Br. J. Pharmacol. (1992), 107, 867-872
'."
Macmillan Press Ltd, 1992
Protection against the effects of anticholinesterases on the latencies of action potentials in mouse skeletal muscles 1S.S. Kelly, C.B. Ferry, J.P. Bamforth & S.K. Das Pharmacological Laboratories, Pharmaceutical Sciences Institute, Aston University, Birmingham B4 7ET 1 Adult male albino mice were injected subcutaneously with an organophosphorous anticholinesterase to initiate excessive variability in the latency of indirectly elicited muscle action potentials (jitter) when assessed 5 days later. 2 Pretreatment of the mice with a single dose of pyridostigmine prevented the development of jitter after subsequent dosing with an organophosphate. 3 Treatment with one dose of pralidoxime (2PAM) prevented the development of jitter if given less than 1 h after treatment with ecothiopate, a reactivatable inhibitor of cholinesterase. Similar treatment with 2PAM after a non-reactivatable inhibitor did not prevent the development of jitter. The repeated administration of 2PAM over 12 h did ameliorate jitter. 4 Pretreatment of mice orally with a-tocopherol and N-acetylcysteine, known to prevent ecothiopateinduced myopathy, did not prevent the development of jitter after ecothiopate. 5 It is concluded that the development of jitter was a consequence of the inhibition of acetylcholinesterase, and although jitter did not develop acutely, the potential for the full development of jitter was achieved about 1 h after intoxication with ecothiopate. The development of jitter did not involve the generation of free radicals. Reduction of the early effects of intoxication with anticholinesterases by pyridostigmine or 2PAM prevented the development of jitter. Keywords: Organophosphorous anticholinesterase; skeletal muscle; action potential conduction; pralidoxime; antioxidants
Introduction It has been reported (Kelly et al., 1990) that the organophosphate (OP) anticholinesterases, ecothiopate (ECO), pinacolyl S- (2-trimethylaminoethyl) methylphosphonothioate (BOS), and diisopropylfluorophosphate (DFP) affected the latencies of indirectly-evoked muscle action potentials (APs). A single dose of any of these OPs increased the variability of the latencies of a train of indirectly-evoked muscle APs, i.e. increased muscle AP jitter. The jitter was manifest as the increased latency which occurred at the beginning of the train and the increased variation of the latency of consecutive APs later in the train. These effects of ECO, BOS or DFP were dose-dependent and lasted for several days after the acetylcholinesterase (AChE) activities of muscle homogenates had returned to control values. This increased jitter was present after doses of OPs which also caused damage to muscle originating at the endplate (Townsend, 1988), and it was also seen after smaller, non-necrotizing doses. Although the increased jitter was a consequence of the inhibition of AChE, the size of the increase did not appear to be related directly either to the maximum inhibition of the enzyme or to its inhibition at the time when APs were recorded. Protection against the lethal effects of large doses of OPs may be afforded by prophylaxis with pyridostigmine (Leadbeater et al., 1985); or, provided that the enzyme-inhibitor complex has not 'aged', by treatment with oximes to reactivate the AChE. In previous experiments done in this laboratory, protection against the myopathy induced by ECO could be provided by prophylaxis with pyridostigmine, or by treatment with pralidoxime (2PAM) (Townsend, 1988), or by prophylaxis with vitamin E and other antioxidants (Das, 1989). The amelioration by drugs of the toxic effects of OPs gives rise to the following questions which are addressed in the ' Author for correspondence at present address: Department of Environmental & Occupational Medicine, The Medical School, University of Newcastle upon Tyne, NE2 4HH.
study described here: (1) Does prophylaxis with pyridostigmine or with vitamin E ameliorate the increased jitter produced by ECO? (2) Does treatment with 2PAM prevent development of increased jitter, and is there a period for optimal treatment? (3) Does 2PAM affect the increase in jitter after BOS, an OP which produces a non-reactivatable inhibition of AChE?
