Gen. Pharmac. Vol. 23, No. 4, pp. 689-692, 1992 Printed in Great Britain. All rights reserved
0306-3623/92 $5.00+ 0.00 Copyright ~ 1992Pergamon Press Lid
SELECTED BIOLOGICAL ACTIVITIES OF NOVEL SELENONIUM CHOLINE ANALOGS A. V. TERRY JR, ~ LOUIS A. SILKS III, 3 R. B. DUNLAP,2 J. D. ODOM2 and J. W. Kosrt ~* tCollege of Pharmacy and 2Department of Chemistry, University of South Carolina, Columbia, SC 29208 and 3Los Alamos National Laboratory, Biochemistry and Spectroscopy Section, INC-4: MS C346, Los Alamos, NM 87545, U.S.A. (Received 25 November 1991)
Abstract--l. The novel choline analogs selenonium choline (SeCh) and acetylselenoniumcholine (ASeCh) have been examined for selected biological activities. 2. ASeCh was found to be an alternative substrate for acetylcholin esterase with K~ and Vm~ values similar to acetylcholine. 3. ASeCh and SeCh inhibited acetylthiocholine hydrolysis by acetylcholinesterase with ICs0 values similar to acetylcholine and choline. 4. SeCh exerted a protective action against physostigmine and DFP induced toxicity. 5. SeCh (85 mg/kg) was found to be 3 times more toxic in mice than choline.
INTRODUCTION A n u m b e r of analogs of choline and acetylcholine have been utilized for studying the kinetics and mechanisms of acetylcholine turnover and metabolism (Barker and Mittag, 1975; Collier et al., 1979; Meyer et al., 1982). M a n y of these compounds have been shown to possess significant biological and pharmacological activity, including the ability to alter the kinetics of acetylcholinesterase (Hedlund et al., 1982; Frankenberg et al., 1973; Roepke and Welch, 1936; Patterson et al., 1989). We have recently reported that the choline analogs monoethylcholine (MEC) and N - a m i n o d e a n o l ( N A D ) also have a potential use in preventing acetylcholinesterase (ACHE) inhibitor toxicity (Patterson et aL, 1989). The data suggested an interaction of the analogs with acetylcholinesterase based on their ability to decrease the velocity of acetylcholine hydrolysis. Therefore, during the binding of the choline analog to ACHE, the rate of carbamylation or phosphorylation by the inhibitor will be slowed. The resulting decrease in toxicity observed with M E C and N A D may also occur in the presence of other analogs of choline, but has not been studied. A novel selenium-containing choline analog "selenonium choline" [(CH3)2Se+-CH2-CH:OH], was recently introduced in this laboratory (Terry et al., 1991a,b). It was the purpose of the present study to evaluate the acute toxicity of this compound, its ability to influence the kinetics of acetylcholinesterase, and to examine its potential to prevent acetylcholinesterase toxicity. MATERIALS AND METHODS
Animals Male ICR mice (Harlan Industries, Prattville, Ala) weighing 20-30 g were used in all studies requiring mice. The
* To whom all correspondence should be addressed.
animals were housed in a temperature-controlled environment (25°C) with a 12 hr light/dark cycle and allowed food and water ad libitum. Acute toxicity determination An estimation of the toxicity of selenonium choline (SeCh) in vivo was assessed in mice. Selenonium choline was administered i.p. in increasing doses (n = 1) until lethality was observed. The dose of SeCh was then adjusted to obtain a lethality of 50*/, using a group of 6 animals. The acute toxicity observation period was 24 hr. Prevention of acute acetylcholinesterase inhibitor toxicity Selenonium choline was examined for its ability to prevent acute acetylcholinesterase inhibitor toxicity in ICR mice. Physostigmine (a carbamate) and diisopropylfluorophosphate (DFP, an organophosphate) were chosen as examples of reversible and irreversible inhibitors, respectively. Acute toxicity was induced using physostigmine (0.8 mg/kg i.p.) or DFP (5 mg/kg s.c.). Physostigmine and DFP solutions were freshly prepared daily in 0.15 M NaCI. Antidotal activity was determined by administering selenonium choline (i.p.) either-simultaneously or 5 rain prior to the induction of acute toxicity. The acute toxicity observation period was 30 rain for physostigmine and 6 hr for DIP. Acetylcholinesterase studies Inhibition. The ability of selenonium choline and acetylselenonium choline to influence the kinetics of acetylcholinesterase was investigated and compared to choline and acetylcholine using a modification of the spectrophotometric method of Ellman et al. (1961). Various concentrations of selenonium choline, acetylselenonium choline, acetylcholine, or choline were incubated in a cuvette at 37°C for 2 rain containing the following in a total volume of 3 ml: acetylthiocholine, 7.5 x 10-s M (Sigma); DTNB, 0.01 M (Aldrich); sodium potassium phosphate buffer, 0.1 M, pH 7.9. After the incubation period enzyme was added (bovine erythrocyte cholinesterase, Type XII-S, Sigma) and the velocity of the reaction recorded at 412nm with a Beckman DU speetrophotometer adapted with a Gilford (model 222A) photometer and 2220 adapter. Velocity was expressed as pmoles of substrate hydrolyzed per min.
