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Cyanide-induced neurotoxicity: Role of neuronal calcium
TOXICOLOGY AND APPLIED PHARMACOLOGYM464-469
Cyanide-Induced JERRY D. JOHNSON,
(1986)
Neurotoxicity: TIMOTHY
Role of Neuronal
L. MEISENHEIMER,*
Calcium
AND GARY E. 1~0~’
Department of Pharmacology and Toxicology, School of Pharmacy and Pharmacal Sciences, and *Department of Physics, School of Science, Purdue University, West Lafayette, Indiana 47907
Received November 21, 1985; accepted March 11, I986 Cyanide-Induced Neurotoxicity: Role of Neuronal Calcium. JOHNSON,J. D., MEISENHEIMER, T. L., AND ISOM, G. E. (1986). Toxicol. Appl. Pharmacol. 84,464-469. The effect of cyanide on whole-brain calcium levels was determined in mice administered KCN and correlated with the neurotoxic signs manifested during acute cyanide poisoning. KCN (10 mg/kg, sc) significantly increased whole-brain total calcium levels from 48.1 + 1.8 to 66.5 f 3.9 &/g dry wt within 15 min after administration. The levels remained elevated for 3 hr and returned to control readings after 12 hr. Dose-response studies revealed KCN, at doses of lo- 15 mg/kg, produced significant elevations of whole-brain calcium 30 min after administration. No measureable effect was obtained from lower doses which suggested a threshold effect. Pretreatment 15 min before KCN with diltiazem, a calcium channel blocker, prevented the cyanide-induced rise in whole-brain total calcium. Cyanide-induced tremors, which are centrally mediated symptoms of intoxication, were quantified and correlated with the observed changes in whole-brain calcium. Tremors were detected at 10 and 12 mg/kg KCN and peak intensity was observed at 15 min postcyanide. Pretreatment with diltiazem markedly attenuated the cyanide-induced tremors. It appears that a correlation exists between cyanide-induced change in whole-brain calcium and tremors. This study suggests that intraneuronal calcium may play an important role in mediating cyanide neurotoxicity and calcium channel blocking agents may be useful in limiting the severity of the centrally mediated symptoms of acute cyanide intoxication. 0 1986 Academic Press, Inc.
It is generally accepted that acute cyanide toxicity is produced by inhibition of cytochrome oxidase uu3, the terminal enzyme of the electron transport chain (Albaum et al., 1946; Schubert and Brill, 1968; Isom and Way, 1984). Little is known about the sequence of events that follow inhibition of oxidative metabolism and the resultant injury at the cellular level. Acute cyanide intoxication is manifested clinically by cardiac arrhythmias, respiratory arrest, cardiovascular collapse, changes in EEG recordings, seizures, tremors, and other centrally mediated effects (Burrows et al., 1982; Persson et al., 1985). The central nervous system is especially sensitive to cyanide, apparently due to its limited anaerobic metabolic capacity and high energy dependence. Chronic ’ To whom correspondence should be addressed. 0041-008X/86
$3.00
Copyright Q 1986 by Academic Pres, Inc. All ti&.s of reproduction in any form reserved.
464
cyanide exposure is associated with a variety of neuronal disorders including tropical ataxic neuropathy (Osuntokun, 1968), Leber’s optic atrophy (Wilson, 1965) tobacco amblyopia (Foulds, 1969) and amyotrophic lateral sclerosis (Kameyama, 1980). The neural lesions induced by cyanide parallel those produced by &hernia and anoxia (Reempts, 1984). Recently, calcium ion has been demonstrated to play an integral role in mediating cellular injury following an ischemit or anoxic insult (Trump et al., 1984; Dienel, 1984). The cessation of energy-dependent calcium regulatory pathways within the cell results in intracellular calcium accumulation. This calcium disequilibrium alters numerous neurophysiological processes resulting in cellular damage (Campbell, 1984). The objective of this study was to investigate
CYANIDE-INDUCED
the effect of cyanide on the accumulation of calcium in the brain, and to assess the relationship of changes in brain calcium levels to the CNS-mediated symptoms of toxicity. METHODS Animals. Male, Swiss-Webster mice (Laboratory Supply, Indianapolis, Ind.), weighing 25-30 g, were used throughout the duration of the studies. The mice were housed in groups of 10 to 12 mice per cage, exposed to a 12-hr light-and-dark cycle, and had accessto standard laboratory chow and tap water ad libitum. Chemicals. Potassium cyanide. lanthanum chloride (Fisher Scientific Co., Fair Lawn, N.J.) and sodium dichromate (Mallinckrodt, Paris, Ky.) were of the highest chemical grade commercially available. Dextran solution was obtained from Cutter Laboratories (Berkeley, Calif.), double-distilled nitric acid from G. Frederick Smith Chemical Company (Columbus, Ohio), and diltiazem HCI was generously provided by Dr. Ronald Browne (Marion Laboratory, Kansas City, MO.). Deionized water, 18 megohms was used as a solvent system and for glassware prep aration. Whole-brain total calcium analysis. The glassware was scrubbed vigorously in Alconox, rinsed with double-distilled water six times, and soaked overnight in nitric acid (12%, v/v) and in deionized water for an additional 24 hr prior to use. The Kjehldahl flasks, 30 ml, were bathed in sodium chromate-concentrated sulfuric acid solution (Skoog and West, 1974) for 10 min to remove