Methods Male albino mice aged 6-7 months were used in all experiments. The OP anticholinesterases, ECO 0.5 mol kg-', or BOS 8molkg-', were given with atropine 0.7molkg'I by subcutaneous injection in the scruff of the neck. At various times after intoxication with the OP, animals were killed by section of the spinal cord in the neck. ECO and BOS are quaternary organophosphates selected because these watersoluble compounds do not penetrate the CNS and exert effects only peripherally (Koelle & Steiner, 1956). The inclusion of atropine in the injection prevents muscarinic effects so that the main action of these drugs is at the neuromuscular junction. The acute toxic effect of a subcutaneous injection of a mixture of atropine sulphate and the quaternary organophosphates ECO and BOS used in these experiments was fasciculation which began about 20 min after the injection. With ECO, the fasciculations lasted 1-2 h and during this period animals were able to move around, but usually remained quiet with reduced exploratory behaviour. By 3 h after ECO, behaviour was undistinguishable from normal. Dosing was in the morning so that the reduced mobility did not coincide with the normal feeding/activity pattern. With BOS, the onset and time course of drug action was slower, but mobility had returned to normal by 10-12 h. None of the many human studies refer to any association of pain with fasciculation after organophosphate poisoning.
868
S.S. KELLY et al.
Electrophysiology The left hemidiaphragm and phrenic nerve was rapidly removed from each animal and pinned to Sylgard 184 (Dow Corning) in a Perspex bath through which flowed physiological saline of the following composition (mM): NaCl 137, NaHCO3 12, NaH2PO4 1, KCI 5, CaCl2 2, MgCl2 1 and glucose 25. The saline was gassed with a mixture of 95% 02 and 5% CO2 and the temperature was 37.0 ± 0.50C. The phrenic nerve was stimulated via a suction electrode by supramaximal pulses of 0.05 ms duration at 30 Hz. Glass capillary microelectrodes filled with 3 M KCI were used intracellularly to record resting membrane potentials (RMPs) and action potentials (APs). When recording action potentials, many pins were used to fix the muscle to the Sylgard and, if necessary, some muscle fibres were cut to minimize movement of muscle in response to nerve stimulation. Action potentials recorded at the ends of uncut muscle fibres near the tendon were displayed on an oscilloscope and recorded on an FM tape recorder (Racal Store 4) with a tape speed of 30 inches per second (ips). These recordings were then replayed at 15/16 ips and analysed with analogue-todigital (AD) converter in a PDP 11/03 minicomputer. With an AD sampling rate of 20 kHz and a record/replay ratio of 32, the effective sampling rate of the recordings was 640 kHz. Trains of 30 APs were recorded from each muscle fibre and approximately 10 fibres were sampled from each muscle. Data from a fibre were rejected if, during the train, the RMP fell by more than 5 mV or the amplitude of the AP fell by more than 10%.
Analysis of AP recordings Computer programmes were devised which measured the amplitude, time course, and latency of each AP as the interval between the stimulus and a point on the AP's rising phase at 10% of peak amplitude. The latency of the first 10 APs in the train usually increased gradually and subsequent APs showed about the same latency, but with some variability. The data relating to individual APs were used to calculate the Mean Consecutive Difference (MCD) of latencies of APs 11-30 (i.e. at the plateau) and the individual latencies of the first 16 APs with respect to the first AP of the train. The formula used to calculate the MCD was: MCD=[IL11-L121 + 1L12-L131 . ...... L29-L301]/19 Where Ln is the latency of AP number n. Use of the MCD as a measure of variability is considered to reduce the effects of any long-term drifts in latency, but might hide any initial changes at the beginning of trains of APs (Ekstedt et al., 1974). In our experiments, the initial change in latency is measured as the 'delay'of the sixteenth AP relative to the first AP (i.e. latency of sixteenth AP minus latency of first AP). Using a pulse generator accurate to 1 in 106 (Digitmer) to simulate APs at 10 Hz, the value obtained for MCD was 2.4 ps, which represents the intrinsic error of the process of record/replay/analysis.
Measurement of cholinesterase activity To estimate the cholinesterase activity of muscle, strips of the junctional region were made by cutting across the hemidiaphragms approximately 2mm either side of the visible intramuscular nerve. The muscle was then weighed and homogenized in 5 ml of 0.1 M sodium phosphate buffer (pH 8.0). The homogenate was incubated for 30 min with 50 mM ethopropazine, a selective inhibitor of butyrylcholinesterase (Bayliss & Todrick, 1953), then sonicated and centrifuged at 1500 g for 15 min at 4°C. The clear supernatant was assayed for cholinesterase activity using the method of Ellman et al. (1961). Cholinesterase activity was expressed in nmol acetylthiocholine hydrolysed per minute per milligram of muscle and is considered to be due substantially to the acetyl-
cholinesterase activity of the endplate (Das, 1989). The anticholinesterases used were: ecothiopate, (S-(2-trimethylammoniumethyl) phosphorothioate iodide) or BOS (pinacolyl S-(2-trimethylaminoethyl) methylphosphonothioate). Ecothiopate (ECO) was made up from Phospholine Eyedrops (Ayerst) which contained 12.5 mg ECO and a potassium acetate/boric acid buffer. The amount of potassium injected in the standard dose of ECO was negligible (approximately 6.0 gmol kg-'). Solutions of BOS for injection were made up shortly before use.
