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Comparative studies of two organophosphorus compounds in the mouse
Toxicology Letters ELSEVIER
Toxicology Letters 81 (1995) 45-53
Comparative studies of two organophosphorus the mouse
compounds in
Elaine Mutch*, Sean S. Kelly, Peter G. Blain, Faith M. Williams Toxicology Unit. Department of Environmental and Occupational Medicine. The Medical School, Newcastle University, Newcastle NE.2 4HH. UK
Received 14 October 1994; revision received 9 May 1995; accepted I I May 1995
A rodent model, the albino mouse, was used to investigate the in vitro and in vivo capacity of 2 organophosphate (OP) compounds, mipafox and ecothiopate, to inhibit enzymes considered to be involved in the mechanisms of OP toxicity. Mipafox and ecothiopate were chosen as model compounds because the former can produce a delayed neuropathy whereas the latter does not. Mipafox (110 pmol/kg, s.c.) inhibited brain acetylcholinesterase (AChE), neuropathy target esterase (NTE) and phenylvalerate hydrolases by 58, 64 and 65%, while diaphragm AChE and phenylvalerate hydrolases were inhibited by 66 and 80%, respectively. In contrast, ecothiopate (0.5 pmol/kg) had no effect on brain NTE or on brain or diaphragm phenylvalerate hydrolases. At the same time, diaphragm AChE was inhibited by 60% while brain AChE activity had increased by 15% of control. Mipafox was a potent inhibitor of AChE and NTE in vitro. Although ecothiopate was a highly potent anti-ChE in vitro, it had no inhibitory effect on NTE. Keywords:
Organophosphates;
Mouse; AChE; Neuropathy
1. Introduction The acute toxicity of organophosphates (OPs) follows inhibition ‘of acetylcholinesterase (AChE, EC 3.1.1.7) in neural tissue and at the neuromuscular junction. This results in accumulation of acetylcholine and hyperactivity within the cholinergic pathways. Acute toxicity of anticholinesterase (anti-ChE) compounds in man is mainly due to the peripheral effects of acetylcholine, although central nervous system (CNS) symp* Corresponding autihor.
target esterase; Phenylvalerate hydrolase
toms may also be apparent. Another syndrome, organophosphorus-induced delayed neuropathy (OPIDN), is believed to be initiated by inhibition and ‘aging’ of a membrane-bound carboxylesterase called neuropathy target esterase (NTE) found in neural tissue [ 11. Aging is a timedependent reaction which involves the loss of an alkyl group from the phosphorylated enzyme. When this reaction takes place, usually within minutes of inhibition, the enzyme becomes irreversibly phosphorylated. NTE is believed to be one of several carboxylesterases present in neural tissue determined as that fraction of and is
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E. Mulch et al. / Toxicology Letters 81 (1995) 45-53
phenylvalerate esterase activity which is paraoxonresistant and mipafox-sensitive. OPIDN may develop lo-21 days post exposure to some OPs regardless of any antiChE effects. The hen is currently the animal of choice when screening for the neurotoxic potential of OP compounds, with greater than 70% inhibition of NTE correlating well with development of OPIDN [2]. However, less sensitive species such as rodent have proved to be a good model for investigating biochemical [3], neuropathological [4] and electrophysiological [5] changes following OP exposure. Mipafox (NJV-diisopropylphosphorodiamidofluoridate) was implicated in the manifestation of both acute (antiChE) and neurotoxic symptoms (OPIDN) in man following an accident in the early stages of its development as a pesticide [6]. It was banned from commercial use soon after the incident but has been used more recently as a tool to study anti-ChE and neurotoxic effects in experimental animals. Ecothiopate o,o-diethyl (S-(2trimethyl-ammoniumethyl)phospHorothioate iodide) is a cationic compound which will quickly diethylphosphorylate the serine active centre of an enzyme, thereby inactivating it. Its reversible antiChE action is made use of in the clinical management of glaucoma. Although some OPs are direct AChE inhibitors, many require metabolic activation through oxidative desulphuration by microsomal cytochrome P-450 for toxicity. Both mipafox and ecothiopate are direct AChE inhibitors. Detoxification of OPs occurs both by hydrolysis via the phosphoric triester hydrolases (EC 3.1.8.1 and 3.1.8.2) and following phosphorylation of serine-containing carboxylesterases (EC 3.1.1.1.) present in blood, liver and other tissues. The aims of the present study were to investigate: (1) the inhibition profiles of AChE (diaphragm and brain), NTE (brain) and carboxylesterases (diaphragm and brain) with respect to time following dosing with mipafox (110 PmoYkg) or ecothiopate (0.5 pmol/kg); (2) the dose-response inhibition profiles for the same enzymes at 2 h post mipafox (27.5- 110 Fmol/kg) administration; (3) in vitro inhibition of brain homogenate by mipafox and ecothiopate; (4) plasma carboxylesterase inhibition in vivo.
2. Materials and methods 2.1. Organophospha tes Mipafox, was obtained from Chemsyn Science Labs (Lenexa, KS) and diluted from a stock solution (pH 6.0) shortly before use. Ecothiopate was prepared from Phospholine Eyedrops (Ayerst). Paraoxon (O,O-diethyl p-nitrophenyl phosphate) was obtained from Sigma (Poole, Dorset, UK) and used in the NTE assay. 2.2. Treatment and condition of animals Male albino mice aged 6-7 months (body weight 40-50 g), obtained from Bantin and Kingman (Hull, UK) were used in all experiments. Atropine (1.4 pmol/kg) was included in the injection to reduce the muscarinic effects caused by inhibition of AChE. The acute effect of a subcutaneous dose of the OPs used in these studies was skeletal muscle fasciculation which occured usually with ecothiopate (0.5 pmol/kg) and occasionally with mipafox (110 pmol/kg) concomitant with tremor. With mipafox, if fasciculation did occur, the onset was slower than with ecothiopate (at about 20 mm), but mobility had usually returned to normal by about 2 h. 2.3. Preparation of tissue homogenates Brain tissues were removed from the -70°C freezer, halved in the longitudinal plane, weighed and sufficient ice-cold 50 mM Tris-0.2 mM EDTA buffer (pH 8.0) added to produce a 10% (w/v) homogenate. The tissues were minced finely using scissors and then homogenised for 5 s using an icecold Polytron or Ultra Turrex Cell Homogeniser. The preparations were centrifuged at 12 000 x g for 15 min to remove cell debris, nuclei and mitochondria and stored at -70°C until analysis within a week. There was no loss of enzyme activity on freezing and thawing (data not shown). Diaphragm post mitochondrial fraction (lo%, w/v) was prepared in a similar manner. 2.4. In vitro studies Control animals were killed by cervical dislocation, the brain removed and 10% homogenates prepared as described previously. Mipafox inhibition of AChE was determined by preincubating brain homogenate (5 mg tissue) in O.,l M phos-
E. Murch et al. / Toxicology Leriers 81 (1995) 45-53