689
690
A. V. TERRYJR et al.
Role as a substrate. Acetylselenonium choline (ASeCh) was examined as a possible substrate of acetylcholinesterase using a modification of the methods of Wilson and Cabib (1954) and Frankenberg et al. (1973). Hydrolysis of ASeCh by acetylcholinesterase was measured by titrating the protons released from the acetic acid formed in the reaction with 0.01 N sodium hydroxide. The assay was carried out in a final volume of 5 ml in an aqueous solution containing 0.1 M KCI and 0.001 M MgC12 at 20~C. The enzyme preparation used was bovine erythrocyte cholinesterase Type XII-S (Sigma). During titration the solution was continuously stirred and maintained at a pH of 7.00 (+ 0.05) by the addition of base using a 50pl syringe. Various concentrations of substrate were evaluated and the results plotted in the Lineweaver-Burk format. All velocities were corrected for aqueous hydrolysis or any non-specific pH change by repeating the assay in the absence of enzyme. RESULTS
Acute toxicity
The dose of selenonium choline producing a 50% lethality in 6 animals was 85 mg/kg. The average time to death (n = 3) was 20 +_ i.1 hr. The symptoms of toxicity consisted of labored breathing with apnea, salivation, decreased motor activity, and ataxia. Cyanosis was evident from the blue appearance of the tail and a strong garlicky odor was also noted in the respired air. Prevention o f acetylcholinesterase inhibitor toxicity by selenonium choline
Table l summarizes the ability of selenonium choline (SeCh) to prevent the acute toxicity of a reversible (physostigmine) and an irreversible acetyicholinesterase inhibitor (DFP). Physostigmine (0.8mg/kg) produced fatalities in 83.3% of the mice tested with an average time to death of 7.8 + 0.80 min. Simultaneous administration of SeCh (200mg/kg) reduced the deaths to 16.7%. SeCh (200 mg/kg) administered 5 min prior to physostigmine also reduced deaths to 16.7%. D F P (5 mg/kg) was lethal in 100% of the mice tested with an average time to death of 9 . 9 + 0.60rain. SeCh (300mg/kg) administered simultaneously failed to reduce the percentage of deaths. Table I. Prevention of acetylcholinesterase inhibitor-toxicity by
selenonium choline Observation
Treatment
n
Physostigmine ( 0 . g m g / k g ) + Saline 12
DFP (Smg/kg) + Saline + ScCh (300 mg/kg) simultaneous + ScCh (300 mg/kg)
pre-treatment
Deaths
(%)
30
7.8 _+ 0.80
83.3
6
30
10*
16.7
6
30
9.2 *
16.7
7
360
9.9 _+ 0.60
6
360
35 _+ 14t
100
6
360
14 _+ 2.21"
100
+ SeCh (200mg/kg) simultaneous + ScCh (200 mg/kg) pre-treatment
Time to
period(rain) death(rain)
100
Mice w o e administered acetylcholin~teras¢ inhibitor alone or with s©lenonium choline (SeCh) simultaneously or as a 5 min pretreatmvnt. SeCh and physostigmine were administered i.p. and D F P ( diisopropylfluorophosphate ) was administered s.c. *n = I, t P < 0.05 compared to D F P + saline group.