Administration ofpossible protective agents Single doses of pralidoxime methiodide (Sigma), 110 gmol
kg-' (2PAM), were given subcutaneously to mice at the first appearance of fasciculations i.e. at 10-15 min after the injection of ECO or of BOS. Two protocols were used for multiple dosing with 2PAM after BOS. The shorter protocol (7 x 2PAM) involved seven injections over a period of 12 h; the first was 10 Lmol kg-', the next 5 injections were of 55 gmol kg-' at 2 h intervals, with a final injection of 10 ptmol kg-' 12 h after the first. The longer protocol (12 x 2PAM) began as the shorter protocol, plus further injections of 110 gmol kg-' at 24, 30, 36, 42 and 48 h after the first. In experiments with pyridostigmine, a single injection (0.38 gmol kg-') of pyridostigmine bromide (Mestinon; Roche) was given 30 min before 0.5 mol kg-' ECO. In experiments with antioxidants, mice were dosed by gavage with vitamin E (Sigma) and/or with N-acetylcysteine solution (Parvolex; Duncan Flockhart). Both were given by gavage once daily for 7 days, vitamin E (VitE, 1.2 mmol kg-') dissolved in 0.1 ml corn oil; N-acetylcysteine (NAC, 6.2 mmol kg-'). Some mice received both at different times in the day. ECO (0.5 mol kg-' or 0.4 mol kg-') was injected on the seventh day. In another experiment, the same dose of vitamin E alone was given for 12 days, with ECO (0.5 mol kg-') being injected on the seventh day.
Statistical analysis Unless otherwise stated, all results are expressed as mean ± 1 s.d. of values from 2 to 5 animals, with the number of muscle fibres in parentheses. To test for significances of differences between groups of data, non-parametric tests were used and differences were taken to be significant if P
'."
Macmillan Press Ltd, 1992
Protection against the effects of anticholinesterases on the latencies of action potentials in mouse skeletal muscles 1S.S. Kelly, C.B. Ferry, J.P. Bamforth & S.K. Das Pharmacological Laboratories, Pharmaceutical Sciences Institute, Aston University, Birmingham B4 7ET 1 Adult male albino mice were injected subcutaneously with an organophosphorous anticholinesterase to initiate excessive variability in the latency of indirectly elicited muscle action potentials (jitter) when assessed 5 days later. 2 Pretreatment of the mice with a single dose of pyridostigmine prevented the development of jitter after subsequent dosing with an organophosphate. 3 Treatment with one dose of pralidoxime (2PAM) prevented the development of jitter if given less than 1 h after treatment with ecothiopate, a reactivatable inhibitor of cholinesterase. Similar treatment with 2PAM after a non-reactivatable inhibitor did not prevent the development of jitter. The repeated administration of 2PAM over 12 h did ameliorate jitter. 4 Pretreatment of mice orally with a-tocopherol and N-acetylcysteine, known to prevent ecothiopateinduced myopathy, did not prevent the development of jitter after ecothiopate. 5 It is concluded that the development of jitter was a consequence of the inhibition of acetylcholinesterase, and although jitter did not develop acutely, the potential for the full development of jitter was achieved about 1 h after intoxication with ecothiopate. The development of jitter did not involve the generation of free radicals. Reduction of the early effects of intoxication with anticholinesterases by pyridostigmine or 2PAM prevented the development of jitter. Keywords: Organophosphorous anticholinesterase; skeletal muscle; action potential conduction; pralidoxime; antioxidants
Introduction It has been reported (Kelly et al., 1990) that the organophosphate (OP) anticholinesterases, ecothiopate (ECO), pinacolyl S- (2-trimethylaminoethyl) methylphosphonothioate (BOS), and diisopropylfluorophosphate (DFP) affected the latencies of indirectly-evoked muscle action potentials (APs). A single dose of any of these OPs increased the variability of the latencies of a train of indirectly-evoked muscle APs, i.e. increased muscle AP jitter. The jitter was manifest as the increased latency which occurred at the beginning of the train and the increased variation of the latency of consecutive APs later in the train. These effects of ECO, BOS or DFP were dose-dependent and lasted for several days after the acetylcholinesterase (AChE) activities of muscle homogenates had returned to control values. This increased jitter was present after doses of OPs which also caused damage to muscle originating at the endplate (Townsend, 1988), and it was