol was far lower than in membrane from both brain and spinal cord of untreated hens (Table 1). In membrane preparations, the most potent compounds in inhibiting the AChE activity in brain and spinal cord 24 h after pretreatment were DFP and paraoxon, and the activities were signi®cantly inhibited (Fig. 3A and B). PMSF and chlorpyrifos did not signi®cantly inhibit
the activity in membranes from either tissue. The activities in both brain and spinal cord membranes were recovered and were not signi®cantly different Table 1 Acetylcholinesterase (AChE) and neuropathy target esterase (NTE) activities in membrane and cytosol preparations from brain and spinal cord of untreated hensa Tissue
Preparation
AChE
NTE
Brain
Membrane Cytosol
442.33 19.40 45.11 6.88**
143.27 19.27 26.88 3.93**
Spinal cord
Membrane Cytosol
190.38 13.97 21.89 0.16**
88.34 2.61 12.71 3.41**
a Activity of AChE is expressed as mmol acetylthiocholine hydrolyzed/ min/g protein, mean S.E.M. of four expremenents for each preparation. Activity of NTE is expressed as nmol phenyl valerate hydrolyzed/min/mg protein, mean S.E.M. of four experiments for each preparation. ** Asterisks indicate experimental values significantly different from the values of respective membrane preparation (P < 0:01).
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Fig. 3. Effects of organophosphates and PMSF on AChE activity in membrane preparations from brain (A) and spinal cord (B) 24 h after treatment, and brain (C) and spinal cord (D) 8 days after treatment. Data are shown as means with standard error bars for four experiments. Asterisks indicate experimental values that are significantly different from the respective control (P < 0:05, P < 0:01).
from those of control at 8 days, regardless of pretreatment compound (Fig. 3C and D). In cytosol preparations, the most potent AChE inhibitors in both tissues 24 h after pretreatment were, like membrane, DFP and paraoxon, which produced signi®cant inhibition (Fig. 4A and B). PMSF produced no signi®cant inhibition in cytosol from either brain or spinal cord but chlorpyrifos produced slight but statistically signi®cant inhibition in spinal cord. The AChE activities in cytosol from both brain and spinal cord were recovered and were not signi®cantly different from those of control at 8 days, regardless of pretreatment compound (Fig. 4C and D). Neuropathy Target Esterase Activities The results of in vivo pretreatments with organophosphates and PMSF on NTE activity in membrane
and cytosol preparations are shown in Figs. 5 and 6, respectively. The NTE activity in cytosol was far lower than in membrane from both brain and spinal cord of untreated hens (Table 1). In membrane preparations, the most potent inhibition of NTE activity in brain and spinal cord 24 h after pretreatment was produced by DFP and PMSF. In these cases, the activities were very low, under 30% of control value, the proposed threshold level for OPIDN (Fig. 5A and B). However, the NTE activity in both brain and spinal cord membranes recovered to far above this proposed threshold by 8 days (Fig. 5C and D), even though slight but signi®cant inhibition remained in spinal cord. Paraoxon and chlorpyrifos did not signi®cantly inhibit NTE activity in membranes in either brain or spinal cord either 24 h or 8 days after pretreatment. In cytosol preparations, the most potent inhibitors of NTE activity in both brain and spinal cord 24 h after
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Fig. 4. Effects of organophosphates and PMSF on AChE activity in cytosol preparations from brain (A) and spinal cord (B) 24 h after treatment, and brain (C) and spinal cord (D) 8 days after treatment. Data are shown as means with standard error bars for four experiments. Asterisks indicate experimental values that are significantly different from the respective control (P < 0:05, P < 0:01).