any organic residue, rinsed with copious amounts of double-distilled water, and then submitted to the above cleaning protocol. Following treatment, mice were sacrificed by cervical dislocation. A small incision was made in the right atrium and 3 ml of 10% dextran solution was infused into the left ventricle to perfuse the brain of blood. The brains were dissected, freeze dried at -50°C (Virtis Freeze Drier, Gardener, N.Y.) to a constant weight (36-48 hr), weighed for dry weight determination, and wet ashed with IO- 15 ml of double-distilled nitric acid in Kjehldahl flasks. The residual salts were reconstituted with 2.0 ml of diluent [deionized water:double-distilled nitric acidlanthanum chloride (0.5%, w/v) in a volume ratio of 15: 1:4]. The calcium concentrations in the IIasks were determined by atomic absorption spectrophotometry (PerkinElmer, Model 290B) using a calcium-magnesium intensitron hollow cathode lamp (Perkin-Elmer, Model 3036092). All samples were assayed in the standard air-acetylene flame with a 4-in. single-slot flat-head burner. The absorption wavelength and slit width settings were 459 nm and 20 A, respectively. Tremor analysis. Tremors were measured using a technique previously described (Conroy et al., 1985). Briefly, a Plexiglas box (6 X 6 X 8 in.) enclosed a free-floating Teflon platform (54 X 54 X 4 in.) which was attached to an aluminum bar by a recessed flat-head screw. The alu-
465
NEUROTOXICITY
minum bar was bolted to the wall and a strain gauge (Model FAE-25-S 13ES, BLH Electronics, Waltham, Mass.) attached to the surface. A change in resistance induced by mechanical displacement of the platform due to animal movement was measured as a voltage differential by a Wheatstone bridge. The electronic signal was amplified and transmitted to an oscilloscope for visualization and standardization of the signal, and to an analog digital converter (Deck 12 bit) for computer acquisition, retrieval, and graphic illustration of the dam. The conversion from a voltage per unit time domain to a frequency-intensity domain was made using a discrete Fourier transformation. The data are expressed as the peak frequency, which was that frequency with the greatest intensity. Treatment protocol. In the whole-brain total calcium or tremor frequency experiments, 10 or 12 mg/kg of KCN was administered SCand mice were either sacrificed and brain calcium levels determined or a recording of tremors taken over a I- to 180-min time period. Control mice received saline (SC)and were sacrificed immediately (0 min) for the whole-brain calcium study. In dose-response studies, saline (control) or KCN in a dose range of 0.5 to I5 mg/kg was administered (SC),and the mice sacrificed or a tremor recording taken 30 min after injection. Tremors were quantified by measuring the intensity-frequency profiles of mice at each time point and repeating the measurement at each subsequent time point with the same group of mice. The effects of diltiazem on whole-brain total calcium or tremors were studied in the following treatment groups: (1) 15 min saline pretreatment, iv, followed by KCN, 10 or 12 mg/kg, sc; (2) 15 min diltiazem pretreatment, 600 rg/kg, iv, followed by saline, sc; and (3) 15 min diltiazem pretreatment, 600 &kg, iv, followed by KCN, IO or 12 mg/kg, sc. The mice were then either sacrificed for the calcium studies or a tremor recording taken after 0, 15, 30, or 60 min. Statistics. Data are expressed as X + SE. All treatment groups were composed of four or more animals. Analysis of variance (ANOVA) was used to determine statistical differencesbetween treatment groups at a significance level of 0.05. If multiple comparisons were not a factor, a Student’s t test (two tail) was employed and the means were ranked using a Neuman-Keuls multiple range test.
RESULTS KCN (10 mg/kg, SC)produced a significant decrease (p < 0.05) in whole-brain total calcium within 5 min after administration, followed by a significant increase (p < 0.05) after 15 min (Fig. 1). The whole-brain total calcium level peaked at 140- 145% of control at 30 min after injection of cyanide. The calcium levels remained elevated for 3 hr, and returned to control values after 12 hr. The effect of gra-
466
JOHNSON. MEISENHEIMER.
AND ISOM TOP
*
l
20
10
I t
1.
. 30
.
. 60
.
90 Tim*
. 120
.
. 150
, 180
(mln)
FIG. I. KCN (IO mg/kg, sc) was administered at zero time and whole-brain total calcium determined at varying time periods after injection. Each point reflects the X f SE of (4-9) mice. Asterisks denote a significant difference (p < 0.05) from saline-treated mice sacrificed at 0 min (control).
dient doses of KCN on whole-brain total calcium, 30 min after administration, is shown in Fig. 2. The three lower doses did not alter calcium levels whereas the 10, 12, and 15 mg/ kg doses produced a significant elevation (p < 0.001) as compared to saline treatment (control). The effect of KCN, diltiazem, or a combination of both agents on brain calcium is illustrated in Fig. 3. KCN, 10 mg/kg SC, (pretreated 15 min before cyanide with saline, iv) produced an increase in whole-brain calcium
FIG. 2. Total whole-brain calcium measured 30 min after administration of gradient doses of KCN. Each bar reflects the X f SE of at least 6 mice. Asterisks denote a significant difference (p < 0.05) from saline-treated mice (C, control).