8
Oh
4o
~C:a
ANALOGCONCeN'mATION(M)
Fig. I. Inhibition of acctylcholinesterascby choline analogs. Inhibitory activity was determined by the method of Ellman et al. (196I) using bovine erythrocyte acetylcholinesterase (Sigma, type XII-S). Final acetylthiocholine concentration was 5 x l0 -~ M. Analogs were added simultaneously with acetylthiocholine and the reaction initiated by addition of enzyme. ACh, acetylcholine; ASeCh, acetylselenonium choline; Ch, choline; SeCh, selenonium choline. However, the average time to death was significantly (P-
0306-3623/92 $5.00+ 0.00 Copyright ~ 1992Pergamon Press Lid
SELECTED BIOLOGICAL ACTIVITIES OF NOVEL SELENONIUM CHOLINE ANALOGS A. V. TERRY JR, ~ LOUIS A. SILKS III, 3 R. B. DUNLAP,2 J. D. ODOM2 and J. W. Kosrt ~* tCollege of Pharmacy and 2Department of Chemistry, University of South Carolina, Columbia, SC 29208 and 3Los Alamos National Laboratory, Biochemistry and Spectroscopy Section, INC-4: MS C346, Los Alamos, NM 87545, U.S.A. (Received 25 November 1991)
Abstract--l. The novel choline analogs selenonium choline (SeCh) and acetylselenoniumcholine (ASeCh) have been examined for selected biological activities. 2. ASeCh was found to be an alternative substrate for acetylcholin esterase with K~ and Vm~ values similar to acetylcholine. 3. ASeCh and SeCh inhibited acetylthiocholine hydrolysis by acetylcholinesterase with ICs0 values similar to acetylcholine and choline. 4. SeCh exerted a protective action against physostigmine and DFP induced toxicity. 5. SeCh (85 mg/kg) was found to be 3 times more toxic in mice than choline.
INTRODUCTION A n u m b e r of analogs of choline and acetylcholine have been utilized for studying the kinetics and mechanisms of acetylcholine turnover and metabolism (Barker and Mittag, 1975; Collier et al., 1979; Meyer et al., 1982). M a n y of these compounds have been shown to possess significant biological and pharmacological activity, including the ability to alter the kinetics of acetylcholinesterase (Hedlund et al., 1982; Frankenberg et al., 1973; Roepke and Welch, 1936; Patterson et al., 1989). We have recently reported that the choline analogs monoethylcholine (MEC) and N - a m i n o d e a n o l ( N A D ) also have a potential use in preventing acetylcholinesterase (ACHE) inhibitor toxicity (Patterson et aL, 1989). The data suggested an interaction of the analogs with acetylcholinesterase based on their ability to decrease the velocity of acetylcholine hydrolysis. Therefore, during the binding of the choline analog to ACHE, the rate of carbamylation or phosphorylation by the inhibitor will be slowed. The resulting decrease in toxicity observed with M E C and N A D may also occur in the presence of other analogs of choline, but has not been studied. A novel selenium-containing choline analog "selenonium choline" [(CH3)2Se+-CH2-CH:OH], was recently introduced in this laboratory (Terry et al., 1991a,b). It was the purpose of the present study to evaluate the acute toxicity of this compound, its ability to influence the kinetics of acetylcholinesterase, and to examine its potential to prevent acetylcholinesterase toxicity. MATERIALS AND METHODS
Animals Male ICR mice (Harlan Industries, Prattville, Ala) weighing 20-30 g were used in all studies requiring mice. The
* To whom all correspondence should be addressed.
animals were housed in a temperature-controlled environment (25°C) with a 12 hr light/dark cycle and allowed food and water ad libitum. Acute toxicity determination An estimation of the toxicity of selenonium choline (SeCh) in vivo was assessed in mice. Selenonium choline was administered i.p. in increasing doses (n = 1) until lethality was observed. The dose of SeCh was then adjusted to obtain a lethality of 50*/, using a group of 6 animals. The acute toxicity observation period was 24 hr. Prevention of acute acetylcholinesterase inhibitor toxicity Selenonium choline was examined for its ability to prevent acute acetylcholinesterase inhibitor toxicity in ICR mice. Physostigmine (a carbamate) and diisopropylfluorophosphate (DFP, an organophosphate) were chosen as examples of reversible and irreversible inhibitors, respectively. Acute toxicity was induced using physostigmine (0.8 mg/kg i.p.) or DFP (5 mg/kg s.c.). Physostigmine and DFP solutions were freshly prepared daily in 0.15 M NaCI. Antidotal activity was determined by administering selenonium choline (i.p.) either-simultaneously or 5 rain prior to the induction of acute toxicity. The acute toxicity observation period was 30 rain for physostigmine and 6 hr for DIP. Acetylcholinesterase studies Inhibition. The ability of selenonium choline and acetylselenonium choline to influence the kinetics of acetylcholinesterase was investigated and compared to choline and acetylcholine using a modification of the spectrophotometric method of Ellman et al. (1961). Various concentrations of selenonium choline, acetylselenonium choline, acetylcholine, or choline were incubated in a cuvette at 37°C for 2 rain containing the following in a total volume of 3 ml: acetylthiocholine, 7.5 x 10-s M (Sigma); DTNB, 0.01 M (Aldrich); sodium potassium phosphate buffer, 0.1 M, pH 7.9. After the incubation period enzyme was added (bovine erythrocyte cholinesterase, Type XII-S, Sigma) and the velocity of the reaction recorded at 412nm with a Beckman DU speetrophotometer adapted with a Gilford (model 222A) photometer and 2220 adapter. Velocity was expressed as pmoles of substrate hydrolyzed per min.