phate buffer, pH 8.0 (control) and with lo-850 pM mipafox (freshly prepared from stock in triscitrate buffer, pH 6.0, and added to incubations in a small volume) for 8 min at 30°C. Acetylthiocholine was added and AChE activity measured at 30°C as described below. Since preliminary experiments showed that control rat brain AChE activity decreased by 20% when incubated at 30°C for 20 min in the absence of inhibitor it was particularly important to relate results to controls under the same assay conditions. Inhibition of brain AChE by ecothiopate was determined following preincubation with the OP (range O-l PM) for 3.0 min at 30°C and then residual enzyme activity calculated by comparison to control incubations. The relationship log % control activity vs. OP concentration is expected to be linear for a singlecompartment kinetic reaction [7]. This plot was used to calculate the mipafox and ecothiopate concentrations which inhibit 50% control AChE activity (IC&. A mipafox inhibition profile was also determined for paraoxon-resistant phenylvalerate hydrolases. Brain homolgenate (equivalent to 6 mg tissue) was preincubated with either 40 PM paraoxon (A tube) or 40 PM paraoxon plus 20-420 pM mipafox (B tubes) for 20 min at 37°C prior to addition of phenylvalerate (1.4 mM). Phenylvalerate hydrolase activity was then measured in all tubes for 30 min at 37°C as described below. The mipafox inhibition profile of paraoxon-resistant hydrolases was obtained by subtracting the activity obtained in each B tube from that in tube .4. Each value was then compared to the control value (A tube) and expressed as a percentage. Inhibition of brain NTE and other phenylvalerate hydrolases with ecothiopate in vitro was determined by preincubation for 10 and 20 min at 37°C at an OP concentration of 1 FM. Data for mipafox inhibition of NTE and other phenylvalerate hydrolases was plotted as log % control activity vs. ,concentration of OP. The data was also fitted by iterative non-linear regression to a two-compartment equation as used by Sogorb et al. [8]: 100 x EIE, = A,e-‘K” + AZeVml + A, (A, t A2 + A, = 1)
41
Where E is enzyme activity at OP concentration I and E, is control activity in the absence of inhibitor. A, and A2 (expressed as percentages) are the amplitudes of the sensitive components 1 and 2 and K, and K2 are the exponential components of each component. A, is the amplitude of the inhibitor-resistant component. From this the ICso of the inhibitor sensitive component(s) were calculated: ICso = Ln 2/Ki 2.5. In vivo studies 2.51. Time profile of enzyme inhibition by mipafox and ecothiopate. Mipafox 20 mgfkg (110 pmovkg) or ecothiopate (0.5 pmol/kg) were given with atropine (1.4 pmol/kg) by S.C.injection in the nape of the neck. At 2 h, 3 days and 7 days animals were killed by cervical dislocation. In some studies with mipafox animals were killed at 0.5, 1, 2, 24 and 72 h in order to define the early time course of inhibition. Animals treated with atropine (1.4 pmol/kg) alone were used for control measurements. Whole brain and diaphragm were removed, washed in 0.1 M KCl/O.l M phosphate buffer (pH 7.4), blotted and then stored at -70°C until analysis of AChE (diaphragm and brain), NTE (brain) and carboxylesterase (brain and diaphragm). In some studies blood was collected by cardiac puncture from animals anaesthetised with Hypnorm/ Medazolam. The blood was placed into heparinised tubes and the plasma separated by centrifugation prior to analysis of carboxylesterase. 2.5.2. Dose profile of enzyme inhibition by mipafox. Mice were dosed with 27.5, 55, 82.5 or 110 rmollkg mipafox (s.c.) together with atropine (1.4 pmol/kg) and killed 2 h after the dose. Control animals were given atropine alone. Tissues were collected as above prior to measurement of AChE, NTE and carboxylesterase. 2.6. Enzyme assays 2.6.1. AChE activity. Activity was measured in brain or diaphragm homogenate by following the hydrolysis of acetylthiocholine (500 PM) at 30°C. Diaphragm homogenates were preincubated for 1 h at 0°C in 0.1 M phosphate buffer (pH 8.0) containing 50 FM ethopropazine in order to inhibit
E. Mutch et al. / Toxicology Letters 81 (1995) 45-53
48
non-specific cholinesterases [9] and thereby make the assay more specific for AChE. Hydrolysis of acetylthiocholine (500 PM) was measured at 30°C in 3-ml incubation volumes containing approximately 2.5 mg brain or 5 mg diaphragm. The method used was that described by Ellman et al. [lo] in which the liberated thiocholine reacts with 5,5’-dithiobis-(2-nitrobenzoic acid) to form 5thio-Znitrobenzoate and is then measured at 412 nm. Enzyme activity was expressed as nmol min-’ mg-’ tissue. 2.6.2. NTE activity. Brain NTE activity was measured by a micro-modification [l l] of the method of Johnson [ 121 using phenylvalerate (1.4 mM) as substrate. Homogenate (50 ~1, equivalent to approximately 6 mg tissue) was incubated in a total volume of 200 ~1 for 20 min at 37°C with either 40 PM paraoxon (Tube A) or 40 PM paraoxon + 50 PM mipafox (Tube B). Phenylvalerate was added and incubated for a further 30 min at 37°C and then the phenol produced determined by spectroscopy. NTE activity was defined as those phenylvalerate hydrolases which are resistant to inhibition by paraoxon (Tube A) but sensitive to inhibition by mipafox (Tube B), that is Tube A - Tube B. Enzyme activity was expressed as nmol min-’ mg-’ tissue. 2.6.3. Phenylvalerate hydrolase activity. Carboxylesterase activities in brain homogenate, diaphragm homogenate and plasma were determined by measuring formation of phenol from phenylvalerate (as described for NTE but in the absence of inhibitors). Tissue homogenate (2.5 mg tissue) or plasma (0.25 ~1) was incubated in 400-~1 volumes at 37°C in the presence of phenylvalerate (1.4 mM) for 10 and 2 min, respectively. Results were expressed as nmol min-’ mg-i tissue or nmol min-’ ~1~’ plasma. 2.7. Statistical analysis Results were expressed as mean f 1 S.D. Groups of treated animals were compared to control values by analysis of variance followed by Tukey’s HSD test. 3. Results 3.1. Control enzyme levels
Baseline enzyme levels for AChE, NTE and
phenylvalerate hydrolase were consistant with published values [13- 151 and are shown in Table 1. Results are the mean f 1 S.D. and are expressed in terms of wet weight of tissue. AChE activity in the brain was 1.39 i 0.10 nmol min-’ mg-’ tissue while brain phenylvalerate hydrolase activity was 2.5 f 0.22 nmol min-t mg-’ tissue. Brain NTE (0.11 f 0.03 nmol min-’ mg-’ tissue) represented 4.5% of the total phenylvalerate hydrolase activity. However, NTE activity was below the limit of detection in diaphragm (
and mipafox on enzyme
The in vitro inhibition of brain AChE at 30°C following preincubation with mipafox for 8 min (Table 2) ranged from 10% at 10 PM to 95% at 850 PM. The I& range was 95-125 PM. Fig. 1
Table 1 Control enzyme activities of brain, diaphragm and plasma for BKW mouse Enzyme activity n
Parameter Brain phenylvalerate Brain NTE
hydrolases
Brain AChE Diaphragm phenylvalerate hydrolases Diaphragm AChE Plasma phenylvalerate hydrolases
2.46 0.11 1.30 11.6 0.59 25.7
0.22 f 0.03 f 0.10 f 1.93 l 0.12 ztz l.OOa l
8 4 11 5 10 8
Enzyme activities are expressed as nmol min-’ mg-’ tissue and ‘nmol min-’ pl-’ plasma. Values are the mean * 1 SD.
E. Mutch et al. / Toxicology Letters 81 (1995) 45-53
Table 2 The mipafox and ecothiopate concentrations required to inhibit 500/aof BKW mouse brain AChE activity (ICSo AChE) at 30°C in vitro OP
IC,, AChE (FM)
Mipafox Ecothiopate
95.0-125 0.074-O. 100
Values are the IC, ranges (n = 4 mice).
