Protection against cyanide-induced convulsions with α-ketoglutarate

Toxicology, 61 (1990) 221--228 Elsevier Scientific Publishers Ireland Ltd.

Protection

against cyanide-induced with a-ketoglutarate

convulsions

Hiro-Aki Yamamoto Department of Environmental Medicine, Institute of Community Medicine, The university of Tsukuba, Tsukuba, Ibaraki 305 (Japan) (Received October 31st, 1989; accepted December 13th, 1989)

Summary Protection against convulsions induced by cyanide was observed after treatment with a-ketoglutarate, either alone or in combination with sodium thiosulfate, a classical antagonist for cyanide intoxication. However, sodium thiosulfate alone did not protect against cyanide (30 mg/kg)-induced convulsions. ),-Aminobutyric acid (GABA) levels in brain were decreased by 31% in KCN-treated mice exhibiting convulsions. The combined administration of a-ketoglutarate and sodium thiosulfate completely abolished the decrease of GABA levels induced by cyanide. Furthermore, sodium thiosulfate alone also completely abolished the decrease of GABA levels. These results suggest that the depletion of brain GABA levels may not directly contribute to the development of convulsions induced by cyanide. On the other hand, cyanide increased calcium levels by 32% in brain crude mitochondrial fractions in mice with convulsions. The increased calcium levels were completely abolished by the combined administration of a-ketoglutarate and sodium thiosulfate, but not affected by sodium thiosulfate alone. These findings support the hypothesis proposed by Johnson et al. (Toxicol. Appl. Pharmacol., 84 (1986) 464) and Robinson et al. (Toxicology, 35 (1985) 59) that calcium may play an important role in mediating cyanide neurotoxicity. Key words." Convulsions; Cyanide; a-Ketoglutarate; Calcium; GABA; Brain

Introduction A c u t e p o i s o n i n g with c y a n i d e p r o d u c e s several clinical signs in m a n an d animals. C o n v u l s i o n s are k n o w n as o n e o f these signs [ 3 - - 5 ] . C o n v u l s i o n s c o u l d be direct or indirect m a n i f e s t a t i o n s o f c y a n id e toxicity in the central n e r v o u s system. P e r s s o n et al. [6] o b s e r v e d that c y a n i d e - i n d u c e d c o n v u l s i o n s o c c u r r e d c o n c o m i tantly with changes in r e g i o n a l b r a in (cerebellum, f r o n t a l c o r t e x a n d h i p p o c a m pus) n e u r o t r a n s m i t t e r s such as G A B A . T h e ch an g es in G A B A in b r ai n were in a g r e e m e n t with earlier o b s e r v a t i o n s in the brains o f c y a n i d e - t r e a t e d a n i m a l s [7]. Recently, S c h w a r t z et al. [8] h a v e r e p o r t e d that p y r u v a t e p r o t e c t e d against the i n d u c t i o n o f c o n v u l s i o n s i n d u c e d by c y a n i d e an d the lethal effects o f cy an i d e in 0300-483X/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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mice. Furthermore, Moore et al. [9] and Yamamoto [10] have found that a-ketoglutarate protects against the lethal effects of cyanide. Green and Williamson [1 l] ascertained that pyruvate and, a-ketoglutarate, reacts with cyanide in vitro to form cyanohidrins. In our preliminary studies, we found that a-ketoglutarate also prevented the induction of convulsions induced by cyanide. Therefore, in this experiment, we tested whether or not there is a relationship between GABA levels in brain and cyanide-induced convulsions with or without a-ketoglutarate. Recently, a number of reports have suggested that calcium may play an important role as a cellular mediator of cyanide neurotoxicity [1,2]. We also tested the relationship between brain calcium contents and cyanide-induced convulsions with and without a-ketoglutarate, an inhibitor of cyanide-induced convulsions. Materials and methods

Potassium cyanide, a-ketoglutarate and sodium thiosulfate were obtained from Wako Pure Chemical Industries, Ltd., Japan. Sodium nitrite was obtained from Nacalai Chemical Industries, Ltd., Japan. Solutions of potassium cyanide, sodium nitrite, sodium thiosulfate, and a-ketoglutarate were prepared in 0.9% NaC1 immediately before use. Male ddy mice (body wt 14--18 g ) were purchased from Ishikawa Pref. Animal Laboratories (Ishikawa, Japan). The animals were housed in a temperature(24 _.+ 1 °C) and light- (12 h dark/light) controlled room. They were given a diet of standard laboratory chow (oriental kobo) and water ad libitum. Mice received a subcutaneous injection of KCN a n d / o r an intraperitoneal injection of a-ketoglutarate, sodium thiosulfate a n d / o r sodium nitrite. The median lethal dose was determined for potassium cyanide either alone or in combination with a-ketoglutarate, sodium thiosulfate a n d / o r sodium nitrite. The experimental values were obtained for three or more groups containing 6--12 mice in each group. LDs0 values based on 24-h mortality were calculated by the method of Litchifield-Wilcoxon [12] with confidence limits of 95% probability.