15
30 Time
45
80
(mid
FIG. 3. Whole-brain total calcium was measured after administration of saline, iv, 15 min pretreatment, followed by KCN, 10 mg/kg, x: (0); diltiazem, 600 &kg, iv, 15 min pretreatment, followed by saline, sc (A); diltiazem, 600 &kg, iv, 15 min pretreatment, followed by KCN, 10 mg/kg, SC(Cl). Each point reflects the X f SE of at least 4 mice. Asterisks denote a significant difference (p -c 0.05) between treatment groups at the same time point.
levels over a 60-min period as previously described (Fig. 1). Diltiazem pretreatment (15 min before KCN), 600 pg/kg, iv, significantly decreased (p < 0.05) the KCN-induced rise in whole-brain total calcium that occurred at 15 and 30 min. Thirty minutes after 12 mg/kg KCN, the whole-brain calcium was elevated to 70.5 + 1.67 pg/g. In mice pretreated with diltiazem, 12 mg/kg KCN did not significantly elevate brain calcium levels (56.5 f 1.3 @g/g). Diltiazem pretreatment, followed by saline, produced no significant change from pretreatment control values or between relative time points of the other treatment groups. KCN ( 12 mg/kg, SC)induced a visible, sustained high-frequency tremor (Fig. 4). Control mice exhibited a low-frequency movement, 0.48 Hz, which reflected exploratory and grooming behavior. A slight increase in frequency, 2.97 + 0.77 Hz, was observed at 5 min after cyanide and corresponded to the observed respiratory rate of 150-200 breaths/ min. After 15 min, a high-frequency tremor, 20.59 f 0.95 Hz, was observed and was significantly greater (p < 0.01) than control movement. The tremors lasted 30 to 45 min and exhibited a frequency ranging from 17.23 + 1.71 to 11.58 + 1.61 Hz. By 60 min postcyanide, tremors were not detected. The slight
CYANIDE-INDUCED
467
NEUROTOXICITY
tion and complete loss of motor and behavioral activity. I 20. Diltiazem treatment produced marked c changes in the peak frequency of cyanide-in:$ 15duced tremor incidence and frequency (Fig. ? 6). Diltiazem, 600 pg/kg iv, administered 15 2 lom min before KCN ( 12 mg/kg), produced a sig: 5nificant decrease (p < 0.05) in the cyanideinduced tremor frequencies at all time periods. 0 C 30 60 90 120 150 160 Diltiazem did not completely block the tremTime tminl ors, but significantly delayed the onset, duration, and peak of the cyanide-induced tremors. FTC. 4. Peak tremor frequency measured in Hz at varying times following administration of KCN (12 mg/kg, SC). Diltiazem completely blocked the tremors Each point reflects the X + SE of 6 mice. Asterisks denote produced following KCN, 10 mg/kg. The peak a significant difference (p < 0.05) from saline-treated mice frequency 30 min after 10 mg/kg KCN was (control). 13.46 + 1.20 Hz and for mice pretreated with diltiazem, 1.14 k 0.65 Hz. Diltiazem alone increase in frequency, 4.53 +- 0.96 Hz, reflects produced no observable change in the behavior or motor activity of the mice. the approximate respiratory rate, 250-300 breaths/min, of the mice as they recovered from the cyanide. After 120- 180 min the mice DISCUSSION had frequency values equal to control and exploratory and grooming behavior returned to The primary biochemical lesion produced normal. Tremors were not detected following by cyanide is inhibition of cytochrome oxidase 3,5, or 7 mg/kg KCN (Fig. 5). However, KCN, (Schubert and Brill, 1968; Isom and Way, 10 and 12 mg/kg, produced tremors with fre- 1984) which results in histotoxic anoxia. Anquencies of 13.35 -+ 1.3 1 and 18.56 + 1.29, aerobic metabolism predominates in a cyarespectively, which were significantly greater nide-intoxicated cell producing a decrease in than control (p < 0.01). The 15-mg/kg dose the ATP/ADP ratio or energy charge (Isom et of KCN incapacitated the mice and approximately 50% died. These animals were severely 25 r intoxicated and exhibited depressed respira25
r
l
Ti
25 I
C
15
30 Tlme
FIG. 5. The effect of gradient doses of KCN on peak tremor frequency measured in Hz 30 min after cyanide administration. Each bar reflects the X Ifr SE of 6 mice. Asterisks denote a significant difference (p < 0.05) from saline-treated mice (control).
45
60
(mln)
FIG. 6. Peak frequency was measured on a time basis after administration of saline, iv, 15 mitt pretreatment, followed by KCN, 12 mgjkg, sc (0); diltiazem, 600 fig/ kg, iv, IS min pretreatment, followed by saline, sc (A); or diltiazem, 600 &kg, iv, followed by KCN, 12 mg/kg, sc (cl). Each point reflects the X +_ SE of 6 mice. Asterisks denote a significant difference (p < 0.05) between treatment groups at the same respective time point.