the pretreatment were, like membrane, DFP and PMSF. Again, the NTE activity in cytosol after pretreatment with DFP and PMSF were very low, under 30% of control (Fig. 6A and B). However, the activities in both brain and spinal cord cytosol 8 days after the exposure to each compound had recovered far above the proposed threshold for OPIDN (30% of control) (Fig. 6C and D), even though signi®cant inhibition remained in brain pretreated with both DFP and PMSF, and in spinal cord pretreated with DFP, respectively. Paraoxon and chlorpyrifos did not signi®cantly inhibit NTE activity in either brain or spinal cord either 24 hr or 8 days after pretreatment. DISCUSSION To ®nd and clarify the biochemical and pharmacological characteristics of [3H]DFP binding site(s) other than membrane components, we used cytosol
preparations from brain and spinal cord of hens and compared the results with the membrane preparations. In the present study, [3H]DFP bound not only to the membrane preparation but also to the cytosol preparation of brain and spinal cord. The existence of the DFP binding sites in cytosol may support the existence of new target site(s) of OPIDN. However, both the number of speci®c [3H]DFP binding sites and the af®nity of the binding to cytosol were lower than those of membrane, even though the cytosol contains a large number of proteins. This indicates that there is weak interaction between cytosolic structures and DFP. On the other hand, the af®nities in spinal cord were higher than those in brain, and the number of binding sites were similar in brain and spinal cord. The higher af®nity in spinal cord is consistent with the distribution of neuropathologic lesions on OPIDN, which are not seen in higher brain but in spinal cord and peripheral nerves (Carrington et al., 1988).
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Fig. 5. Effects of organophosphates and PMSF on NTE activity in membrane preparations from brain (A) and spinal cord (B) 24 h after treatment, and brain (C) and spinal cord (D) 8 days after treatment. Data are shown as means with standard error bars for four experiments. Sharps indicate experimental values that are under 30% of respective control value, as a threshold level, because it has been proposed that OPIDN is caused at 70±80% inhibition of NTE. Asterisks indicate experimental values that are significantly different from the respective control (P < 0:05, P < 0:01).
DFP produced severe reduction of speci®c [3H]DFP binding to both membrane and cytosol preparations 24 h after pretreatment in vivo. However, the effects of pretreatment with DFP on speci®c [3H]DFP binding in cytosol even 8 days after treatment were still marked, especially in spinal cord. In contrast to cytosol, the effects of DFP pretreatment on membrane at 8 days were slight or not signi®cant. The latent period of OPIDN after a single dose of organophosphates is approximately 1±3 weeks (Lotti, 1992). In the present study, hens pretreated with DFP also exhibited the early clinical signs of OPIDN 8 days later (data not shown). These observations indicate that the continuous binding sites of DFP are present in cytosol rather than on membrane, whose region inhibited continuously (spinal cord) is consistent with the neuropathology of OPIDN. Moreover, the effect of DFP on cytosol
continues to the onset of OPIDN or longer. These data suggest, therefore, target site(s) for the initiation of DFP induced delayed toxicity may be in cytosol rather than on membrane, especially in spinal cord. Although paraoxon produced strong reductions on speci®c [3H]DFP binding to membrane in vitro (Konno et al., 1994; Kamata et al., 2001), there was hardly any effect from in vivo pretreatment in either membrane or cytosol in the present study. Pretreatment in vivo with chlorpyrifos and PMSF also produced no reduction in speci®c [3H]DFP binding to either membrane or cytosol. Paraoxon, chlorpyrifos and PMSF, and their reactive metabolites are expected to have no effect on speci®c [3H]DFP binding after in vivo pretreatment in either brain or spinal cord, because of detoxication mechanisms such as carboxylesterase and cytochromes P450 (Atterberry et al., 1997). It is interesting to note,
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Fig. 6. Effects of organophosphates and PMSF on NTE activity in cytosol preparations from brain (A) and spinal cord (B) 24 h after treatment, and brain (C) and spinal cord (D) 8 days after treatment. Data are shown as means with standard error bars for four experiments. Sharps indicate experimental values that are under 30% of respective control value, as a threshold level, because it has been proposed that OPIDN is caused at 70±80% inhibition of NTE. Asterisks indicate experimental values that are significantly different from the respective control (P < 0:05, P < 0:01).
however, that these compounds still produced inhibition of AChE and NTE activity in the present study. Our results also show that the activities of AChE and NTE in cytosol preparation are far lower than those in membrane, in agreement with a previous study (Sogorb et al., 1994). The results are consistent with the idea that AChE and NTE are membrane-bound proteins. DFP and paraoxon produced marked inhibition of AChE activity in both membranes and cytosol 24 h after pretreatment in vivo. Pretreatment with DFP and PMSF in vivo also produced strong inhibition of NTE activity as measured at 24 h. If the initiation and progression of OPIDN results from inhibition of AChE and/or NTE activity, it is expected that some moderate inhibition of these esterases would continue at least until the onset of OPIDN or thereafter. However, AChE
activity exhibited good recovery in both membrane and cytosol by 8 days after in vivo pretreatment. This suggests that AChE inhibition by DFP or paraoxon is acute and does not correlate with the progression of OPIDN. Similarly, NTE activity also exhibited good recovery in both membrane and cytosol by 8 days after in vivo pretreatment, even though some inhibition (above the proposed threshold) remained. The half-life of recovery of NTE in hen brain after treatment with neuropathic compounds has been reported to be between 4 and 6 days (Johnson, 1974), which is similar to data from spinal cord and sciatic nerve (Caroldi and Lotti, 1982). And then, NTE activity gradually recovers. However, the clinical symptoms of OPIDN progress more and more in a time-dependent manner and are not necessarily consistent with NTE inhibition (Kamata et al., 1999).