49
from 6% at 10 nM to complete inhibition at 1000 nM with an IC,, range of 0.074-0.100 PM (Table 2). There was no inhibition of brain NTE or the other phenylvalerate hydrolases by ecothiopate at 37°C using a 1pM concentration for up to 30 min. 3.3. Effects of mipafox and ecothiopate on enzyme activity in vivo 3.3.1. Time profde of enzyme inhibition. Two h
shows the inhib:ition of paraoxon-resistant phenylvalerate hydrolases at 37°C by mipafox. Inhibition ranged from 3% at 2 PM to 25% at 50 PM with a very modest increase in inhibition with rising mipafox concentration up to 420 PM. Therefore about 70% of the paraoxon-resistant phenylvalerate hydrolases were also resistant to mipafox inhibition. Visual inspection of the linear portion (4-20 PM mipafox) of the semi-log plot estimated the I& at 10 PM although it was apparent that there was contribution from a low affinity component. Data was fitted to the two-compartment non-linear equation as described earlier, but with high errors due to the small number of points. The ICsO for the low affinity component thus derived (32.9 PM) was in good agreement with the visual estimate. An I&-, of 12.5 @I was calculated for the highaftinity component by using the one exponential mathematical model. Inhibition of brain AChE at 30°C following preincubation with ecothiopate for 3 min ranged
after injection of mipafox (110 pmolkg), AChE activity in brain and diaphragm was inhibited by 58 and 66%, respectively (Fig. 2). At the same time, brain phenylvalerate hydrolases were inhibited by 65% but NTE, which was inhibited by 64% at 2 h, was further inhibited at 24 h and below the limit of detection (0.01 nmol min-’ mg-’ tissue) at 3 days post dose (Fig. 3). Three days after mipafox there had been some recovery of AChE activity in brain and diaphragm to 22 and 58% inhibition, respectively. At 2 h post dose diaphragm phenylvalerate hydrolases were inhibited to a greater extent than those of the brain (Fig. 3). Observation of these enzymes at 3 days indicated that recovery was more rapid in diaphragm than brain. By 7 days after treatment diaphragm AChE had completely recovered although it was still inhibited by 26% in brain tissue. Brain NTE activity had also recovered by 7 days post dose. Three h after treatment with ecothiopate (0.5 pmolikg) diaphragm AChE was inhibited by 60% (Fig. 4), which was similar to diaphragm AChE inhibition at 2 h after treatment with mipafox (110 pmolkg). Seven days after dosing with ecothiopate the activity of diaphragm AChE was
Fig. 1. The in vitro inhibhion of brain paraoxon (40 PM) resistant phenylvalerate hydrolyses by mipafox for 20 min at 37°C. Results are the mean * I SD. (n = 3).
Fig. 2. Brain and diaphragm AChE levels following mipafox (110 ~olkg, s.c.) in the mouse. Results are the mean f 1SD. (n = 4).
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E. Mutch et al. / Toxicology Letters 81 (1995) 45-53
Fig. 3. Brain NTE, and brain and diaphragm phenylvalerate hydrolase levels following mipafox (I 10 pmollkg, s.c.) in the mouse. Results are the mean + I S.D. (n = 4).
Fig. 5. Brain AChE, NTE. phenylvalerate hydrolase and plasma phenylvalerate hydrolase levels 2 h after a single dose of mipafox in the mouse. Results are the mean f I S.D. (n = 4).
not different from control values. However, ecothiopate did not produce any inhibition of brain AChE at either 3 h or 7 days post dose and in fact there appeared to be a slight (ca. 15%) increase in the activity of this enzyme at these times. Brain and diaphragm phenylvalerate hydrolases were unaffected by ecothiopate at any time interval. 3.3.2. Dose response of enzyme inhibition. In the mipafox dose-related study, inhibition of brain AChE at 2 h increased from 28% at 27.5 pmol/kg to 60% at 110 pmol/kg (Fig. 5). Brain NTE activity measured at 2 h was inhibited more than 70% at 27.5 and 55 PmoYkg and completely abolished at 82.5 and 110 pmollkg. Approximately 50 and 90%
inhibition of brain and plasma phenylvalerate hydrolase activity, respectively, was observed at all dosage levels (Fig. 5).
Fig. 4. Brain and diaphragm AChE levels following ecothiopate (0.5 pmohkg, s.c.) in the mouse. Results are the mean l I SD. (n = 4).
4. Discussion The experiments to determine ICss values in vitro were carried out using post mitochondrial brain homogenate. I& AChE values were 100 PM (mipafox) and 0.08 PM (ecothiopate), a ratio of lOO/O.OB = 1250. However, in vivo a similar degree of diaphragm AChE inhibition was observed at 2 h following 110 pmol/kg mipafox and 0.5 PmoYkg ecothiopate (110/0.5 = 220). Ecothiopate was therefore 6 times more potent as an antiChE than mipafox in vitro compared to measurements in vivo. In vivo differences in distribution, uptake, metabolism and elimination between the 2 OPs will affect the free local concentration available to inhibit target enzymes such as AChE. This may account for the difference observed in vivo between the 2 OPs and could confound the I& values determined in vitro. AChE activity was measured in the present in vitro and in vivo experiments using rough homogenate which will consist of various molecular forms of acetyl cholinesterase. This homogenate will contain both functional and non-functional forms of AChE, and therefore data obtained using these preparations may not exactly reflect the activity of functional AChE at the neuromuscular
E. Mulch et al. / Toxicology Letters BI (1995) 45-53
junction [16]. Isolation and measurement of individual forms of AChE may provide a more meaningful tool to evaluate toxicity following dosing with some OPs. The in vivo results suggest that ecothiopate, a quarternary ammonium compound, was unable to cross the blood-brain barrier and did not inhibit brain AChE although it was a highly potent anti-ChE in vitro. Diaphragm AChE was inhibited by about 60% at 3 h following ecothiopate (0.5 pmol/kg). The slight in vivo increase (ca. 15%) in brain AChE with ecothiopate is unexplained, but it is possible that inhibition of peripheral AChE stimulated production of the enzyme in central neural cell bodies. In contrast, both brain and diaphragm AChE were inhibited by about 60% following mipafox (110 PmoVkg). Central tremor was not expected, nor observed, with ecothiopate since brain AChE was not inhibited in vivo, but muscle fasciculation was apparent soon after dosing. Mice dosed with mipafox demonstrated central tremor and peripheral fasciculation, indicating CNS involvment but mobility had geneially returned to normal by about 2 h post dose. There was no inhibition of brain phenylvalerate hydrolases, including NTE, in vitro using ecothiopate (1 PM for up to 30 min). Ecothiopate (0.5 PmoYkg) did not inhibit diaphragm phenylvalerate hyclrolases, brain phenylvalerate hydrolases or brain NTE, in vivo. This suggests that ecothiopate ha.s poor affinity for these isoenzymes even if it could cross the blood-brain barrier. In contrast, mipafox was a potent inhibitor of brain paraoxon-resistant phenylvalerate hydrolases in vitro and in vivo with > 70% inhibition of brain NTE at 24 h post dose. Diaphragm and brain phenylvalera.te hydrolases were inhibited by >70 and 50%, respectively, at 2 h post dose (110 pmol/kg). Differences in detoxification between OPs will greatly influence the concentration at target enzymes. The tissue and plasma carboxylesterases are an important detoxification pathway in rodents as they provide alternative phosphorylation sites to remove circulating OPs before they reach target AChE or NTE. The total number of plasma carboxylesterase binding sites has been shown to be high in the rat [ 171 which
51