468
JOHNSON, MEISENHEIMER,
al., 1975). As a result of this effect, cyanide in sublethal doses can alter important energy-dependent processes including cellular calcium homeostasis. Calcium plays a key role as an intracellular regulator and in the central nervous system small changes in calcium homeostasis can produce marked physiological alterations. For instance, the release of neurotransmitters is a calcium-dependent process (Miledi, 1973; Llinas and Heuser, 1977; Henkart, 1980a,b). Recently, Persson et al. ( 1985) observed that cyanide-induced convulsions were concomitant with changes in regional brain neurotransmitter levels. These observations appear to have important implications with regard to the neurotoxicity associated with acute cyanide intoxication. In the present study, cyanide-induced rise in brain calcium levels corresponded to the induction of tremors. The role of calcium in mediating the cyanide-induced tremors is supported by the observation that diltiazem pretreatment blocked both the generation of tremors and the increases in whole-brain calcium. Diltiazem is a specific calcium channel blocker which can penetrate the blood-brain barrier and block voltage-dependent calcium channels in the plasma membrane and the Ca/ Na antiporter in the mitochondria (Matlib and Schwartz, 1983; White et al., 1983; Fitzpatrick and Karmazyn, 1984; Spedding and Cavero, 1984). Diltiazem would block calcium influx from extracellular pools and prevent accumulation of intracellular calcium. The effect of cyanide on whole-brain total calcium levels may be explained by activation or inactivation of various neuronal calcium homeostatic mechanisms. For instance, during the first 5 min after administration, cyanide decreased the whole-brain total calcium levels. Blaustein and Hodgkin ( 1968) demonstrated that cyanide-induced release of calcium from intracellular organelles results in activation of Na/Ca antiports in the plasma membrane of the squid axon. As a consequence, total cellular calcium levels dropped. The sudden increase in whole-brain total calcium after 5 min suggests that calcium accumulation is occurring. Since many of the neuronal processes
AND ISOM
which regulate intracellular calcium are energy dependent, the depletion of ATP would severely compromise the active transport systems in the plasma membrane and organelles (Brinley, 1980). The rise in whole-brain total calcium paralleled the onset of the tremors; both were initiated at 15 min after cyanide administration. However, the duration of the whole-brain total calcium elevation was longer than the duration of the tremors. It is unlikely that the duration of the two events would coincide since the neurogenic active calcium pool in the cytosol was not assessedby measuring the whole-brain total calcium levels. It is possible the cytosolic calcium pools returned to normal within 60 min and the calcium buffering capacity was restored to the cellular organelles. In support, l-3 hr after cyanide, the animals did not exhibit detectable tremors and respiration, behavioral and motor activity appeared normal. Both the alterations in brain calcium and tremor responses to cyanide exhibited a threshold effect. At the IO-mg/kg dose of cyanide, a significant rise in whole-brain total calcium levels and whole-body tremors were detected. Subtremor doses (0.5-7 mg/kg) were ineffective in altering the whole-brain total calcium concentrations. Even though the 15mg/kg dose of cyanide produced a significant elevation of whole-brain total calcium, the absence of tremors at this dose may be the result of the severely compromised state of the mice (over 50% died within l-2 hr after administration), depletion of neurotransmitters, neuronal fatigue, and/or neuronal hyperpolarization (Isenberg et al., 1983; Van der Hayden, 1985). Prolonged accumulation of free intraneuronal calcium could induce hyperpolarization by activating calcium-dependent potassium channels. The ensuing efflux of potassium would increase the membrane potential and decrease neuronal excitability. Such a mechanism would also explain the EEG “silence” typically observed following an acute cyanide intoxication (Burrows et al., 1973). The role of calcium as an intraneuronal mediator of cyanide intoxication, may have
CYANIDE-INDUCED
far-reaching implications with regard to the signs of intoxication and, possibly, chronic disease states attributed to cyanide. Presently, studies are being initiated to determine the mechanism of the observed effect of cyanide on whole-brain calcium.
of sublethal doses of cyanide on glucose catabolism. Biochem.
Pharmacol.
Clin. Neural.
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J., AND
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(1946). The in vivo inactivation by cyanide of brain cytochrome oxidase and its effects on glycolysis and on the high energy phosphorous compounds in brain. J. Biol. Chem.
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BLAUSTEIN, M. P., AND HODGKIN, A. L. (1968). The effect of cyanide on the efflux of calcium from squid axon. J. Physiol.
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F. J., JR., (1980). Regulation of intracellular calcium in squid axons. Fed. Proc. 39, 2778-2782. BURROWS,G. E., LIU, D. H. W., AND WAY, J. L. (1973). Effect of oxygen on cyanide intoxication. V. Physiologic effects.J. Pharmacol. Exp. Ther. 184, 739-148. BURROWS,G. E., Lru, D. H. W., ISOM, G. E., AND WAY, J. L. (1982). Effect of antagonists on the physiologic disposition of sodium cyanide. J. Taxicol. Environ. BRINLEY,
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CAMPBELL, A. K. (1984). Intracellular Calcium: Its Universal Role as Regulator. Wiley, New York. CONROY,W. G., MEISENHEIMER, T. L., JOHNSON,J. D., AND ISOM, G. E. (1985). A quantitative method for characterizing whole body tremors in mice. Pharmacologist
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ISOM, G. E., AND WAY, J. L. (1984). Effects of oxygen on the antagonism of cyanide intoxication: Cytochrome oxidase, in vitro. Toxicol. Appt. Pharmacol. 74, 57-62. KAMEYAMA, M. (1980). Cyanide metabolism in nervous disorders, with special reference to motor neuron disease. 20,999-
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LLINAS, R. R., AND HEUSER,J. E. (1977). Depolarizationreleasecoupling systemsin neurons. Neurosci. Res. Prog,
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DIENEL, G. A. (1984). Regional accumulation of calcium in post ischemic rat brain. J. Neurochem. 43.9 13-925. FITZPATRICK, D. B., AND KARMAZYN, M. (1984). Comparative effectsof calcium channel blocking agents and varying extracellular calcium concentration on hypoxia/ reoxygenation and ischemia/reperfusion-induced cardiac injury. J. Pharmacol. Exp. Ther. 228, 761-767. FOULDS, W. S. (1969). Visual disturbances in systemic disorders, optic neuropathy, and systemicdisease. Trans. Opthalmol. Sot. UK 89, 125- 146. HENKART, M. (1980a). Identification and function of intracellular calcium stores in neurons. Fed. Proc. 39, 2776-2777.
HENKART, M. (1980b). Identification and function of intracellular calcium stores in axons and bodies of neurons. Fed. Proc. 39, 2783-2789.