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NTE has been reported to account for only a small proportion of total phenyl valerate hydrolyzing activity in hen brains (Abou-Donia and Lapadula, 1990; Lotti, 1992). Therefore, NTE is only a portion of many carboxylesterases which hydrolyze phenyl valerate as an arti®cial substrate. It is important to note that PMSF can irreversibly inhibit carboxylesterase, including NTE, without inducing neuropathy (CeÂspedes et al., 1997). Moreover, neural tissue, including glial and satellite cells, has a high level ChEs such as AChE (Lefkkowitz et al., 1996). Paraoxon, as an organophosphate, inhibits both carboxylesterases and ChE (Maxwell, 1992). However, because NTE is de®ned as phenyl valerate esterase activity resistant to paraoxon and sensitive to mipafox (Johnson, 1977), paraoxon does not, by de®nition, inhibit NTE. In this study, the inhibition of ChE and NTE does not account for the reduction of speci®c [3H]DFP binding produced by in vivo pretreatment with these ChE and NTE inhibitors. Reductions in speci®c [3H]DFP binding does not correlate to ChE and NTE inhibition either with regard to the degree of inhibition or the course, even though paraoxon and PMSF are ChE and NTE inhibitors, respectively. This suggests that the target site(s) for DFP are different from the active sites of ChE and NTE (and carboxylesterase). Chlorpyrifos has been reported to cause OPIDN in man (Lotti and Moretto, 1986) and in hens (Capodicasa et al., 1991). Capodicasa and coworkers (1991) concluded that the minimal neuropathic dose of chlorpyrifos was 60±90 mg/kg p.o., corresponding to four to six times the estimated LD50, even though pralidoxime (2PAM) in conjunction with atropine was necessary to reverse AChE inhibition and cholinergic toxicity. This single oral dose represented an anomaly in which the inhibition of NTE reached threshold (greater than 70%) within 5±6 days but the high inhibition of AChE (greater than 90%) was measured within hours after dosing. However, in contrast, with continuous dosing of hens (either orally or dermally), Francis et al. (1985) reported that chlorpyrifos did not produce typical OPIDN. The syndrome produced differed in the delay of onset after dosing and in the apparent reversibility of the ataxia. Chlorpyrifos may be a dif®cult compound to experimentally detect evident symptoms of OPIDN. Moreover, the acute toxicity of chlorpyrifos results from its reactive metabolite, termed `oxon-form' (Sultatos et al., 1984), therefore, chlorpyrifos may exhibit the inhibitory effects in later periods. Although chlorpyrifos was used at a comparatively low dose in the present study, it hardly had any effect on either the [3H]DFP binding or the activities of AChE and NTE at
either 24 h or 8 days, except for the reduced activity of AChE in spinal cord cytosol 24 h after pretreatment. Therefore, it appears that chlorpyrifos may have delayed neurotoxicity at a very high dose without the interaction with ChE or NTE. In this decade, Abou-Donia and coworkers have reported ultrastructural changes at cellular level during OPIDN. These were characterized by enhanced phosphorylation of various cytoskeletal proteins and autophosphorylation of Ca2/calmodulin dependent protein kinase II (CaM kinase II) (Abou-Donia and Lapadula, 1990; Gupta and Abou-Donia, 1994). OPs decreased Ca2 pump activity and would result in increased total nerve intracellular calcium level (Sharma and Bhattacharya, 1995). CaM kinase II has been proposed to correlate with increased Ca2 in axoplasm and to result in enhanced Ca2-activated proteolysis of cytoskeletal proteins and a later degeneration of the axon (AbouDonia and Lapadula, 1990). In deed, recently, enhanced CaM kinase II activity in DFP-treated hen brain supernatant has been reported to cause increased phosphorylation of cytoskeletal proteins, such as neuro®lament and tau, and to be consistent with the observed anomalous aggregations (Gupta and AbouDonia, 1995, 1998, 1999). Moreover, DFP