demonstrates their quantitative importance in detoxification. Although the plasma carboxylesterases were not measured following ecothiopate administration they were totally inhibited after mipafox, even at 27.5 pmollkg. It is unlikely that ecothiopate had the capacity to inhibit plasma carboxylesterases since brain and diaphragm carboxylesterases were not inhibited either in vitro or in vivo. Taken in isolation this could be expected to result in higher OP levels. However, since the initial reversible complex formed between AChE and ecothiopate is analogous to that between the enzyme and its normal substrate acetylcholine, reasonably rapid recovery of the enzyme and hydrolysis of ecothiopate could be expected. The spontaneous reactivation half-life of ecothiopate on AChE has been determined experimentally as 27 h [18] and this essentially amounts to a detoxification pathway for the OP. A study by Chemnitius and Zech [ 191estimated that about 40% of hen brain phenylvalerate hydrolases were resistant to mipafox inhibition while at least 1 isoenzyme was extremely sensitive to both mipafox and paraoxon. In the present study using mouse brain, calculation of an ICsOvalue towards mipafox for the paraoxon-resisitant phenylvalerate hydrolases was difficult since about 70% of the hydrolases were inhibitor resistant (i.e. resistant to 40 PM paraoxon and up to 420 PM mipafox). ICsO for the paraoxon-resistant and mipafox-sensitive hydrolases were calculated at 12.5 and 32.9 PM by fitting the data to 1 and 2 exponentials and a resistant component. Similar experiments by Novak and Padilla [20] defined the inhibitor-resistant phenylvalerate hydrolases for rat and hen as 40 and 20% of the paraoxon-resistant hydrolases (I& 11.6 and 7.3 PM, respectively). Sogorb et al. (81 have recently reported the inhibitor-resistant hydrolases of bovine brain to be 28% of the paraoxon-resistant population. Comparative studies between hen and cat (species sensitive to OPIDN) and rat and chicken (species resistant to OPIDN) NTE and other phenylvalerate hydrolases have discounted the hypothesis that the distribution of NTE or related hydrolases could be associated with sensitivity to OPIDN [21]. Our in vitro data generally supports our in vivo dose response study which
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E. Mulch et al. / Toxicology Letters 81 (1995) 45-53
demonstrated that about 40% of brain phenylvalerate hydrolases were highly resistant to mipafox, even at 110 pmol/kg. Greater than 70% inhibition of brain NTE was observed in vivo over the whole mipafox concentration range, and 70% is the inhibition threshold believed to be associated with OPIDN for sensitive species such as the hen. In another study [5] which electrophysiological to biochemical related changes, 65% inhibition of brain NTE (2 h after a 110 pmol mipafox/kg dose) was associated with electrophysiological effects after 7 days at a time when NTE had recovered. None of the mice in this study showed any clinical signs of delayed neuropathy up to 77 days after a single dose of mipafox (110 pmol/kg). Histological examination of peripheral nerves and the CNS was not carried out in these mice. However, mipafox (82.5 rmol/ kg) has been shown to produce clinical signs of acute toxicity and brain NTE inhibition which correlated with histological changes in the spinal cord and hind brain of rats 14-21 days after a single dose [22]. Because of synthesis of fresh protein, NTE activity recovers markedly during the period between exposure and development of OPIDN, with hen brain requiring 5-7 days to recover 50% activity following irreversible inhibition with an OP capable of aging [23]. Experiments with hen have also shown that there is no correlation between neuropathy and brain NTE inhibition measured at the time when clinical symptoms reach their peak [24]. Mipafox is believed to irreversibly inhibit and ‘age’ on NTE, and probably also on AChE, and therefore recovery will depend entirely on synthesis of new protein (de novo synthesis) which will vary between tissues. In this study brain AChE, diaphragm and brain phenylvalerate hydrolases had not returned to control levels at 7 days following dosing with mipafox (110 pmol/kg). In contrast, ecothiopate can be considered to be reversibly bound to AChE. Analyses in vitro indicate that the diethylphosphorylated enzyme reactivates spontaneously with a half-time of 27 h [18] and recovery of AChE following ecothiopate exposure will therefore be a balance between spontaneous reactivation and de novo synthesis. In order to achieve a good correlation between
physiological effect and biochemical measurements it is necessary to investigate the form(s) of AChE and NTE which are of physiological importance. Until recently, many laboratories have measured AChE and NTE using only post mitochondrial fraction tissue homogenate, but this may not be the most appropriate method when relating enzyme inhibition to other physiological parameters [25]. Although tissue homogenate gives a reasonable indication of enzyme inhibition and recovery, measurement of AChE molecular forms may be more accurate following dosing with some OPs. Interestingly, a study by Carrera et al. [26] using hen sciatic nerve described the in vivo inhibition of soluble and particulate (membrane) forms of NTE by mipafox and compared the results to inhibition in vitro. The authors concluded that the 2 forms show different responses in vivo and in vitro. Post mitochondrial fraction homogenate, containing soluble and particulate forms of NTE, was used in the present study although measurement of both forms in parallel may provide a better marker of effect in vivo. These results using a rodent model indicate that manifest OP toxicity may be a balance between access, metabolism (including detoxification and affinity) and other in vivo parameters, and that this balance will differ between OPs. However, long-term studies to relate biochemical and physiological effects using a multi-dose regimen may be more relevant both occupationally and in order to fully precipitate dysfunction in our rodent model. Acknowledgements
The authors thank C. Charlton for excellent technical assistance and the MRC for project Grant support. References
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131 Carrington, C.D. and Abou-Donia, M.B. (1988) Variation between three strains of rat: inhibition of NTE and acetylcholinesterase by tri-o-cresyl phosphate. J. Toxicol. Environ. Health 25, 259-268. 141 Veronesi, B. (1984) A rodent model of OPIDN: distribution of central (spinal cord) and peripheral nerve damage. Neuropathol. Appl. Neurobiol. 20, 357-368. [51 Kelly, S.S., Mutch, E., Williams, F.M. and Blain, P.G. (1994) Electrophysiological and biochemical effects following single domsesof organophosphates in the mouse. Arch. Toxicol. 68, 459-466. 161Bidstrup, P.L., Bonnel, J.A. and Beckett, A.G. (1953) Paralysis following poisoning by a new organic phosphorus insecticide (mipafox). Br. Med. J. 1, 1068-1071. I71 Reiner, E., Aldridge, W.N. and Hoskin, C.G. (Eds.) (1989) Enzymes Hydrolysing Organophosphorus Compounds. Ellis Horwood Publishers. I81Sogorb, M.A., Viniegra, S., Reig, J.A. and Vilanova, E. (1994) Partial characterisation of NTE and related phenylvalerate esterases from bovine adrenal medulla. J. Biochem. Toxicol. 9(3), 145- 152. [91 Todrik, A. (1954) The inhibition of cholinesterases by antagonists of acetylcholine and histamine. Br. J. Pharmacol. 41(l), 139-141. [lOI Ellman, G.L., Courtney, K. and Featherstone, R. (1961) A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 89-95. [ill Mutch, E., Blain, P.G. and Williams, F.M. (1992) Interindividual varia.tions in enzymes controlling organophosphate toxicity in man. Hum. Exp. Toxicol. 11, 109-l 16. WI Johnson, M.K. (1977) Improved assay of NTE for screening organophosphates for delayed neurotoxic potential. Arch. Toxicol. 37, 113-115. H31 Kelly, S.S., Ferry C.B., Bamforth, J.P. and Das, S.K. (1992) Protection against the effects of anticholinesterases on the latencies of action potentials in mouse skeletal muscles. Br. J. Pharmacol. 107, 867-872. [I41 Veronesi, B., Padilla, S., Blackman, K. and Pope, C. (1991) Murine susceptibility to OPIDN. Toxicol. Appl. Pharmacol. 107, 3 1I-324. [I51 Lapadula, D.M., Patton, S.E., Campbell, G.A. and Abou-Donia, M.B. (1985) Characterisation of delayed neurotoxicity in the mouse. following chronic oral administration of TOCP Toxicol. Appl. Pharmacol. 79, 83-90.