ISENBERG,G., VEREECKE, J., VAN DERHEYDEN, G., AND CARMELIET, E. (1983). The shortening of the action potential by DNP in guinea pig ventricular myocytes is mediated by an increase of a time-independent K conductance. PfIuegers Arch. 397, 25 l-259. ISOM, G. E., Lru, D. H. W., AND WAY, J. L. (1975). Effect
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MATLIB, M. A., AND SCHWARTZ, A. (1983). Selective effects of diltiazem, a benzothiazepine calcium channel blocker, and diazepam, and other benzodiazepines on the Na+/Ca*+ exchange carrier systemof heart and brain mitochondria. Life Sci. 32, 2837-2842. MILEDI, R. (1973). Transmitter release induced by the injection of calcium ions into nerve terminals. Proc. R. Sot. London
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OSUNTOKUN, B. 0. (1968). An ataxic neuropathy in Nigeria: A clinical, biochemical and electrophysiological study. Brain 91, 215-248. PERSSON,S. A., CASSEL,G., AND SELLSTROM, A. (1985). Acute cyanide intoxication and central transmitter systems. Fundam. Appl. Toxicol. 5, S150-S159. REEMPTS, J. V. (1984). The hypoxic brain: Histological and ultrastructural aspects. Behav. Brain Res. 14, 99108. SCHUBERT,J., AND BRILL, W. A. (1968). Antagonism of experimental cyanide toxicity in relation to the in vivo activity of cytochrome oxidase. J. Pharmacol. Exp. Ther. 162,352-359.
SKOOG, D. A., AND WEST, D. M. (1974). In Analytical Chemistry An Introduction, 2nd ed. Holt, Rinehart and Winston, New York. SPEDDING, M., AND CAVERO, I. (1984). Calcium antagonists: A class of drugs with a bright future. Part II. Determination of basic pharmacological properties. Life Sci. 35, 575-587.
TRUMP, B. F., BEREZESKY,I. K., SATO, T., LAIHO, K. U., PHELPS,P. C., ANDDECLARIS, N. (1984). Cell calcium, cell injury and cell death. Environ. Health Perspect. 57, 281-287. WHITE, B.C., WINEGAR, C. D., WILSON, R. T., HOCHNER, P. J., AND TROMBLEY, J. H. (1983). Possible role of calcium blockers in cerebral resuscitation: A review of the literature and synthesis for future studies. Crit. Cure Med.
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WILSON, J. (1965). Leber’s hereditary optic atrophy: A possible defect of cyanide metabolism. Clin. Sci. 29, 505-5 15. VAN DER HAYDEN, G., VEREECKE, J., AND CARMELIET, E. (1985). The effect of cyanide on the K-current in guinea-pig ventricular myocytes. Basic Res. Cardiol. (Suppl. I) 80, 93-96.
Cyanide-Induced JERRY D. JOHNSON,
(1986)
Neurotoxicity: TIMOTHY
Role of Neuronal
L. MEISENHEIMER,*
Calcium
AND GARY E. 1~0~’
Department of Pharmacology and Toxicology, School of Pharmacy and Pharmacal Sciences, and *Department of Physics, School of Science, Purdue University, West Lafayette, Indiana 47907
Received November 21, 1985; accepted March 11, I986 Cyanide-Induced Neurotoxicity: Role of Neuronal Calcium. JOHNSON,J. D., MEISENHEIMER, T. L., AND ISOM, G. E. (1986). Toxicol. Appl. Pharmacol. 84,464-469. The effect of cyanide on whole-brain calcium levels was determined in mice administered KCN and correlated with the neurotoxic signs manifested during acute cyanide poisoning. KCN (10 mg/kg, sc) significantly increased whole-brain total calcium levels from 48.1 + 1.8 to 66.5 f 3.9 &/g dry wt within 15 min after administration. The levels remained elevated for 3 hr and returned to control readings after 12 hr. Dose-response studies revealed KCN, at doses of lo- 15 mg/kg, produced significant elevations of whole-brain calcium 30 min after administration. No measureable effect was obtained from lower doses which suggested a threshold effect. Pretreatment 15 min before KCN with diltiazem, a calcium channel blocker, prevented the cyanide-induced rise in whole-brain total calcium. Cyanide-induced tremors, which are centrally mediated symptoms of intoxication, were quantified and correlated with the observed changes in whole-brain calcium. Tremors were detected at 10 and 12 mg/kg KCN and peak intensity was observed at 15 min postcyanide. Pretreatment with diltiazem markedly attenuated the cyanide-induced tremors. It appears that a correlation exists between cyanide-induced change in whole-brain calcium and tremors. This study suggests that intraneuronal calcium may play an important role in mediating cyanide neurotoxicity and calcium channel blocking agents may be useful in limiting the severity of the centrally mediated symptoms of acute cyanide intoxication. 0 1986 Academic Press, Inc.
It is generally accepted that acute cyanide toxicity is produced by inhibition of cytochrome oxidase uu3, the terminal enzyme of the electron transport chain (Albaum et al., 1946; Schubert and Brill, 1968; Isom and Way, 1984). Little is known about the sequence of events that follow inhibition of oxidative metabolism and the resultant injury at the cellular level. Acute cyanide intoxication is manifested clinically by cardiac arrhythmias, respiratory arrest, cardiovascular collapse, changes in EEG recordings, seizures, tremors, and other centrally mediated effects (Burrows et al., 1982; Persson et al., 1985). The central nervous system is especially sensitive to cyanide, apparently due to its limited anaerobic metabolic capacity and high energy dependence. Chronic ’ To whom correspondence should be addressed. 0041-008X/86
$3.00
Copyright Q 1986 by Academic Pres, Inc. All ti&.s of reproduction in any form reserved.
