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Excretion of 14C-labeled cyanide in rats exposed to chronic intake of potassium cyanide
TOXlCOLOCY
AND
APPLIED
PHARMACOLOGY
70, 335-339 (1983)
SHORT COMMUNICATION Excretion of “C-labeled Cyanide in Rats Exposed to Chronic Intake of Potassium Cyanide Excretion of “C-labeled Cyanide in Rats Exposed to Chronic Intake of Potassium Cyanide. P. N. (1983). Toxicol. Appl. Pharmocol. 70, 335-339. The excretion of an acute dose of “C-labeled cyanide in urine, feces,and expired air was studied in rats exposed to daily intake of unlabeled KCN in the diet for 6 weeks. Urinary excretion was the main route of elimination of cyanide carbon in these rats, accounting for 83% of the total excreted radioactivity in 12 hr and 89% of the total excreted radioactivity in 24 hr. The major excretion metabolite of cyanide in urine was thiocyanate, and this metabolite accounted for 71 and 79% of the total urinary activity in 12 hr and 24 hr, respectively. The mean total activity excreted in expired air after 12 hr was only 4%, and this value did not change after 24 hr. Of the total activity in expired air in 24 hr, 90% was present as carbon dioxide and 9% as cyanide. When these results were compared with those observed for control rats, it was clear that the mode of elimination of cyanide carbon in both urine and breath was not altered by the chronic intake of cyanide. OKOH,
Several routes of metabolism of cyanide have been described; these routes have been reviewed by Williams (1959) Oke (1969), and Baumeister et al. (1975). The best known mechanism for detoxification of cyanide is the conversion of cyanide to the relatively nontoxic thiocyanate which is excreted largely in the urine (Boxer and Rickards, 1952). Another route is the reaction with cystine to form 2iminothiazolidine-4-carboxylic acid (Wood and Cooley, 1956). Following injection of Na14CN in dogs and rats, r4C appears in urine as free cyanide, as a constituent of cyanocobalamin, and in thiocyanate. A part of the cyanide and thiocyanate is oxidized directly to form carbon dioxide (Boxer and Rickards, 1952). The chronic intake of cyanide has been implicated in the pathogenesis of a number of diseases. Although it has been suggested that a diminution of cyanide metabolism is probably responsible (Baumeister et al., 1975), the exact cause of the clinical state has not been established. Smith and Foulkes ( 1966) claimed that urinary excretion of thiocyanate is decreased in the rat following chronic administration of cyanide. These workers suggested that the main excretory pathway for cyanide excretion was affected by chronic but unphys335
iologic cyanide stimulation. They concluded that some unknown degradation product of cyanide contributed to the observed clinical state in chronic cyanide intoxication. Mehta and McGinity (1977) failed to confirm this observation of altered cyanide metabolism made by Smith and Foulkes (1966) by the same chemical technique. Confirmation of any change in the metabolism of cyanide as a result of chronic intake will be of value in understanding the nature of chronic cyanide toxicity in humans. By studying the pattern of excretion of 14Clabeled cyanide in rats chronically exposed to cyanide, it was hoped that any effect of chronic intake of cyanide on the urinary elimination of cyanide carbon would be readily observed by the more sensitive radiochemical technique. The effect of chronic intake of cyanide on the respiratory excretion of cyanide carbon was also studied. METHODS Twenty male albino rats (from the stock colony of the Department of Biochemistry, University of Liverpool, England) weighing about 100 g each were randomly assigned to individual cages and divided into two groups of 10 rats each. They were maintained on a semisynthetic diet containing extracted soyabean meal as the main protein source (Okoh, 1978; Okoh and Pitt, 1982). Potassium 0041-008X/83
$3.00
Copyright 0 1983 by Academic Press Inc. All rights of reproductmn in any form reserved.