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1161 Younkin, S.G., Rosenstein, C., Collins, P.L. and Rosenberry, T.L. (1982) Cellular localisation of the molecular forms of acetylcholinesterase in rat diaphragm. J. Biol. Chem. 257(22), 13630-13637. 1171 Maxwell, D.M., Lenz, D.E., Groff, W.A., Kaminskis, A. and Froelich, H.L. (1987) The effect of blood flow and detoxification on in vivo cholinesterase inhibition by soman in rats. Toxicol. Appl. Pharmacol. 88, 66-76. [I81 Newman, J.R., Virgin, J.B., Younkin, L.H. and Younkin, S.G. (1984) Turnover of acetylcholinesterase in innervated and denervated rat diaphragm. J. Physiol. 352, 305-318. [I91 Chemnitius, J.G. and Zech, R. (1983) Inhibition of brain carboxylesterases by neurotoxic and non-neurotoxic organophosphorus compounds. Mol. Pharmacol. 23, 717-722. PO1 Novak, R. and Padilla, S. (1986) An in vitro comparison of rat and chicken brain neurotoxic esterase. Fundam. Appl. Toxicol. 6, 464-47 1. 1211 Tonno, N., Gimeno, J.R., Sogorb, M.A., Diaz-Alejo, N. and Vilanova, E. (1993) Soluble and particulate organophosphorus neuropathy target esterase in brain and sciatic nerve of the hen, cat, rat and chick. J. Neurochem. 61, 2164-2168. PI Veronesi, B., Padilla, S. and Lyerly, D. (1986) The correlation between NTE inhibition and mipafox-induced neuropathic damage in rats. Neurotoxicology 7(l), 207-216. 1231 Barril, J.B., Vilanova, E. and Pellin, MC. (1988) Sciatic nerve neuropathy target esterase. Methods of assay, proximo-distal distribution and regeneration. Toxicology 49, 107-l 14. 1241 Johnson, M.K. (1975b) Structure-activity relationships for substrates and inhibitors of hen brain NTE. B&hem. Pharmacol. 24, 797-805. 1251 Cabezas-Herrera. J., Campoy, F.J. and Vidal, C.J. (1992) Differential effects of ethanol on membrane-bound and soluble acetylcholinesterase from sarcoplasmic reticulum membranes. Neurochem. Res. 17(7) 717-722. WI Carrera, V., Diaz-Alejo, N., Sogorb, M.A., Vicedo, J.L. and Vilanova, E. (I 994) In vivo inhibition by mipafox of soluble and particulate forms of organophosphorus neuropathy target esterase (NTE) in hen sciatic nerve. Toxicol. Lett. 71. 47-51.
Toxicology Letters 81 (1995) 45-53
Comparative studies of two organophosphorus the mouse
compounds in
Elaine Mutch*, Sean S. Kelly, Peter G. Blain, Faith M. Williams Toxicology Unit. Department of Environmental and Occupational Medicine. The Medical School, Newcastle University, Newcastle NE.2 4HH. UK
Received 14 October 1994; revision received 9 May 1995; accepted I I May 1995
A rodent model, the albino mouse, was used to investigate the in vitro and in vivo capacity of 2 organophosphate (OP) compounds, mipafox and ecothiopate, to inhibit enzymes considered to be involved in the mechanisms of OP toxicity. Mipafox and ecothiopate were chosen as model compounds because the former can produce a delayed neuropathy whereas the latter does not. Mipafox (110 pmol/kg, s.c.) inhibited brain acetylcholinesterase (AChE), neuropathy target esterase (NTE) and phenylvalerate hydrolases by 58, 64 and 65%, while diaphragm AChE and phenylvalerate hydrolases were inhibited by 66 and 80%, respectively. In contrast, ecothiopate (0.5 pmol/kg) had no effect on brain NTE or on brain or diaphragm phenylvalerate hydrolases. At the same time, diaphragm AChE was inhibited by 60% while brain AChE activity had increased by 15% of control. Mipafox was a potent inhibitor of AChE and NTE in vitro. Although ecothiopate was a highly potent anti-ChE in vitro, it had no inhibitory effect on NTE. Keywords:
Organophosphates;
Mouse; AChE; Neuropathy
1. Introduction The acute toxicity of organophosphates (OPs) follows inhibition ‘of acetylcholinesterase (AChE, EC 3.1.1.7) in neural tissue and at the neuromuscular junction. This results in accumulation of acetylcholine and hyperactivity within the cholinergic pathways. Acute toxicity of anticholinesterase (anti-ChE) compounds in man is mainly due to the peripheral effects of acetylcholine, although central nervous system (CNS) symp* Corresponding autihor.
target esterase; Phenylvalerate hydrolase
toms may also be apparent. Another syndrome, organophosphorus-induced delayed neuropathy (OPIDN), is believed to be initiated by inhibition and ‘aging’ of a membrane-bound carboxylesterase called neuropathy target esterase (NTE) found in neural tissue [ 11. Aging is a timedependent reaction which involves the loss of an alkyl group from the phosphorylated enzyme. When this reaction takes place, usually within minutes of inhibition, the enzyme becomes irreversibly phosphorylated. NTE is believed to be one of several carboxylesterases present in neural tissue determined as that fraction of and is
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E. Mulch et al. / Toxicology Letters 81 (1995) 45-53
phenylvalerate esterase activity which is paraoxonresistant and mipafox-sensitive. OPIDN may develop lo-21 days post exposure to some OPs regardless of any antiChE effects. The hen is currently the animal of choice when screening for the neurotoxic potential of OP compounds, with greater than 70% inhibition of NTE correlating well with development of OPIDN [2]. However, less sensitive species such as rodent have proved to be a good model for investigating biochemical [3], neuropathological [4] and electrophysiological [5] changes following OP exposure. Mipafox (NJV-diisopropylphosphorodiamidofluoridate) was implicated in the manifestation of both acute (antiChE) and neurotoxic symptoms (OPIDN) in man following an accident in the early stages of its development as a pesticide [6]. It was banned from commercial use soon after the incident but has been used more recently as a tool to study anti-ChE and neurotoxic effects in experimental animals. Ecothiopate o,o-diethyl (S-(2trimethyl-ammoniumethyl)phospHorothioate iodide) is a cationic compound which will quickly diethylphosphorylate the serine active centre of an enzyme, thereby inactivating it. Its reversible antiChE action is made use of in the clinical management of glaucoma. Although some OPs are direct AChE inhibitors, many require metabolic activation through oxidative desulphuration by microsomal cytochrome P-450 for toxicity. Both mipafox and ecothiopate are direct AChE inhibitors. Detoxification of OPs occurs both by hydrolysis via the phosphoric triester hydrolases (EC 3.1.8.1 and 3.1.8.2) and following phosphorylation of serine-containing carboxylesterases (EC 3.1.1.1.) present in blood, liver and other tissues. The aims of the present study were to investigate: (1) the inhibition profiles of AChE (diaphragm and brain), NTE (brain) and carboxylesterases (diaphragm and brain) with respect to time following dosing with mipafox (110 PmoYkg) or ecothiopate (0.5 pmol/kg); (2) the dose-response inhibition profiles for the same enzymes at 2 h post mipafox (27.5- 110 Fmol/kg) administration; (3) in vitro inhibition of brain homogenate by mipafox and ecothiopate; (4) plasma carboxylesterase inhibition in vivo.