464
cyanide exposure is associated with a variety of neuronal disorders including tropical ataxic neuropathy (Osuntokun, 1968), Leber’s optic atrophy (Wilson, 1965) tobacco amblyopia (Foulds, 1969) and amyotrophic lateral sclerosis (Kameyama, 1980). The neural lesions induced by cyanide parallel those produced by &hernia and anoxia (Reempts, 1984). Recently, calcium ion has been demonstrated to play an integral role in mediating cellular injury following an ischemit or anoxic insult (Trump et al., 1984; Dienel, 1984). The cessation of energy-dependent calcium regulatory pathways within the cell results in intracellular calcium accumulation. This calcium disequilibrium alters numerous neurophysiological processes resulting in cellular damage (Campbell, 1984). The objective of this study was to investigate
CYANIDE-INDUCED
the effect of cyanide on the accumulation of calcium in the brain, and to assess the relationship of changes in brain calcium levels to the CNS-mediated symptoms of toxicity. METHODS Animals. Male, Swiss-Webster mice (Laboratory Supply, Indianapolis, Ind.), weighing 25-30 g, were used throughout the duration of the studies. The mice were housed in groups of 10 to 12 mice per cage, exposed to a 12-hr light-and-dark cycle, and had accessto standard laboratory chow and tap water ad libitum. Chemicals. Potassium cyanide. lanthanum chloride (Fisher Scientific Co., Fair Lawn, N.J.) and sodium dichromate (Mallinckrodt, Paris, Ky.) were of the highest chemical grade commercially available. Dextran solution was obtained from Cutter Laboratories (Berkeley, Calif.), double-distilled nitric acid from G. Frederick Smith Chemical Company (Columbus, Ohio), and diltiazem HCI was generously provided by Dr. Ronald Browne (Marion Laboratory, Kansas City, MO.). Deionized water, 18 megohms was used as a solvent system and for glassware prep aration. Whole-brain total calcium analysis. The glassware was scrubbed vigorously in Alconox, rinsed with double-distilled water six times, and soaked overnight in nitric acid (12%, v/v) and in deionized water for an additional 24 hr prior to use. The Kjehldahl flasks, 30 ml, were bathed in sodium chromate-concentrated sulfuric acid solution (Skoog and West, 1974) for 10 min to remove any organic residue, rinsed with copious amounts of double-distilled water, and then submitted to the above cleaning protocol. Following treatment, mice were sacrificed by cervical dislocation. A small incision was made in the right atrium and 3 ml of 10% dextran solution was infused into the left ventricle to perfuse the brain of blood. The brains were dissected, freeze dried at -50°C (Virtis Freeze Drier, Gardener, N.Y.) to a constant weight (36-48 hr), weighed for dry weight determination, and wet ashed with IO- 15 ml of double-distilled nitric acid in Kjehldahl flasks. The residual salts were reconstituted with 2.0 ml of diluent [deionized water:double-distilled nitric acidlanthanum chloride (0.5%, w/v) in a volume ratio of 15: 1:4]. The calcium concentrations in the IIasks were determined by atomic absorption spectrophotometry (PerkinElmer, Model 290B) using a calcium-magnesium intensitron hollow cathode lamp (Perkin-Elmer, Model 3036092). All samples were assayed in the standard air-acetylene flame with a 4-in. single-slot flat-head burner. The absorption wavelength and slit width settings were 459 nm and 20 A, respectively. Tremor analysis. Tremors were measured using a technique previously described (Conroy et al., 1985). Briefly, a Plexiglas box (6 X 6 X 8 in.) enclosed a free-floating Teflon platform (54 X 54 X 4 in.) which was attached to an aluminum bar by a recessed flat-head screw. The alu-
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minum bar was bolted to the wall and a strain gauge (Model FAE-25-S 13ES, BLH Electronics, Waltham, Mass.) attached to the surface. A change in resistance induced by mechanical displacement of the platform due to animal movement was measured as a voltage differential by a Wheatstone bridge. The electronic signal was amplified and transmitted to an oscilloscope for visualization and standardization of the signal, and to an analog digital converter (Deck 12 bit) for computer acquisition, retrieval, and graphic illustration of the dam. The conversion from a voltage per unit time domain to a frequency-intensity domain was made using a discrete Fourier transformation. The data are expressed as the peak frequency, which was that frequency with the greatest intensity. Treatment protocol. In the whole-brain total calcium or tremor frequency experiments, 10 or 12 mg/kg of KCN was administered SCand mice were either sacrificed and brain calcium levels determined or a recording of tremors taken over a I- to 180-min time period. Control mice received saline (SC)and were sacrificed immediately (0 min) for the whole-brain calcium study. In dose-response studies, saline (control) or KCN in a dose range of 0.5 to I5 mg/kg was administered (SC),and the mice sacrificed or a tremor recording taken 30 min after injection. Tremors were quantified by measuring the intensity-frequency profiles of mice at each time point and repeating the measurement at each subsequent time point with the same group of mice. The effects of diltiazem on whole-brain total calcium or tremors were studied in the following treatment groups: (1) 15 min saline pretreatment, iv, followed by KCN, 10 or 12 mg/kg, sc; (2) 15 min diltiazem pretreatment, 600 rg/kg, iv, followed by saline, sc; and (3) 15 min diltiazem pretreatment, 600 &kg, iv, followed by KCN, IO or 12 mg/kg, sc. The mice were then either sacrificed for the calcium studies or a tremor recording taken after 0, 15, 30, or 60 min. Statistics. Data are expressed as X + SE. All treatment groups were composed of four or more animals. Analysis of variance (ANOVA) was used to determine statistical differencesbetween treatment groups at a significance level of 0.05. If multiple comparisons were not a factor, a Student’s t test (two tail) was employed and the means were ranked using a Neuman-Keuls multiple range test.