689
690
A. V. TERRYJR et al.
Role as a substrate. Acetylselenonium choline (ASeCh) was examined as a possible substrate of acetylcholinesterase using a modification of the methods of Wilson and Cabib (1954) and Frankenberg et al. (1973). Hydrolysis of ASeCh by acetylcholinesterase was measured by titrating the protons released from the acetic acid formed in the reaction with 0.01 N sodium hydroxide. The assay was carried out in a final volume of 5 ml in an aqueous solution containing 0.1 M KCI and 0.001 M MgC12 at 20~C. The enzyme preparation used was bovine erythrocyte cholinesterase Type XII-S (Sigma). During titration the solution was continuously stirred and maintained at a pH of 7.00 (+ 0.05) by the addition of base using a 50pl syringe. Various concentrations of substrate were evaluated and the results plotted in the Lineweaver-Burk format. All velocities were corrected for aqueous hydrolysis or any non-specific pH change by repeating the assay in the absence of enzyme. RESULTS
Acute toxicity
The dose of selenonium choline producing a 50% lethality in 6 animals was 85 mg/kg. The average time to death (n = 3) was 20 +_ i.1 hr. The symptoms of toxicity consisted of labored breathing with apnea, salivation, decreased motor activity, and ataxia. Cyanosis was evident from the blue appearance of the tail and a strong garlicky odor was also noted in the respired air. Prevention o f acetylcholinesterase inhibitor toxicity by selenonium choline
Table l summarizes the ability of selenonium choline (SeCh) to prevent the acute toxicity of a reversible (physostigmine) and an irreversible acetyicholinesterase inhibitor (DFP). Physostigmine (0.8mg/kg) produced fatalities in 83.3% of the mice tested with an average time to death of 7.8 + 0.80 min. Simultaneous administration of SeCh (200mg/kg) reduced the deaths to 16.7%. SeCh (200 mg/kg) administered 5 min prior to physostigmine also reduced deaths to 16.7%. D F P (5 mg/kg) was lethal in 100% of the mice tested with an average time to death of 9 . 9 + 0.60rain. SeCh (300mg/kg) administered simultaneously failed to reduce the percentage of deaths. Table I. Prevention of acetylcholinesterase inhibitor-toxicity by
selenonium choline Observation
Treatment
n
Physostigmine ( 0 . g m g / k g ) + Saline 12
DFP (Smg/kg) + Saline + ScCh (300 mg/kg) simultaneous + ScCh (300 mg/kg)
pre-treatment
Deaths
(%)
30
7.8 _+ 0.80
83.3
6
30
10*
16.7
6
30
9.2 *
16.7
7
360
9.9 _+ 0.60
6
360
35 _+ 14t
100
6
360
14 _+ 2.21"
100
+ SeCh (200mg/kg) simultaneous + ScCh (200 mg/kg) pre-treatment
Time to
period(rain) death(rain)
100
Mice w o e administered acetylcholin~teras¢ inhibitor alone or with s©lenonium choline (SeCh) simultaneously or as a 5 min pretreatmvnt. SeCh and physostigmine were administered i.p. and D F P ( diisopropylfluorophosphate ) was administered s.c. *n = I, t P < 0.05 compared to D F P + saline group.
8
Oh
4o
~C:a
ANALOGCONCeN'mATION(M)
Fig. I. Inhibition of acctylcholinesterascby choline analogs. Inhibitory activity was determined by the method of Ellman et al. (196I) using bovine erythrocyte acetylcholinesterase (Sigma, type XII-S). Final acetylthiocholine concentration was 5 x l0 -~ M. Analogs were added simultaneously with acetylthiocholine and the reaction initiated by addition of enzyme. ACh, acetylcholine; ASeCh, acetylselenonium choline; Ch, choline; SeCh, selenonium choline. However, the average time to death was significantly (P-