2. Materials and methods 2.1. Organophospha tes Mipafox, was obtained from Chemsyn Science Labs (Lenexa, KS) and diluted from a stock solution (pH 6.0) shortly before use. Ecothiopate was prepared from Phospholine Eyedrops (Ayerst). Paraoxon (O,O-diethyl p-nitrophenyl phosphate) was obtained from Sigma (Poole, Dorset, UK) and used in the NTE assay. 2.2. Treatment and condition of animals Male albino mice aged 6-7 months (body weight 40-50 g), obtained from Bantin and Kingman (Hull, UK) were used in all experiments. Atropine (1.4 pmol/kg) was included in the injection to reduce the muscarinic effects caused by inhibition of AChE. The acute effect of a subcutaneous dose of the OPs used in these studies was skeletal muscle fasciculation which occured usually with ecothiopate (0.5 pmol/kg) and occasionally with mipafox (110 pmol/kg) concomitant with tremor. With mipafox, if fasciculation did occur, the onset was slower than with ecothiopate (at about 20 mm), but mobility had usually returned to normal by about 2 h. 2.3. Preparation of tissue homogenates Brain tissues were removed from the -70°C freezer, halved in the longitudinal plane, weighed and sufficient ice-cold 50 mM Tris-0.2 mM EDTA buffer (pH 8.0) added to produce a 10% (w/v) homogenate. The tissues were minced finely using scissors and then homogenised for 5 s using an icecold Polytron or Ultra Turrex Cell Homogeniser. The preparations were centrifuged at 12 000 x g for 15 min to remove cell debris, nuclei and mitochondria and stored at -70°C until analysis within a week. There was no loss of enzyme activity on freezing and thawing (data not shown). Diaphragm post mitochondrial fraction (lo%, w/v) was prepared in a similar manner. 2.4. In vitro studies Control animals were killed by cervical dislocation, the brain removed and 10% homogenates prepared as described previously. Mipafox inhibition of AChE was determined by preincubating brain homogenate (5 mg tissue) in O.,l M phos-
E. Murch et al. / Toxicology Leriers 81 (1995) 45-53
phate buffer, pH 8.0 (control) and with lo-850 pM mipafox (freshly prepared from stock in triscitrate buffer, pH 6.0, and added to incubations in a small volume) for 8 min at 30°C. Acetylthiocholine was added and AChE activity measured at 30°C as described below. Since preliminary experiments showed that control rat brain AChE activity decreased by 20% when incubated at 30°C for 20 min in the absence of inhibitor it was particularly important to relate results to controls under the same assay conditions. Inhibition of brain AChE by ecothiopate was determined following preincubation with the OP (range O-l PM) for 3.0 min at 30°C and then residual enzyme activity calculated by comparison to control incubations. The relationship log % control activity vs. OP concentration is expected to be linear for a singlecompartment kinetic reaction [7]. This plot was used to calculate the mipafox and ecothiopate concentrations which inhibit 50% control AChE activity (IC&. A mipafox inhibition profile was also determined for paraoxon-resistant phenylvalerate hydrolases. Brain homolgenate (equivalent to 6 mg tissue) was preincubated with either 40 PM paraoxon (A tube) or 40 PM paraoxon plus 20-420 pM mipafox (B tubes) for 20 min at 37°C prior to addition of phenylvalerate (1.4 mM). Phenylvalerate hydrolase activity was then measured in all tubes for 30 min at 37°C as described below. The mipafox inhibition profile of paraoxon-resistant hydrolases was obtained by subtracting the activity obtained in each B tube from that in tube .4. Each value was then compared to the control value (A tube) and expressed as a percentage. Inhibition of brain NTE and other phenylvalerate hydrolases with ecothiopate in vitro was determined by preincubation for 10 and 20 min at 37°C at an OP concentration of 1 FM. Data for mipafox inhibition of NTE and other phenylvalerate hydrolases was plotted as log % control activity vs. ,concentration of OP. The data was also fitted by iterative non-linear regression to a two-compartment equation as used by Sogorb et al. [8]: 100 x EIE, = A,e-‘K” + AZeVml + A, (A, t A2 + A, = 1)
41
Where E is enzyme activity at OP concentration I and E, is control activity in the absence of inhibitor. A, and A2 (expressed as percentages) are the amplitudes of the sensitive components 1 and 2 and K, and K2 are the exponential components of each component. A, is the amplitude of the inhibitor-resistant component. From this the ICso of the inhibitor sensitive component(s) were calculated: ICso = Ln 2/Ki 2.5. In vivo studies 2.51. Time profile of enzyme inhibition by mipafox and ecothiopate. Mipafox 20 mgfkg (110 pmovkg) or ecothiopate (0.5 pmol/kg) were given with atropine (1.4 pmol/kg) by S.C.injection in the nape of the neck. At 2 h, 3 days and 7 days animals were killed by cervical dislocation. In some studies with mipafox animals were killed at 0.5, 1, 2, 24 and 72 h in order to define the early time course of inhibition. Animals treated with atropine (1.4 pmol/kg) alone were used for control measurements. Whole brain and diaphragm were removed, washed in 0.1 M KCl/O.l M phosphate buffer (pH 7.4), blotted and then stored at -70°C until analysis of AChE (diaphragm and brain), NTE (brain) and carboxylesterase (brain and diaphragm). In some studies blood was collected by cardiac puncture from animals anaesthetised with Hypnorm/ Medazolam. The blood was placed into heparinised tubes and the plasma separated by centrifugation prior to analysis of carboxylesterase. 2.5.2. Dose profile of enzyme inhibition by mipafox. Mice were dosed with 27.5, 55, 82.5 or 110 rmollkg mipafox (s.c.) together with atropine (1.4 pmol/kg) and killed 2 h after the dose. Control animals were given atropine alone. Tissues were collected as above prior to measurement of AChE, NTE and carboxylesterase. 2.6. Enzyme assays 2.6.1. AChE activity. Activity was measured in brain or diaphragm homogenate by following the hydrolysis of acetylthiocholine (500 PM) at 30°C. Diaphragm homogenates were preincubated for 1 h at 0°C in 0.1 M phosphate buffer (pH 8.0) containing 50 FM ethopropazine in order to inhibit
E. Mutch et al. / Toxicology Letters 81 (1995) 45-53
48
non-specific cholinesterases [9] and thereby make the assay more specific for AChE. Hydrolysis of acetylthiocholine (500 PM) was measured at 30°C in 3-ml incubation volumes containing approximately 2.5 mg brain or 5 mg diaphragm. The method used was that described by Ellman et al. [lo] in which the liberated thiocholine reacts with 5,5’-dithiobis-(2-nitrobenzoic acid) to form 5thio-Znitrobenzoate and is then measured at 412 nm. Enzyme activity was expressed as nmol min-’ mg-’ tissue. 2.6.2. NTE activity. Brain NTE activity was measured by a micro-modification [l l] of the method of Johnson [ 121 using phenylvalerate (1.4 mM) as substrate. Homogenate (50 ~1, equivalent to approximately 6 mg tissue) was incubated in a total volume of 200 ~1 for 20 min at 37°C with either 40 PM paraoxon (Tube A) or 40 PM paraoxon + 50 PM mipafox (Tube B). Phenylvalerate was added and incubated for a further 30 min at 37°C and then the phenol produced determined by spectroscopy. NTE activity was defined as those phenylvalerate hydrolases which are resistant to inhibition by paraoxon (Tube A) but sensitive to inhibition by mipafox (Tube B), that is Tube A - Tube B. Enzyme activity was expressed as nmol min-’ mg-’ tissue. 2.6.3. Phenylvalerate hydrolase activity. Carboxylesterase activities in brain homogenate, diaphragm homogenate and plasma were determined by measuring formation of phenol from phenylvalerate (as described for NTE but in the absence of inhibitors). Tissue homogenate (2.5 mg tissue) or plasma (0.25 ~1) was incubated in 400-~1 volumes at 37°C in the presence of phenylvalerate (1.4 mM) for 10 and 2 min, respectively. Results were expressed as nmol min-’ mg-i tissue or nmol min-’ ~1~’ plasma. 2.7. Statistical analysis Results were expressed as mean f 1 S.D. Groups of treated animals were compared to control values by analysis of variance followed by Tukey’s HSD test. 3. Results 3.1. Control enzyme levels
Baseline enzyme levels for AChE, NTE and
phenylvalerate hydrolase were consistant with published values [13- 151 and are shown in Table 1. Results are the mean f 1 S.D. and are expressed in terms of wet weight of tissue. AChE activity in the brain was 1.39 i 0.10 nmol min-’ mg-’ tissue while brain phenylvalerate hydrolase activity was 2.5 f 0.22 nmol min-t mg-’ tissue. Brain NTE (0.11 f 0.03 nmol min-’ mg-’ tissue) represented 4.5% of the total phenylvalerate hydrolase activity. However, NTE activity was below the limit of detection in diaphragm (
and mipafox on enzyme
The in vitro inhibition of brain AChE at 30°C following preincubation with mipafox for 8 min (Table 2) ranged from 10% at 10 PM to 95% at 850 PM. The I& range was 95-125 PM. Fig. 1
Table 1 Control enzyme activities of brain, diaphragm and plasma for BKW mouse Enzyme activity n
Parameter Brain phenylvalerate Brain NTE
hydrolases
Brain AChE Diaphragm phenylvalerate hydrolases Diaphragm AChE Plasma phenylvalerate hydrolases
2.46 0.11 1.30 11.6 0.59 25.7
0.22 f 0.03 f 0.10 f 1.93 l 0.12 ztz l.OOa l
8 4 11 5 10 8
Enzyme activities are expressed as nmol min-’ mg-’ tissue and ‘nmol min-’ pl-’ plasma. Values are the mean * 1 SD.