RESULTS KCN (10 mg/kg, SC)produced a significant decrease (p < 0.05) in whole-brain total calcium within 5 min after administration, followed by a significant increase (p < 0.05) after 15 min (Fig. 1). The whole-brain total calcium level peaked at 140- 145% of control at 30 min after injection of cyanide. The calcium levels remained elevated for 3 hr, and returned to control values after 12 hr. The effect of gra-
466
JOHNSON. MEISENHEIMER.
AND ISOM TOP
*
l
20
10
I t
1.
. 30
.
. 60
.
90 Tim*
. 120
.
. 150
, 180
(mln)
FIG. I. KCN (IO mg/kg, sc) was administered at zero time and whole-brain total calcium determined at varying time periods after injection. Each point reflects the X f SE of (4-9) mice. Asterisks denote a significant difference (p < 0.05) from saline-treated mice sacrificed at 0 min (control).
dient doses of KCN on whole-brain total calcium, 30 min after administration, is shown in Fig. 2. The three lower doses did not alter calcium levels whereas the 10, 12, and 15 mg/ kg doses produced a significant elevation (p < 0.001) as compared to saline treatment (control). The effect of KCN, diltiazem, or a combination of both agents on brain calcium is illustrated in Fig. 3. KCN, 10 mg/kg SC, (pretreated 15 min before cyanide with saline, iv) produced an increase in whole-brain calcium
FIG. 2. Total whole-brain calcium measured 30 min after administration of gradient doses of KCN. Each bar reflects the X f SE of at least 6 mice. Asterisks denote a significant difference (p < 0.05) from saline-treated mice (C, control).
15
30 Time
45
80
(mid
FIG. 3. Whole-brain total calcium was measured after administration of saline, iv, 15 min pretreatment, followed by KCN, 10 mg/kg, x: (0); diltiazem, 600 &kg, iv, 15 min pretreatment, followed by saline, sc (A); diltiazem, 600 &kg, iv, 15 min pretreatment, followed by KCN, 10 mg/kg, SC(Cl). Each point reflects the X f SE of at least 4 mice. Asterisks denote a significant difference (p -c 0.05) between treatment groups at the same time point.
levels over a 60-min period as previously described (Fig. 1). Diltiazem pretreatment (15 min before KCN), 600 pg/kg, iv, significantly decreased (p < 0.05) the KCN-induced rise in whole-brain total calcium that occurred at 15 and 30 min. Thirty minutes after 12 mg/kg KCN, the whole-brain calcium was elevated to 70.5 + 1.67 pg/g. In mice pretreated with diltiazem, 12 mg/kg KCN did not significantly elevate brain calcium levels (56.5 f 1.3 @g/g). Diltiazem pretreatment, followed by saline, produced no significant change from pretreatment control values or between relative time points of the other treatment groups. KCN ( 12 mg/kg, SC)induced a visible, sustained high-frequency tremor (Fig. 4). Control mice exhibited a low-frequency movement, 0.48 Hz, which reflected exploratory and grooming behavior. A slight increase in frequency, 2.97 + 0.77 Hz, was observed at 5 min after cyanide and corresponded to the observed respiratory rate of 150-200 breaths/ min. After 15 min, a high-frequency tremor, 20.59 f 0.95 Hz, was observed and was significantly greater (p < 0.01) than control movement. The tremors lasted 30 to 45 min and exhibited a frequency ranging from 17.23 + 1.71 to 11.58 + 1.61 Hz. By 60 min postcyanide, tremors were not detected. The slight
CYANIDE-INDUCED
467
NEUROTOXICITY
tion and complete loss of motor and behavioral activity. I 20. Diltiazem treatment produced marked c changes in the peak frequency of cyanide-in:$ 15duced tremor incidence and frequency (Fig. ? 6). Diltiazem, 600 pg/kg iv, administered 15 2 lom min before KCN ( 12 mg/kg), produced a sig: 5nificant decrease (p < 0.05) in the cyanideinduced tremor frequencies at all time periods. 0 C 30 60 90 120 150 160 Diltiazem did not completely block the tremTime tminl ors, but significantly delayed the onset, duration, and peak of the cyanide-induced tremors. FTC. 4. Peak tremor frequency measured in Hz at varying times following administration of KCN (12 mg/kg, SC). Diltiazem completely blocked the tremors Each point reflects the X + SE of 6 mice. Asterisks denote produced following KCN, 10 mg/kg. The peak a significant difference (p < 0.05) from saline-treated mice frequency 30 min after 10 mg/kg KCN was (control). 13.46 + 1.20 Hz and for mice pretreated with diltiazem, 1.14 k 0.65 Hz. Diltiazem alone increase in frequency, 4.53 +- 0.96 Hz, reflects produced no observable change in the behavior or motor activity of the mice. the approximate respiratory rate, 250-300 breaths/min, of the mice as they recovered from the cyanide. After 120- 180 min the mice DISCUSSION had frequency values equal to control and exploratory and grooming behavior returned to The primary biochemical lesion produced normal. Tremors were not detected following by cyanide is inhibition of cytochrome oxidase 3,5, or 7 mg/kg KCN (Fig. 5). However, KCN, (Schubert and Brill, 1968; Isom and Way, 10 and 12 mg/kg, produced tremors with fre- 1984) which results in histotoxic anoxia. Anquencies of 13.35 -+ 1.3 1 and 18.56 + 1.29, aerobic metabolism predominates in a cyarespectively, which were significantly greater nide-intoxicated cell producing a decrease in than control (p < 0.01). The 15-mg/kg dose the ATP/ADP ratio or energy charge (Isom et of KCN incapacitated the mice and approximately 50% died. These animals were severely 25 r intoxicated and exhibited depressed respira25
r
l
Ti
25 I
C
15
30 Tlme
FIG. 5. The effect of gradient doses of KCN on peak tremor frequency measured in Hz 30 min after cyanide administration. Each bar reflects the X Ifr SE of 6 mice. Asterisks denote a significant difference (p < 0.05) from saline-treated mice (control).