treatment induced transient increases in mRNA expression of CaM kinase IIa subunit and neuro®lament subunits in central nervous system of hen (Gupta et al., 1998, 1999). Therefore, the interaction between cytoskeletal proteins and enzymes that regulate the phosphorylation of these proteins, such as CaM kinase, may play a signi®cant role in the induction of OPIDN. How these observations relate to our results is unknown. However, the binding sites of DFP also existed in cytosol, and both the properties of those binding sites in the present study and the reported alterations of cytoskeletal proteins were consistent with a time-dependent manner of OPIDN. Therefore, the target site(s) for DFP toxicity, which is proposed to exist in cytosol in the present study, may not be membrane-bound structures like Ca2 pump and ChE, but intracellular functional protein(s). In summary, we demonstrated the existence of the speci®c binding sites of [3H]DFP not only in membrane but also in cytosol preparations from brain and spinal cord. The assays of speci®c [3H]DFP binding indicated that the DFP binding lasts longer in cytosol, up to at least 8 days. Similar long-lasting DFP binding on membrane was not observed. Furthermore, we attempted to clarify a correlation between the binding sites and ChE and NTE, and found that the binding sites were different from the active sites of ChE and
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carboxylesterase, including NTE. Therefore, the target site(s) for the initiation of DFP induced delayed toxicity may be present in cytosol rather than on membrane, especially in spinal cord. And the speci®c binding sites of DFP in cytosol, which were found in the present study, may play a signi®cant role in the initiation of OPIDN, and may be unrelated to the inhibition of ChE and/or NTE. ACKNOWLEDGEMENTS The authors wish to thank Dr. Michikazu Ota and Dr. Hisayoshi Kofujita for the supply of synthesized phenyl valerate, Dr. Donald E. Moss for reviewing the manuscript and valuable advice, and Ms. Kaori Yotsuya for useful advice on statistical analysis. REFERENCES Abou-Donia MB. Organophosphorus ester-induced delayed neurotoxicity. Annu Rev Pharmacol Toxicol 1981;21:511±48. Abou-Donia MB, Lapadula DM. Mechanisms of organophosphorus ester-induced delayed neurotoxicity: type I and type II. Annu Rev Pharmacol Toxicol 1990;30:405±40. Atterberry TT, Burnett WT, Chambers JE. Age-related differences in parathion and chlorpyrifos toxicity in male rats: target and nontarget esterase sensitivity and cytochrome P450-mediated metabolism. Toxicol Appl Pharmacol 1997;147:411±8. Bruns RF, Lawson-Wendling K, Pugsley TA. A rapid filtration assay for soluble receptors using polyethylenimine-treated filters. Anal Biochem 1983;132:74±81. Capodicasa E, Scapellato ML, Moretto A, Caroldi S, Lotti M. Chlorpyrifos-induced delayed polyneuropathy. Arch Toxicol 1991;65:150±5. Caroldi S, Lotti M. Neurotoxic esterase in peripheral nerve: assay, inhibition, and rate of resynthesis. Toxicol Appl Pharmacol 1982;62:498±501. Carrington CD, Abou-Donia MB. Characterization of [3 H]diisopropyl phosphorofluoridate-binding proteins in hen brain. Rates of phosphorylation and sensitivity to neurotoxic and nonneurotoxic organophosphorus compounds. Biochem J 1985;228:537±44. Carrington CD, Brown HR, Abou-Donia MB. Histopathological assessment of triphenyl phosphite neurotoxicity in the hen. NeuroToxicology 1988;9:223±34. CeÂspedes MV, Escudero MA, Barril J, Sogorb MA, Vicedo JL, Vilanova E. Discrimination of carboxylesterases of chicken neural tissue by inhibition with a neuropathic, non-neuropathic organophosphorus compounds and neuropathy promoter. Chem Biol Interac 1997;106:191±200. Ellman GL, Courtney KD, Andres Jr. V, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88±95. Francis BM, Metcalf RL, Hansen LG. Toxicity of organophosphorus esters to laying hens after oral and dermal administration. J Environ Sci Health 1985;B20:73±95.
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