Assay of GABA in brain Mice were killed by decapitation and the brains were homogenized in a 10-fold volume of ice-cold distilled water immediately after convulsions (tonic seizures and tremors) or 5 min after treatment with saline or the combination of a-ketoglutarate and cyanide. The homogenate was treated with a 9-fold volume of 10% trichloroacetic acid and centrifuged at 1000 x g for 15 min. The supernatant was filtered through Millipore H A W P (0.45 /am pore ) filters. The supernatant was used for the GABA assay by H P L C Twincle System (Jasco, Japan) for highperformance liquid chromatography (HPLC) in a lithium buffer system.

Preparations of crude mitochondrial fraction (P2) Whole brain homogenate (10 vol. of 0.32 M sucrose, 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acids (Hepes) pH 7.5) in mice with and without convulsions were fractionated by a modification of the methods described by Cotman and Matthews [13] and Gurd et al. [14]. The nuclear pellet and the crude

222

mitochondrial fraction were spun down at 1085 X g (5 rain ) and 17 300 X g (10 min), respectively, and the crude mitochondrial fraction, including synaptosomes (P2), was washed three times with 0.32 M sucrose (pH 7.5, 5 mM Hepes buffer), and was resuspended in distilled water for calcium assays.

Determination o f calcium content in crude mitochondrial fractions The calcium content of the homogenate of crude mitochondria was determined by a modification of the method described by Bradbury et al. [15]. One milliliter of the 10% homogenate was mixed with 1 ml of 1 N HNO3, incubated for 24 h at 37°C and centrifuged for 15 min at 3000 × g. Lanthanium chloride (1.5°70) in 0.5 N HN03 (0.5 ml) was added to 1 ml supernatant, and the calcium was measured by atomic absorption flame spectrophotometry.

Assay o f protein The method of Lowry et al. [16] was used to determine the protein content of each crude mitochondrial fraction.

Statistical analysis All data obtained on G A B A and calcium levels were compared by the analysis of variance. When the analysis indicated that a significant difference existed, the means of selected groups were compared by a Student's t-test.

TABLE I EFFECT OF a - K E T O G L U T A R A T E , S O D I U M T H I O S U L F A T E , A N D SODIUM NITRITE ON T H E L E T H A L EFFECTS OF C Y A N I D E IN MICE Treatments (g/kg)

KCN: LDs0 (95% confidence)

PR*

Saline a-Ketoglutarate (0.5) Sodium thiosulfate (1.0) Sodium nitrite (0.1) a-Ketoglutarate (0.5) + Sodium thiosulfate (1.0) a-Ketoglutarate (0.5) + Sodium nitrite (0.1) Sodium thiosulfate (1.0) + Sodium nitrite (0.1) a-Ketoglutarate (0.5) + Sodium thiosulfate (1.0) + Sodium nitrite (0.1)

9.9 19.0 21.0 20.0

(7.9--10.3) (15.5--22.8) (19.4--25.4) (19.4--21.1)

1.0 2.1 2.3 2.2

42.0 (30.1--58.6)

4.7

19.0 0 7 . 4 - - 2 0 . 7 )

2.1

42.0 (34.4--51.2)

4.7

41.0 (32.1--50.2)

4.6

*LDs0 KCN + Antagonist(s)/LDs0 KCN. Each LDs0 value was obtained from three or more graded doses of KCN administered to three or more groups of mice with 6 - - 1 2 mice/group, a-Ketoglutarate, sodium thiosulfate, sodium nitrite a n d / or saline was administered intraperitoneally at the same time to mice treated with subcutaneous injections of KCN.

