Cyanide intoxication—I. An oral chronic animal model

Gen. Pharmac. Vol. 20, No. 3, pp. 323-327, 1989 Printed in Great Britain. All rights reserved

0306-3623/89 $3.00+ 0.00 Copyright © 1989 Pergamon Press pie

CYANIDE INTOXICATION--I. AN ORAL C H R O N I C ANIMAL MODEL ANA MARIA BUZALEH, ELBA SUSANAVAZQUEZ and ALCIRA M. DEL C. BATLLE* Centro de Investigaciones sobre Porfirinas y Portirias (CIPYP), CONICET, Facultad de Ciencias Exactas y Naturales, University of Buenos Aires, 1448 Buenos Aires, Argentina (Received 4 July 1988)

Almraet--The effects of oral chronic cyanide administration to mice were studied. Cyanide intoxication was confirmed by the increased levels of this poison and the concomitant inhibition of cytochrome oxidase activity in liver, brain, heart and blood. The detoxifying enzyme rhodanese was measured. The state of the sulfane sulfur pool was investigated by determination of the cyanide labile-sulfur levels. A clear correlation between rhodanese activity and sulfur content was obtained as a consequence of cyanide action. These results support the belief that rhodanese plays a fundamental role in the detoxification process of cyanide, in preventing cyanide reaching the target tissues.

INTRODUCTION Cyanide and cyanogenic compounds are ubiquitous in nature and so humans and animals are regularly exposed to environmental poisons, with universally known toxic properties. Most of the reports on cyanide are focused on the acute effects, while the chronic effects have been rather ignored. However, there is relevant epidemiological, therapeutic and experimental evidence, indicating that there are important consequences of chronic exposure to cyanide and cyanogens (Wilson, 1983) and a series of signs and symptoms, mostly neurological, can be ascribed to a chronic cyanide syndrome. Perhaps the reason for the scarcity of information on the chronic effects of cyanide intoxication is because most of this literature corresponds to clinical or field reports, making it difficult to attribute only to cyanide the cause of such lesions, due to the lack of suitable experimental controls. Nevertheless accumulating evidence is consistent with the hypothesis that chronic low cyanide exposure is a major source of the observed adverse effects in some diseases, as well as in some occupational, dietary and environmental conditions (Way, 1984). It should also be borne in mind that because of newer industrial advances, a greater exposure to cyanide in the future is expected. Cyanide is a potent, rapidly acting poison of respiration in biological systems and this effect is attributable to the binding and inactivation of cytochrome oxidase, the terminal component of the mitochondrial electron transport chain. However, Isom and Way (1976) reported that cyanide lethality could occur with no apparent inhibition of liver cytochrome oxidase activity. These results were explained by suggesting that brain cytochrome oxidase may be the actual site of lethal action of cyanide, on the basis of

*Address for correspondence: Professor Alcira Batlle, Viamonte 1881 10° "A", 1056--Buenos Aires, Argentina.

the tissue distribution of cyanide, thiosulfate and rhodanese. We should recall that the major route of biological cyanide detoxification is by conversion to its less toxic metabolite, thiocyanate, a reaction requiring a source of sulfane sulfur to react with cyanide (Westley et al., 1983). For many years i t has been believed that the liver mitochondrial rhodanese is the primary responsible enzyme for this biotransformation (Vennesland et al., 1982), although other minor pathways are known to contribute to its detoxification (Boxer and Rickards, 1952; Wood and Cooley, 1956). Thus in vivo sensitivity to cyanide is dependent not only on the affinity of cytochrome oxidase for cyanide but also on the reactivity and distribution of competing detoxification reactions (Solomonson, 1982). Although there are numerous reports on the in vitro inhibition of cytochrome oxidase by cyanide, there have been very few studies conducted at the/n vivo level (Albaum et al., 1946; Schubert and Brill, 1968). On the other hand, a good deal of experimental data reveals the importance of the species, route, dose and duration of cyanide administration in determining both the tissue susceptibility and the pathology to the cyanide effects. As already mentioned, it is important to study the many-fold chronic effects of cyanide intoxication employing a suitable experimental animal model. Except for the work of Ballantyne (1983) who established the influence of different species and routes on the cyanide concentration in various tissues and blood, no information exists on the effects of oral administration of cyanide on other biochemical parameters. On these grounds we have carried out experiments in mice to examine the effect of oral chronic cyanide administration on the activity of the detoxifying enzyme and the state of the sulfane-sulfur pool. To evaluate the validity of the animal model, cytoehrome oxidase activity and tissue cyanide levels were measured and to quantitate the degree of the

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detoxification response rhodanese activity, cyanide labile-sulfur and thiocyanate concentrations were determined.

Thiocyanate determinations The determination of thiocyanate is based on the red color developed by the thiocyanate ferric-ion complex (Himwich and Saunders, 1948).

MATERIALS AND METHODS

Enzymic assays Cytochrome oxidase activity was determined by the method of Yonetani and Ray (1965) and rhodenase activity by the method of S~rbo (1953).

All chemicals used were reagent grade.