E. Mutch et al. / Toxicology Letters 81 (1995) 45-53
Table 2 The mipafox and ecothiopate concentrations required to inhibit 500/aof BKW mouse brain AChE activity (ICSo AChE) at 30°C in vitro OP
IC,, AChE (FM)
Mipafox Ecothiopate
95.0-125 0.074-O. 100
Values are the IC, ranges (n = 4 mice).
49
from 6% at 10 nM to complete inhibition at 1000 nM with an IC,, range of 0.074-0.100 PM (Table 2). There was no inhibition of brain NTE or the other phenylvalerate hydrolases by ecothiopate at 37°C using a 1pM concentration for up to 30 min. 3.3. Effects of mipafox and ecothiopate on enzyme activity in vivo 3.3.1. Time profde of enzyme inhibition. Two h
shows the inhib:ition of paraoxon-resistant phenylvalerate hydrolases at 37°C by mipafox. Inhibition ranged from 3% at 2 PM to 25% at 50 PM with a very modest increase in inhibition with rising mipafox concentration up to 420 PM. Therefore about 70% of the paraoxon-resistant phenylvalerate hydrolases were also resistant to mipafox inhibition. Visual inspection of the linear portion (4-20 PM mipafox) of the semi-log plot estimated the I& at 10 PM although it was apparent that there was contribution from a low affinity component. Data was fitted to the two-compartment non-linear equation as described earlier, but with high errors due to the small number of points. The ICsO for the low affinity component thus derived (32.9 PM) was in good agreement with the visual estimate. An I&-, of 12.5 @I was calculated for the highaftinity component by using the one exponential mathematical model. Inhibition of brain AChE at 30°C following preincubation with ecothiopate for 3 min ranged
after injection of mipafox (110 pmolkg), AChE activity in brain and diaphragm was inhibited by 58 and 66%, respectively (Fig. 2). At the same time, brain phenylvalerate hydrolases were inhibited by 65% but NTE, which was inhibited by 64% at 2 h, was further inhibited at 24 h and below the limit of detection (0.01 nmol min-’ mg-’ tissue) at 3 days post dose (Fig. 3). Three days after mipafox there had been some recovery of AChE activity in brain and diaphragm to 22 and 58% inhibition, respectively. At 2 h post dose diaphragm phenylvalerate hydrolases were inhibited to a greater extent than those of the brain (Fig. 3). Observation of these enzymes at 3 days indicated that recovery was more rapid in diaphragm than brain. By 7 days after treatment diaphragm AChE had completely recovered although it was still inhibited by 26% in brain tissue. Brain NTE activity had also recovered by 7 days post dose. Three h after treatment with ecothiopate (0.5 pmolikg) diaphragm AChE was inhibited by 60% (Fig. 4), which was similar to diaphragm AChE inhibition at 2 h after treatment with mipafox (110 pmolkg). Seven days after dosing with ecothiopate the activity of diaphragm AChE was
Fig. 1. The in vitro inhibhion of brain paraoxon (40 PM) resistant phenylvalerate hydrolyses by mipafox for 20 min at 37°C. Results are the mean * I SD. (n = 3).
Fig. 2. Brain and diaphragm AChE levels following mipafox (110 ~olkg, s.c.) in the mouse. Results are the mean f 1SD. (n = 4).
50
E. Mutch et al. / Toxicology Letters 81 (1995) 45-53
Fig. 3. Brain NTE, and brain and diaphragm phenylvalerate hydrolase levels following mipafox (I 10 pmollkg, s.c.) in the mouse. Results are the mean + I S.D. (n = 4).
Fig. 5. Brain AChE, NTE. phenylvalerate hydrolase and plasma phenylvalerate hydrolase levels 2 h after a single dose of mipafox in the mouse. Results are the mean f I S.D. (n = 4).
not different from control values. However, ecothiopate did not produce any inhibition of brain AChE at either 3 h or 7 days post dose and in fact there appeared to be a slight (ca. 15%) increase in the activity of this enzyme at these times. Brain and diaphragm phenylvalerate hydrolases were unaffected by ecothiopate at any time interval. 3.3.2. Dose response of enzyme inhibition. In the mipafox dose-related study, inhibition of brain AChE at 2 h increased from 28% at 27.5 pmol/kg to 60% at 110 pmol/kg (Fig. 5). Brain NTE activity measured at 2 h was inhibited more than 70% at 27.5 and 55 PmoYkg and completely abolished at 82.5 and 110 pmollkg. Approximately 50 and 90%
inhibition of brain and plasma phenylvalerate hydrolase activity, respectively, was observed at all dosage levels (Fig. 5).
Fig. 4. Brain and diaphragm AChE levels following ecothiopate (0.5 pmohkg, s.c.) in the mouse. Results are the mean l I SD. (n = 4).