223

Results

Effect o f various antagonists on the lethal effects o f cyanide in mice The effects o f a - k e t o g l u t a r a t e a d m i n i s t e r e d either alone or in c o m b i n a t i o n with s o d i u m thiosulfate a n d / o r s o d i u m nitrite in a n t a g o n i z i n g the lethal effects o f p o t a s s i u m cyanide are s h o w n in T a b l e I. The LDs0 value o f p o t a s s i u m cyanide alone was similar to a previously reported value [8,10,17]. C o t r e a t m e n t with cyanide a n d a - k e t o g l u t a r a t e at the same time increased the LDs0 2-fold in mice, as it did in o u r previous report [10] a l t h o u g h M o o r e et al. [9] have reported that p r e t r e a t m e n t with a - k e t o g l u t a r a t e 10 m i n before cyanide i n j e c t i o n increased the LDs0 5-fold in mice. O n the other h a n d , a l t h o u g h the protective effect o f s o d i u m nitrite was n o t e n h a n c e d b y a - k e t o g l u t a r a t e , the effect o f s o d i u m thiosulfate was t r e m e n d o u s l y e n h a n c e d by a - k e t o g l u t a r a t e . This result is similar to a report described b y M o o r e et al. [9]. The c o m b i n e d a d m i n i s t r a t i o n o f a - k e t o g l u t a r a t e a n d s o d i u m thiosulfate was as efficacious as the a d m i n i s t r a t i o n of the classical a n t i d o t a l c o m b i n a t i o n o f s o d i u m thiosulfate a n d s o d i u m nitrite. However, the a d d i t i o n of a - k e t o g l u t a r a t e to the classical a n t i d o t a l c o m b i n a t i o n o f s o d i u m thiosulfate a n d s o d i u m nitrite p r o v i d e d n o a d d i t i o n a l protective effect.

TABLE II EFFECT OF a-KETOGLUTARATE, SODIUM THIOSULFATE AND SODIUM NITRITE ON CYANIDE-INDUCED CONVULSIONS Treatments (g/kg)

N

KCN (20 mg/kg) 070 of convulsed mice

N

KCN (30 mg/kg) % of convulsed mice

Saline Sodium thiosulfate (1.0) Sodium nitrite (0.1) a-Ketoglutarate (0.5) Sodium thiosulfate (1.0) + Sodium nitrite (0.1) a-Ketoglutarate (0.5) + Sodium thiosulfate (1.0) a-Ketoglutarate (0.5) + Sodium nitrite (0.1)

6

100

6

100

8

75

6

100

6

100

6

100 80

6

32*

5

6

17"

12

58**

6

0*

12

17"*

6

32*

6

83

a-Ketoglutarate, sodium thiosulfate, sodium nitrite and/or saline was administered intraperitoneally at the same time to mice treated with subcutaneous injection of KCN. Significant decreased from saline + cyanide (20 mg/kg or 30 mg/kg)-treated mice (*P < 0.01 or **P < 0.01).

224

Effect o f a-ketoglutarate, sodium thiosulfate and sodium nitrite on convulsions induced by cyanide Subcutaneous injections of potassium cyanide (20 or 30 m g / k g ) caused convulsions (tonic seizures and tremors) in all of the treated mice. Sodium thiosulfate or sodium nitrite alone did not prevent the cyanide (30 mg/kg)-induced convulsions. However, a-ketoglutarate alone prevented the convulsions in 68% or 2007o of the cyanide (20 m g / k g or 30 mg/kg)-treated mice. The combination of sodium thiosulfate and sodium nitrite also prevented the incidence of cyanideinduced convulsions. Furthermore, when it was combined with sodium thiosulfate and a-ketoglutarate, preventive effect against cyanide-induced convulsions was further potentiated. The combined administration of a-ketoglutarate and sodium thiosulfate was superior to the classic antidotal combination of sodium thiosulfate and sodium nitrite.

Cyanide-induced convulsions and brain GABA levels Potassium cyanide (10 mg/kg) administration elicited a significant decrease in the G A B A content of brains of mice with convulsions (Table III). The combined administration of a-ketoglutarate and sodium thiosulfate completely abolished the decrease of brain G A B A levels observed in the cyanide-treated mice, and also blocked the induction of convulsions induced by cyanide. However, although administration of sodium thiosulfate alone did not prevent the convulsions induced by injection of potassium cyanide (30 mg/kg), it completely abolished the decrease of G A B A levels in brain induced by cyanide.