336
SHORT COMMUNICATION
cyanide (unlabeled) was added to the diet (mixed with 5% arachis oil) of each rat in one group (cyanide rats) to provide 77 pmole cyanide per rat per day for six weeks. The cyanide was added to 10 g of diet which was given to each rat in the evening. Ten g was about the amount of diet each rat consumed overnight. By the following morning, each rat was given the soyabean diet without cyanide for the rest of the day. Potassium cyanide is stable in the soyabean meal diet when mixed with 5% arachis oil (Okoh, 1978; Okoh and Pitt, 1982). Also the mixing of the diet with arachis oil reduced spilling by the rats. The rats in the second group (control rats) were fed similarly except that no potassium cyanide was added to their diet. At the end of 6 weeks, each rat was given two doses of 8.3 rmol ‘%N- with a 30-min interval by sc injection. The specific activity of the injected material was 1.15 rCi/ rmol. Following injection, each rat was placed in a metabolism unit which allowed for collection of respiratory activity in NaOH absorbers, feces an a fine screen, and urine in the bottom of a dessicator (Boxer and Rickards, 1952). At the end of each study period, any urine in the bladder was forced out of each rat into a stainless-steel tray by gripping the rat firmly at the back of the neck with the thumb and forefingers and then stretching the animal by a slight pull at the tail. The used NaOH absorbers and the collected feces and urine were stored separately at -20°C. To achieve quantitative separation of carbon dioxide and cyanide in the urine and the NaOH absorbers, the following procedure described by Boxer and Rickards (1952) was used. A known volume of the sample was put into a tube with a rubber stopper equipped with gas inlet and outlet tubes. Silicon antifoaming agent (1 ml 8% solution) was added, and the mixture was acidified to pH 3 to 4 by addition of 0.5 M H$O,. Four traps were connected in series with the first and the second containing 6 ml 0.02 M Ag2S04 in 0.05 M HzS04 each, while the third and fourth contained 5 ml 0.1 M NaOH each. Cyanide and carbon dioxide were transferred over a period of 45 min by aerating with oxygen-free nitrogen at a flow rate of between 700 and 800 ml/min. The silver sulfate traps removed all the cyanide while the carbon dioxide liberated was trapped in the NaOH. In the urine samples, thiocyanate left in the mixture after removal of cyanide and carbon dioxide was oxidized to cyanide by the addition of I ml 0.01 M potassium permanganate; oxidation was usually completed in 3 min. Stannous chloride (1 ml 2% solution) was added to reduce excess permanganate, and the cyanide formed was aerated into two tubes containing 5 ml 0.1 M NaOH each by aerating with nitrogen as previously described. Samples were prepared for counting in a toluene scintillator fluid mixed with NCS solubilizer (New England Nuclear, Boston). The amount of radioactivity in each sample was determined in a liquid scintillation counter (Model SL 40 Intertechnique Ltd., Bringhton, England). Appropriate corrections were made for quenching by the
external standard method. Data were subjected to analysis of variance by the least square method with a digital computer.
RESULTS
AND
DISCUSSION
Rats on the KCN diet ate adequate diet and grew like the control rats fed a similar diet without KCN. Furthermore, the addition of KCN (77 pmol/rat/day) to the diet of the rats resulted in increased thiocyanate excretion in the urine. The mean thiocyanate content of 24-hr urine collected from four rats just before the addition of KCN to the diet was 1.69 -+ 0.19 pmol SE. By contrast, the first 24-hr urine collection from the same rats following the addition of KCN to the basal diet was 30.80 + 2.57 pmol SE. Table 1 shows the result of the elimination of 14C activity in urine, expired air, and feces after injection of the Na14CN to normal rats and those exposed to chronic intake of cyanide in the diet (cyanide rats). Urine was the main route of excretion of radioactivity in all the rats, accounting for over 80% of the total excreted radioactivity in 12 hr and over 85% of the total excreted radioactivity in 24 hr. Moreover, the cumulative 24-hr urinary excretion of radioactivity in both control and cyanide rats was significantly higher (p c 0.05) than the total excretion in 12 hr. By contrast, the small increase in the radioactivity excreted in expired air in 24 hr compared with the 12hr period was not statistically significant in either control or cyanide rats. The fecal radioactivity was highly variable (C.V. 94.0% Table 1). This result was due to the large difference in the amount of feces collected from individual rats. However, the radioactivity excreted in feces by the cyanide rats was higher than that excreted by control rats at the same period, but this finding was not statistically significant. Okoh and Pitt (Okoh, 1978; Okoh and Pitt, 1982) have shown that a large amount of radioactive cyanide injected into rats was largely secreted into the stomach contents as thiocyanate but reabsorbed into the body fluid in the lower part of the gut. The higher amount of radioactivity in the feces of
337
SHORT COMMUNICATION TABLE CUMULATIVE
AMOUNT
OF 14C IN URINE,
KCN in diet (woVrac/day)
Time after injection (W 12 12 24 24
0.0
77.0 0.0
77.0 SE cv
EXPIRED
(%)
1
AIR, AND FECES AITER INJECTION
OF Na’%ZN
TO RATS
Radioactivity as % of dose injected”’ Urine
Expired air
24.31' f 1.32 23.91' + 1.48
4.16=-c 0.18 4.32' + 0.49 4.53=+ 0.45 4.90' -+ 0.43 0.41 20.0
57.28*
f 3.40
51.72* f 2.45 2.32 12.0
FeOeS 0.37' 0.48' 1.15"* 1.70* 0.39 94.0
* f -+ f
0.25 0.45 0.24 0.54
a Each value is X k SE for five rats. ’ Means in the same column followed by different letters differ significantly at p G 0.05.