also seen after smaller, non-necrotizing doses. Although the increased jitter was a consequence of the inhibition of AChE, the size of the increase did not appear to be related directly either to the maximum inhibition of the enzyme or to its inhibition at the time when APs were recorded. Protection against the lethal effects of large doses of OPs may be afforded by prophylaxis with pyridostigmine (Leadbeater et al., 1985); or, provided that the enzyme-inhibitor complex has not 'aged', by treatment with oximes to reactivate the AChE. In previous experiments done in this laboratory, protection against the myopathy induced by ECO could be provided by prophylaxis with pyridostigmine, or by treatment with pralidoxime (2PAM) (Townsend, 1988), or by prophylaxis with vitamin E and other antioxidants (Das, 1989). The amelioration by drugs of the toxic effects of OPs gives rise to the following questions which are addressed in the ' Author for correspondence at present address: Department of Environmental & Occupational Medicine, The Medical School, University of Newcastle upon Tyne, NE2 4HH.
study described here: (1) Does prophylaxis with pyridostigmine or with vitamin E ameliorate the increased jitter produced by ECO? (2) Does treatment with 2PAM prevent development of increased jitter, and is there a period for optimal treatment? (3) Does 2PAM affect the increase in jitter after BOS, an OP which produces a non-reactivatable inhibition of AChE?
Methods Male albino mice aged 6-7 months were used in all experiments. The OP anticholinesterases, ECO 0.5 mol kg-', or BOS 8molkg-', were given with atropine 0.7molkg'I by subcutaneous injection in the scruff of the neck. At various times after intoxication with the OP, animals were killed by section of the spinal cord in the neck. ECO and BOS are quaternary organophosphates selected because these watersoluble compounds do not penetrate the CNS and exert effects only peripherally (Koelle & Steiner, 1956). The inclusion of atropine in the injection prevents muscarinic effects so that the main action of these drugs is at the neuromuscular junction. The acute toxic effect of a subcutaneous injection of a mixture of atropine sulphate and the quaternary organophosphates ECO and BOS used in these experiments was fasciculation which began about 20 min after the injection. With ECO, the fasciculations lasted 1-2 h and during this period animals were able to move around, but usually remained quiet with reduced exploratory behaviour. By 3 h after ECO, behaviour was undistinguishable from normal. Dosing was in the morning so that the reduced mobility did not coincide with the normal feeding/activity pattern. With BOS, the onset and time course of drug action was slower, but mobility had returned to normal by 10-12 h. None of the many human studies refer to any association of pain with fasciculation after organophosphate poisoning.
868
S.S. KELLY et al.
Electrophysiology The left hemidiaphragm and phrenic nerve was rapidly removed from each animal and pinned to Sylgard 184 (Dow Corning) in a Perspex bath through which flowed physiological saline of the following composition (mM): NaCl 137, NaHCO3 12, NaH2PO4 1, KCI 5, CaCl2 2, MgCl2 1 and glucose 25. The saline was gassed with a mixture of 95% 02 and 5% CO2 and the temperature was 37.0 ± 0.50C. The phrenic nerve was stimulated via a suction electrode by supramaximal pulses of 0.05 ms duration at 30 Hz. Glass capillary microelectrodes filled with 3 M KCI were used intracellularly to record resting membrane potentials (RMPs) and action potentials (APs). When recording action potentials, many pins were used to fix the muscle to the Sylgard and, if necessary, some muscle fibres were cut to minimize movement of muscle in response to nerve stimulation. Action potentials recorded at the ends of uncut muscle fibres near the tendon were displayed on an oscilloscope and recorded on an FM tape recorder (Racal Store 4) with a tape speed of 30 inches per second (ips). These recordings were then replayed at 15/16 ips and analysed with analogue-todigital (AD) converter in a PDP 11/03 minicomputer. With an AD sampling rate of 20 kHz and a record/replay ratio of 32, the effective sampling rate of the recordings was 640 kHz. Trains of 30 APs were recorded from each muscle fibre and approximately 10 fibres were sampled from each muscle. Data from a fibre were rejected if, during the train, the RMP fell by more than 5 mV or the amplitude of the AP fell by more than 10%.