Cyanide-induced convulsions and calcium levels in brain crude mitochondrial fractions including synaptosomes Administration of potassium cyanide (10 mg/kg) increased by 32% the cal-

TABLE III CYANIDE-INDUCEDCONVULSIONSAND BRAIN GABA CONTENT IN MICE Treatments

°7oof convulsed mice

GABA contents (nmol/g wet brain)

Saline a-Ketoglutarate (0.5 g/kg) Sodium thiosulfate (1.0 g/kg) KCN (10 mg/kg) KCN (30 mg/kg) + Sodium thiosulfate (1.0 g/kg) KCN (30 mg/kg) + a-Ketoglutarate (0.5 g/kg) + Sodium thiosulfate (1.0 g/kg)

0 0 0 100

3397.2 3385.5 3402.4 2357.7

100

3316.2 ± 74.49**

17

± ± ± ±

3443.1 ±

106.93 84.58 89.42 84.64*

15.40"*

*Significantly decreased from saline treated mice (P < 0.001). **Significantly increased from KCN (10 mg/kg)-treated mice (P < 0.001). Data are represented the mean ± S.E.M. (N = 6--12).

225

TABLE IV EFFECT OF CYANIDE, a-KETOGLUTARATE AND SODIUM THIOSULFATE ON CALCIUM CONTENTS OF CRUDE MITOCHONDRIAL FRACTIONS INCLUDING SYNAPTOSOMES IN MICE BRAIN Treatments

N

Calcium contents (nmol/mg protein)

Convulsed mice (%)

Saline a-Ketoglutarate (0.5 g/kg) Sodium thiosulfate (1.0 g/kg) KCN (10 mg/kg) KCN (30 mg/kg) + Sodium thiosulfate (1.0 g/kg) KCN (30 mg/kg) + a-Ketoglutarate (0.5 g/kg) + Sodium thiosulfate (1.0 g/kg)

18 18 18 18

4.51 4.57 4.54 5.95

0.175 0.182 0.170 0.185"

0 0 0 100

18

5.58 __. 0.165"

100

18

4.63 ± 0.256**

11

± _ ± ±

*Significantly increased from saline-treated mice (P < 0.01). **Significantly decreased from KCN-treated mice (P < 0.01). Data are represented the mean ± S.E.M.

c i u m c o n t e n t s in c r u d e m i t o c h o n d r i a l f r a c t i o n s i n c l u d i n g s y n a p t o s o m e s in the b r a i n s o f mice with c o n v u l s i o n s ( T a b l e IV). T h e c o m b i n e d a d m i n i s t r a t i o n o f ak e t o g l u t a r a t e a n d s o d i u m t h i o s u l f a t e c o m p l e t e l y a b o l i s h e d the increase o f b r a i n c r u d e m i t o c h o n d r i a l c a l c i u m c o n t e n t in mice a n d b l o c k e d the c o n v u l s i o n s , while a d m i n i s t r a t i o n o f s o d i u m t h i o s u l f a t e a l o n e d i d n o t p r e v e n t c y a n i d e - i n d u c e d convulsions a n d d i d n o t a b o l i s h t h e increase o f b r a i n c r u d e m i t o c h o n d r i a l calcium levels. Discussion

a - K e t o acids such as p y r u v a t e a n d a - k e t o g l u t a r a t e have been s h o w n to a n t a g o nize the lethal effects o f c y a n i d e [8--10,18,19] a n d to p r e v e n t the incidence o f c y a n i d e - i n d u c e d c o n v u l s i o n s [8] b u t m o r e d e t a i l e d studies have n o t been cond u c t e d . T h e p r e s e n t s t u d y d e m o n s t r a t e d t h a t a - k e t o g l u t a r a t e a n t a g o n i z e d the lethal effects o f c y a n i d e a n d p r o t e c t e d a g a i n s t the i n d u c t i o n o f c o n v u l s i o n s . The effects o f a - k e t o g l u t a r a t e were f u r t h e r p o t e n t i a t e d in c o m b i n a t i o n with s o d i u m t h i o s u l f a t e b u t n o t with s o d i u m nitrite. T h e a b i l i t y o f the c o m b i n e d a d m i n i s t r a t i o n o f a - k e t o g l u t a r a t e a n d s o d i u m t h i o s u l f a t e to a n t a g o n i z e the lethal effects o f c y a n i d e was as e f f i c a c i o u s as t h e classic a n t i d o t a l s o d i u m t h i o s u l f a t e - s o d i u m nitrite c o m b i n a t i o n . H o w e v e r , the a b i l i t y o f the c o m b i n e d a d m i n i s t r a t i o n o f ak e t o g l u t a r a t e a n d s o d i u m t h i o s u l f a t e to p r o t e c t a g a i n s t c o n v u l s i o n s i n d u c e d b y c y a n i d e was m o r e efficacious t h a n t h a t o f the classical a n t i d o t a l c o m b i n a t i o n . S c h w a r t z et al. [8] h a v e r e p o r t e d t h a t p y r u v a t e does p o t e n t i a t e the a n t i d o t a l effect o f s o d i u m t h i o s u l f a t e a n d d o e s p r o v i d e p r o t e c t i o n a g a i n s t c y a n i d e - i n d u c e d convulsions. Since it has been suggested t h a t the a n t a g o n i s t i c effects o f p y r u v a t e m a y be a t t r i b u t e d to its r e a c t i o n with c y a n i d e , which leads to the f o r m a t i o n o f