Animals CF 1 mice weighing 25-30 g were maintained in controlled conditions and allowed free access to food (Purina 3) and water. Intoxications Freshly prepared potassium cyanide was administered to animals in drinking water (I g/ml) during 40 days. Tissue preparation Mice were initially heparinized and then under ether anaesthesia, they were killed by cardiac puncture and bled. Liver, brain and heart previously perfused were removed and immediately processed. 10% w/v crude homogenates were prepared by homogenizing the whole mouse organ in icecold 0.25 M sucrose for 10-15 sec in a glass vessel Teflon pestle-Potter-Elvehjeim homogenizer. The homogenates were centrifuged for 10min at 600g and the supernatant fractions were centrifuged for 5 min at 15,000g. The supernatants were used for rhodanese activity, cyanide, cyanide labile-sulfur and thiocyanate determinations. Cytochrome oxidase activity was determined in the mitochondria fractions. Blood was hemolized with Triton X-100 5% (1:5, v/v). Cyanide determination Tissue cyanide concentration was determined by the method of Guilbault and Kramer (1965). Cyanide labile-sulfur determination The measurement was oarried out according to the method of Koh (1965), slightly modified.

Protein concentrations This was determined by the method of Lowry et al. (195 I). Statistical analysis Student's t-test was used to assess the degree of significance. A probability level of 0.01-0.05 was used in testing for significant differences between experimental groups. RESULTS

Cyanide tissue levels Cyanide concentration was significantly increased (P < 0.01) in all tissues examined. This accumulation varied from tissue to tissue and with time; time dependence being more noticeable in blood (Fig. 1). Cytochrome oxidase activity Figure 2 shows that cytochrome oxidase activity was inhibited by about 50% (P < 0.05); the degree o f inhibition was correlated with the levels o f cyanide accumulated in each case. At longer times o f intoxication, lower inhibition was observed probably due to the reversible union between the cyanide ion and the protein. The dissociation of the cyanide-enzyme complex, in the

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Fig. 1. Cyanide tissue levels. (11) Liver, (1~) brain, (tim) heart, (IS]) blood. The data represent mean values _+ SD of 4 animals and are expressed relative to mean cyanide concentration of control animals. Mean control values (~g/ml): Liver: 7.54 + 2.23 (n = 7); brain: 7.28 _+2.09 (n = 6); heart: 5.56 _+ 1.05 (n = 5) and blood: 0.69_+0.12 (n = 12). n = number of animals. Experimental conditions arc given in the text.

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Fig. 2. Cytochrome oxidase activity in liver ( I ) , brain ([]) and heart (m). The data represent mean values + SD of 4 animals and are expressed relative to mean specific activity of control animals. Mean control values: Liver: 5,38+0.57 ( n - 6 ) ; brain: 1.05_+0.14 (n=6); heart: 65.26+6.94 (n=6). n = number of animals. Experimental conditions are given in the text. presence o f rhodanese would produce reactivation of cytochrome oxidase ( I s o m e t al., 1982).

Rhodanese activity Rhodanese activity was measured with and with-

out cyanide in the incubation system to evaluate the effect of tissue cyanide accumulation. Liver rhodanese (Fig. 3a), measured in the presence o f cyanide, diminished its activity reaching its minimum on the 24th day of intoxication; on the other hand

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Fig. 3. Rhodanese activity measured with (O) and without ( 0 ) the addition of cyanide to the incubation mixture. Each point represents the mean value-t-SD of 4 animals. (a) Liver, mean control value: 2.82 + 0.88 (n = 12) and 0.0123 + 0.0043 (n = 12), in the presence and in the absence of added cyanide respectively. (b) Brain, mean control value: 0.55 +0.13 (n =9) and 0.0053 +0.0013 (n = 11), in the presence and in the absence of added cyanide respectively. (c) Heart, mean control value: 0.52 + 0.10 (n = 12) and 0.0209 -+ 0.0024 (n - 13), in the presence and in the absence of added cyanide respectively. (d) Blood, mean control value: 0.054+0.006 (, = 12) and (5.40+0.44) x 10 -3 (n = 13), in the presence and in the absence of added cyanide respectively, n = number of animals. Experimental conditions are given in the text.

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w h e n activity was determined without adding with rapid urinary excretion of this metabolite, thus cyanide, about 50% enhancement (P R ¢ ¢ substituted form of the enzyme and therefore we m ..J v would not expect its activity to be modified by the 0,05 0.05 E E excess of cyanide detected in this tissue• This is the form of rhodanese which would actually detoxify the O E E:L cyanide. In conclusion, one of the earliest effects of cyanide seems to be an inhibition of the hepatic rhodanese I 0 . 0 0 _1 0.00 which is presumably due to either blockage by excess binding to the active site and/or depletion of the sulfane-sulfur pool. These changes do not seem to [ (b) -!4 occur in blood where rhodanese functions at its maximal rate, thus preventing cyanide reaching the target tissues and therefore exerting its lethal action• 0 O. 1 0

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Fig. 4. Cyanide labile sulfur tissue level• (O) Liver, (A) brain, ([]) heart, (O) blood• Each point represents the mean value + SD of 4 animals• Mean control values ~mol/ml): Liver: 0.060 + 0.008 (n = 15); brain: 0•026 + 0.009 (n = 16); heart: 0.079 -4-0.003 (n = 16); blood: 2•66 5:0.23 (n = 10). n = number of animals• Experimental conditions are given in the text.

Acknowledgements--A. M. del C. BatUe holds the post of Scientific Research in the Argentine National Research Council (CONICET); E. Vfizquez and A. M. Buzaleh are research fellows at the CONICET. This work was supported by grants from the CONICET, the SECYT, SecretafiA de Salud Pfblica del Ministerio de Bienestar Social, UBA and Banco de la N~ci6n Argentina. We wish to express our gratitude to Lic. S. Afonso for the excellent drawings, and to Laboratios Promeco, B. Aires for providing the animals. REFERENCES

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