45
60
(mln)
FIG. 6. Peak frequency was measured on a time basis after administration of saline, iv, 15 mitt pretreatment, followed by KCN, 12 mgjkg, sc (0); diltiazem, 600 fig/ kg, iv, IS min pretreatment, followed by saline, sc (A); or diltiazem, 600 &kg, iv, followed by KCN, 12 mg/kg, sc (cl). Each point reflects the X +_ SE of 6 mice. Asterisks denote a significant difference (p < 0.05) between treatment groups at the same respective time point.
468
JOHNSON, MEISENHEIMER,
al., 1975). As a result of this effect, cyanide in sublethal doses can alter important energy-dependent processes including cellular calcium homeostasis. Calcium plays a key role as an intracellular regulator and in the central nervous system small changes in calcium homeostasis can produce marked physiological alterations. For instance, the release of neurotransmitters is a calcium-dependent process (Miledi, 1973; Llinas and Heuser, 1977; Henkart, 1980a,b). Recently, Persson et al. ( 1985) observed that cyanide-induced convulsions were concomitant with changes in regional brain neurotransmitter levels. These observations appear to have important implications with regard to the neurotoxicity associated with acute cyanide intoxication. In the present study, cyanide-induced rise in brain calcium levels corresponded to the induction of tremors. The role of calcium in mediating the cyanide-induced tremors is supported by the observation that diltiazem pretreatment blocked both the generation of tremors and the increases in whole-brain calcium. Diltiazem is a specific calcium channel blocker which can penetrate the blood-brain barrier and block voltage-dependent calcium channels in the plasma membrane and the Ca/ Na antiporter in the mitochondria (Matlib and Schwartz, 1983; White et al., 1983; Fitzpatrick and Karmazyn, 1984; Spedding and Cavero, 1984). Diltiazem would block calcium influx from extracellular pools and prevent accumulation of intracellular calcium. The effect of cyanide on whole-brain total calcium levels may be explained by activation or inactivation of various neuronal calcium homeostatic mechanisms. For instance, during the first 5 min after administration, cyanide decreased the whole-brain total calcium levels. Blaustein and Hodgkin ( 1968) demonstrated that cyanide-induced release of calcium from intracellular organelles results in activation of Na/Ca antiports in the plasma membrane of the squid axon. As a consequence, total cellular calcium levels dropped. The sudden increase in whole-brain total calcium after 5 min suggests that calcium accumulation is occurring. Since many of the neuronal processes
AND ISOM
which regulate intracellular calcium are energy dependent, the depletion of ATP would severely compromise the active transport systems in the plasma membrane and organelles (Brinley, 1980). The rise in whole-brain total calcium paralleled the onset of the tremors; both were initiated at 15 min after cyanide administration. However, the duration of the whole-brain total calcium elevation was longer than the duration of the tremors. It is unlikely that the duration of the two events would coincide since the neurogenic active calcium pool in the cytosol was not assessedby measuring the whole-brain total calcium levels. It is possible the cytosolic calcium pools returned to normal within 60 min and the calcium buffering capacity was restored to the cellular organelles. In support, l-3 hr after cyanide, the animals did not exhibit detectable tremors and respiration, behavioral and motor activity appeared normal. Both the alterations in brain calcium and tremor responses to cyanide exhibited a threshold effect. At the IO-mg/kg dose of cyanide, a significant rise in whole-brain total calcium levels and whole-body tremors were detected. Subtremor doses (0.5-7 mg/kg) were ineffective in altering the whole-brain total calcium concentrations. Even though the 15mg/kg dose of cyanide produced a significant elevation of whole-brain total calcium, the absence of tremors at this dose may be the result of the severely compromised state of the mice (over 50% died within l-2 hr after administration), depletion of neurotransmitters, neuronal fatigue, and/or neuronal hyperpolarization (Isenberg et al., 1983; Van der Hayden, 1985). Prolonged accumulation of free intraneuronal calcium could induce hyperpolarization by activating calcium-dependent potassium channels. The ensuing efflux of potassium would increase the membrane potential and decrease neuronal excitability. Such a mechanism would also explain the EEG “silence” typically observed following an acute cyanide intoxication (Burrows et al., 1973). The role of calcium as an intraneuronal mediator of cyanide intoxication, may have
CYANIDE-INDUCED
far-reaching implications with regard to the signs of intoxication and, possibly, chronic disease states attributed to cyanide. Presently, studies are being initiated to determine the mechanism of the observed effect of cyanide on whole-brain calcium.
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