226

the n o n - t o x i c p y r u v i c - c y a n h y d r i n [8,18,19], a m e c h a n i s m o f a n t a g o n i s t i c effects o f a - k e t o g l u t a r a t e to c y a n i d e t o x i c i t y m a y be similar to t h a t o f p y r u v a t e . C y a n i d e (10 m g / k g ) s i g n i f i c a n t l y d e c r e a s e d b r a i n G A B A levels in mice with c o n v u l s i o n s as earlier r e p o r t e d [6,7]. T h e d e c r e a s e d G A B A levels p r o d u c e d b y c y a n i d e (30 m g / k g ) was c o m p l e t e l y a b o l i s h e d b y the c o m b i n e d a d m i n i s t r a t i o n o f a - k e t o g l u t a r a t e a n d s o d i u m t h i o s u l f a t e in mice. T h e t r e a t m e n t s also p r o t e c t e d a g a i n s t the c y a n i d e - i n d u c e d c o n v u l s i o n s . H o w e v e r , a l t h o u g h s o d i u m t h i o s u l f a t e a l o n e d i d n o t p r e v e n t c o n v u l s i o n s i n d u c e d b y i n j e c t i o n o f c y a n i d e (30 m g / k g ) , it c o m p l e t e l y a b o l i s h e d the d e c r e a s e d G A B A levels in b r a i n . These results suggested t h a t ' a d e c r e a s e o f b r a i n G A B A levels m a y n o t d i r e c t l y c o n t r i b u t e to the d e v e l o p m e n t o f c o n v u l s i o n s i n d u c e d b y c y a n i d e . M e a n w h i l e , as s h o w n in T a b l e IV, c y a n i d e elicited a n increase o f c a l c i u m levels in b r a i n c r u d e m i t o c h o n d r i a l fractions i n c l u d i n g s y n a p t o s o m e s . T h e i n c r e a s e d c a l c i u m levels were c o m p l e t e l y a b o l ished b y the c o m b i n e d a d m i n i s t r a t i o n o f a - k e t o g l u t a r a t e a n d s o d i u m t h i o s u l f a t e b u t n o t b y s o d i u m t h i o s u l f a t e a l o n e . T h e s e f i n d i n g s s u p p o r t the h y p o t h e s i s p r o p o s e d b y J o h n s o n et al. [1] a n d R o b i n s o n et al. [2], t h a t i n t r a n e u r o n a l c a l c i u m m a y p l a y a n i m p o r t a n t role in m e d i a t i n g c y a n i d e n e u r o t o x i c i t y . H o w e v e r , since P e r s s o n et al. [6] h a v e r e p o r t e d t h a t c e r e b e l l a r cyclic G M P levels m a y p l a y a n i m p o r t a n t role in t h e i n d u c t i o n o f c o n v u l s i o n s i n d u c e d b y c y a n i d e , it is considered t h a t f u r t h e r studies o n the r e l a t i o n s h i p s a m o n g b r a i n r e g i o n a l c a l c i u m c o n t e n t s , cyclic G M P levels, a n d c y a n i d e - i n d u c e d c o n v u l s i o n s are necessary to e l u c i d a t e the m e c h a n i s m s at w o r k .

Acknowledgments This r e s e a r c h was s u p p o r t e d in p a r t b y the research a i d f r o m T h e U n i v e r s i t y of Tsukuba Project Research.

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