Analysis of AP recordings Computer programmes were devised which measured the amplitude, time course, and latency of each AP as the interval between the stimulus and a point on the AP's rising phase at 10% of peak amplitude. The latency of the first 10 APs in the train usually increased gradually and subsequent APs showed about the same latency, but with some variability. The data relating to individual APs were used to calculate the Mean Consecutive Difference (MCD) of latencies of APs 11-30 (i.e. at the plateau) and the individual latencies of the first 16 APs with respect to the first AP of the train. The formula used to calculate the MCD was: MCD=[IL11-L121 + 1L12-L131 . ...... L29-L301]/19 Where Ln is the latency of AP number n. Use of the MCD as a measure of variability is considered to reduce the effects of any long-term drifts in latency, but might hide any initial changes at the beginning of trains of APs (Ekstedt et al., 1974). In our experiments, the initial change in latency is measured as the 'delay'of the sixteenth AP relative to the first AP (i.e. latency of sixteenth AP minus latency of first AP). Using a pulse generator accurate to 1 in 106 (Digitmer) to simulate APs at 10 Hz, the value obtained for MCD was 2.4 ps, which represents the intrinsic error of the process of record/replay/analysis.
Measurement of cholinesterase activity To estimate the cholinesterase activity of muscle, strips of the junctional region were made by cutting across the hemidiaphragms approximately 2mm either side of the visible intramuscular nerve. The muscle was then weighed and homogenized in 5 ml of 0.1 M sodium phosphate buffer (pH 8.0). The homogenate was incubated for 30 min with 50 mM ethopropazine, a selective inhibitor of butyrylcholinesterase (Bayliss & Todrick, 1953), then sonicated and centrifuged at 1500 g for 15 min at 4°C. The clear supernatant was assayed for cholinesterase activity using the method of Ellman et al. (1961). Cholinesterase activity was expressed in nmol acetylthiocholine hydrolysed per minute per milligram of muscle and is considered to be due substantially to the acetyl-
cholinesterase activity of the endplate (Das, 1989). The anticholinesterases used were: ecothiopate, (S-(2-trimethylammoniumethyl) phosphorothioate iodide) or BOS (pinacolyl S-(2-trimethylaminoethyl) methylphosphonothioate). Ecothiopate (ECO) was made up from Phospholine Eyedrops (Ayerst) which contained 12.5 mg ECO and a potassium acetate/boric acid buffer. The amount of potassium injected in the standard dose of ECO was negligible (approximately 6.0 gmol kg-'). Solutions of BOS for injection were made up shortly before use.
Administration ofpossible protective agents Single doses of pralidoxime methiodide (Sigma), 110 gmol
kg-' (2PAM), were given subcutaneously to mice at the first appearance of fasciculations i.e. at 10-15 min after the injection of ECO or of BOS. Two protocols were used for multiple dosing with 2PAM after BOS. The shorter protocol (7 x 2PAM) involved seven injections over a period of 12 h; the first was 10 Lmol kg-', the next 5 injections were of 55 gmol kg-' at 2 h intervals, with a final injection of 10 ptmol kg-' 12 h after the first. The longer protocol (12 x 2PAM) began as the shorter protocol, plus further injections of 110 gmol kg-' at 24, 30, 36, 42 and 48 h after the first. In experiments with pyridostigmine, a single injection (0.38 gmol kg-') of pyridostigmine bromide (Mestinon; Roche) was given 30 min before 0.5 mol kg-' ECO. In experiments with antioxidants, mice were dosed by gavage with vitamin E (Sigma) and/or with N-acetylcysteine solution (Parvolex; Duncan Flockhart). Both were given by gavage once daily for 7 days, vitamin E (VitE, 1.2 mmol kg-') dissolved in 0.1 ml corn oil; N-acetylcysteine (NAC, 6.2 mmol kg-'). Some mice received both at different times in the day. ECO (0.5 mol kg-' or 0.4 mol kg-') was injected on the seventh day. In another experiment, the same dose of vitamin E alone was given for 12 days, with ECO (0.5 mol kg-') being injected on the seventh day.
Statistical analysis Unless otherwise stated, all results are expressed as mean ± 1 s.d. of values from 2 to 5 animals, with the number of muscle fibres in parentheses. To test for significances of differences between groups of data, non-parametric tests were used and differences were taken to be significant if P