4. Discussion The experiments to determine ICss values in vitro were carried out using post mitochondrial brain homogenate. I& AChE values were 100 PM (mipafox) and 0.08 PM (ecothiopate), a ratio of lOO/O.OB = 1250. However, in vivo a similar degree of diaphragm AChE inhibition was observed at 2 h following 110 pmol/kg mipafox and 0.5 PmoYkg ecothiopate (110/0.5 = 220). Ecothiopate was therefore 6 times more potent as an antiChE than mipafox in vitro compared to measurements in vivo. In vivo differences in distribution, uptake, metabolism and elimination between the 2 OPs will affect the free local concentration available to inhibit target enzymes such as AChE. This may account for the difference observed in vivo between the 2 OPs and could confound the I& values determined in vitro. AChE activity was measured in the present in vitro and in vivo experiments using rough homogenate which will consist of various molecular forms of acetyl cholinesterase. This homogenate will contain both functional and non-functional forms of AChE, and therefore data obtained using these preparations may not exactly reflect the activity of functional AChE at the neuromuscular
E. Mulch et al. / Toxicology Letters BI (1995) 45-53
junction [16]. Isolation and measurement of individual forms of AChE may provide a more meaningful tool to evaluate toxicity following dosing with some OPs. The in vivo results suggest that ecothiopate, a quarternary ammonium compound, was unable to cross the blood-brain barrier and did not inhibit brain AChE although it was a highly potent anti-ChE in vitro. Diaphragm AChE was inhibited by about 60% at 3 h following ecothiopate (0.5 pmol/kg). The slight in vivo increase (ca. 15%) in brain AChE with ecothiopate is unexplained, but it is possible that inhibition of peripheral AChE stimulated production of the enzyme in central neural cell bodies. In contrast, both brain and diaphragm AChE were inhibited by about 60% following mipafox (110 PmoVkg). Central tremor was not expected, nor observed, with ecothiopate since brain AChE was not inhibited in vivo, but muscle fasciculation was apparent soon after dosing. Mice dosed with mipafox demonstrated central tremor and peripheral fasciculation, indicating CNS involvment but mobility had geneially returned to normal by about 2 h post dose. There was no inhibition of brain phenylvalerate hydrolases, including NTE, in vitro using ecothiopate (1 PM for up to 30 min). Ecothiopate (0.5 PmoYkg) did not inhibit diaphragm phenylvalerate hyclrolases, brain phenylvalerate hydrolases or brain NTE, in vivo. This suggests that ecothiopate ha.s poor affinity for these isoenzymes even if it could cross the blood-brain barrier. In contrast, mipafox was a potent inhibitor of brain paraoxon-resistant phenylvalerate hydrolases in vitro and in vivo with > 70% inhibition of brain NTE at 24 h post dose. Diaphragm and brain phenylvalera.te hydrolases were inhibited by >70 and 50%, respectively, at 2 h post dose (110 pmol/kg). Differences in detoxification between OPs will greatly influence the concentration at target enzymes. The tissue and plasma carboxylesterases are an important detoxification pathway in rodents as they provide alternative phosphorylation sites to remove circulating OPs before they reach target AChE or NTE. The total number of plasma carboxylesterase binding sites has been shown to be high in the rat [ 171 which
51
demonstrates their quantitative importance in detoxification. Although the plasma carboxylesterases were not measured following ecothiopate administration they were totally inhibited after mipafox, even at 27.5 pmollkg. It is unlikely that ecothiopate had the capacity to inhibit plasma carboxylesterases since brain and diaphragm carboxylesterases were not inhibited either in vitro or in vivo. Taken in isolation this could be expected to result in higher OP levels. However, since the initial reversible complex formed between AChE and ecothiopate is analogous to that between the enzyme and its normal substrate acetylcholine, reasonably rapid recovery of the enzyme and hydrolysis of ecothiopate could be expected. The spontaneous reactivation half-life of ecothiopate on AChE has been determined experimentally as 27 h [18] and this essentially amounts to a detoxification pathway for the OP. A study by Chemnitius and Zech [ 191estimated that about 40% of hen brain phenylvalerate hydrolases were resistant to mipafox inhibition while at least 1 isoenzyme was extremely sensitive to both mipafox and paraoxon. In the present study using mouse brain, calculation of an ICsOvalue towards mipafox for the paraoxon-resisitant phenylvalerate hydrolases was difficult since about 70% of the hydrolases were inhibitor resistant (i.e. resistant to 40 PM paraoxon and up to 420 PM mipafox). ICsO for the paraoxon-resistant and mipafox-sensitive hydrolases were calculated at 12.5 and 32.9 PM by fitting the data to 1 and 2 exponentials and a resistant component. Similar experiments by Novak and Padilla [20] defined the inhibitor-resistant phenylvalerate hydrolases for rat and hen as 40 and 20% of the paraoxon-resistant hydrolases (I& 11.6 and 7.3 PM, respectively). Sogorb et al. (81 have recently reported the inhibitor-resistant hydrolases of bovine brain to be 28% of the paraoxon-resistant population. Comparative studies between hen and cat (species sensitive to OPIDN) and rat and chicken (species resistant to OPIDN) NTE and other phenylvalerate hydrolases have discounted the hypothesis that the distribution of NTE or related hydrolases could be associated with sensitivity to OPIDN [21]. Our in vitro data generally supports our in vivo dose response study which
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demonstrated that about 40% of brain phenylvalerate hydrolases were highly resistant to mipafox, even at 110 pmol/kg. Greater than 70% inhibition of brain NTE was observed in vivo over the whole mipafox concentration range, and 70% is the inhibition threshold believed to be associated with OPIDN for sensitive species such as the hen. In another study [5] which electrophysiological to biochemical related changes, 65% inhibition of brain NTE (2 h after a 110 pmol mipafox/kg dose) was associated with electrophysiological effects after 7 days at a time when NTE had recovered. None of the mice in this study showed any clinical signs of delayed neuropathy up to 77 days after a single dose of mipafox (110 pmol/kg). Histological examination of peripheral nerves and the CNS was not carried out in these mice. However, mipafox (82.5 rmol/ kg) has been shown to produce clinical signs of acute toxicity and brain NTE inhibition which correlated with histological changes in the spinal cord and hind brain of rats 14-21 days after a single dose [22]. Because of synthesis of fresh protein, NTE activity recovers markedly during the period between exposure and development of OPIDN, with hen brain requiring 5-7 days to recover 50% activity following irreversible inhibition with an OP capable of aging [23]. Experiments with hen have also shown that there is no correlation between neuropathy and brain NTE inhibition measured at the time when clinical symptoms reach their peak [24]. Mipafox is believed to irreversibly inhibit and ‘age’ on NTE, and probably also on AChE, and therefore recovery will depend entirely on synthesis of new protein (de novo synthesis) which will vary between tissues. In this study brain AChE, diaphragm and brain phenylvalerate hydrolases had not returned to control levels at 7 days following dosing with mipafox (110 pmol/kg). In contrast, ecothiopate can be considered to be reversibly bound to AChE. Analyses in vitro indicate that the diethylphosphorylated enzyme reactivates spontaneously with a half-time of 27 h [18] and recovery of AChE following ecothiopate exposure will therefore be a balance between spontaneous reactivation and de novo synthesis. In order to achieve a good correlation between
physiological effect and biochemical measurements it is necessary to investigate the form(s) of AChE and NTE which are of physiological importance. Until recently, many laboratories have measured AChE and NTE using only post mitochondrial fraction tissue homogenate, but this may not be the most appropriate method when relating enzyme inhibition to other physiological parameters [25]. Although tissue homogenate gives a reasonable indication of enzyme inhibition and recovery, measurement of AChE molecular forms may be more accurate following dosing with some OPs. Interestingly, a study by Carrera et al. [26] using hen sciatic nerve described the in vivo inhibition of soluble and particulate (membrane) forms of NTE by mipafox and compared the results to inhibition in vitro. The authors concluded that the 2 forms show different responses in vivo and in vitro. Post mitochondrial fraction homogenate, containing soluble and particulate forms of NTE, was used in the present study although measurement of both forms in parallel may provide a better marker of effect in vivo. These results using a rodent model indicate that manifest OP toxicity may be a balance between access, metabolism (including detoxification and affinity) and other in vivo parameters, and that this balance will differ between OPs. However, long-term studies to relate biochemical and physiological effects using a multi-dose regimen may be more relevant both occupationally and in order to fully precipitate dysfunction in our rodent model. Acknowledgements
The authors thank C. Charlton for excellent technical assistance and the MRC for project Grant support. References
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