cyanide rats may therefore be due to a decrease in the efficiency of reabsorption of cyanide carbon in the lower region of the gut. Regardless of this small increase in the fecal r4C of cyanide rats, it is clear from Table 1 that feces are not a major route of excretion of cyanide carbon by either group of rats. The quantitative distribution of the injected cyanide carbon in the various fractions of the urine is illustrated in Table 2. The urinary cyanide includes Bi2 cyanide, while the CO* fraction includes carbonate and bicarbonate. Neither cyanide nor carbon dioxide plays a major role as an excretory pathway. In each group of rats, the largest urinary activity was the thiocyanate fraction, and the amount in 24 hr was significantly higher (p -L 0.05) than
in the 12-hr collection. This increase in the proportion of thiocyanate in urine with time is likely associated with increase in the amount of this radical formed from cyanide by the tissue rhodanese enzyme (Lang, 1933). The proportion of urinary radioactivity in cyanide and carbon dioxide fractions did not significantly change with time in either control or cyanide rats. The distribution of respiratory activity between cyanide and carbon dioxide fraction is given in Table 3. Of the total activity in expired air in 24 hr, 90% Was present as carbon dioxide and only 9% as cyanide. This distribution is not significantly different from that observed for control rats, not on chronic cyanide intake. However, in both groups of rats, the propor-
TABLE 2 CUMULATIVE
DISTRIBUTION
KCN in diet (rmol/rat/day) 0.0 77.0 0.0 77.0 SE cv (o/o)
OF RADIOACTIVITY
Time after injection (hd 12 12 24 24
IN VARIOUS
URINE
FRACTIONS
AFTER INJECTION
OF Na’%N
Radioactivity as % of activity in Urinea~b SCN-
CN-
69.50' +- 1.74 70.86' k 1.85 80.28*~ 2.87 78.80* zk 1.97 2.16 6.0
1.08' -+ 0.20 1.13c + 0.15 1.41'? 0.10 1.31'2 0.13 0.14 26.0
u Each value is X _t SE for five rats. b Means in the same column followed by different letters differ significantly at p < 0.05.
co2 5.10' + 5.34' f 5.51=_+ 6.15'+ 0.37 15.0
0.54 0.34 0.31 0.27
338
SHORT COMMUNICATION TABLE 3 CUMULATIVE
DISTRIBUTION OF RADIOA~TIVW IN VARIOUS FFMXIONS OF THE EXPIRED AIR AFTER INJECI-ION OF Na?ZN
Radioactivity as % of total activity in expired aiFb
Time after injection (hd
KCN in diet bmobat/W) 0.0 77.0 0.0 77.0 SE cv (%b)
CN-
co2
12 12 24 24
85.59’ + 0.12 85.69’ f 1.52 91.15’f 0.93 89.55’ + 1.88 1.34 3.0
13.05’ f 14.12’-c 9.58d f 9.44d f 0.96 18.0
0.41 1.71 0.64 0.41
’ Each value X f SE for 5 rats. b Means in the same column followed by different letters differ significantly at p G 0.05.
tion of cyanide in expired air significantly (p < 0.05) decreased in 24 hr compared with the 12-hr collection. This result is because the increase in activity in the expired air at 24 hr compared with the 12-hr period (Table 1) is largely associated with an increase in the carbon dioxide fraction and not in cyanide. The rate of excretion of radioactivity in the urine and expired air was studied in four groups of rats previously exposed to daily cyanide intake in the diet for 3 weeks. The elimination of the 14C activity in the urine and expired air up to 1 hr after sc injection of Na14CN is summarized in Table 4. No radioactivity was detected in the urine of rats 10 min after the injection of the Na14CN. However, urine collected by 20 min contained de-
tectable activity in all rats. In contrast, excretion of radioactivity by the expired air was detectable 10 min after injection of the dose (Table 4). The urinary excretion of radioactivity was more variable than respiratory excretion. When urinary cyanide and thiocyanate were separated from the total urinary excretion, it was observed that the latter radical was the major excretory component at each period (20, 30 and 60 min). Excretion of cyanide carbon has been examined in a number of animal species not exposed to chronic cyanide intake (Boxer and Rickards, 1952; Crawley and Goddard, 1977). One such study in rats (Crawley and Goddard, 1977) showed that the amount of cyanide carbon excreted in 24 hr was 54% of the injected
TABLE 4 CUMULATIVE
RATE OF APPEARANCE OF RADIOACWITY IN EXPIRED AIR, URINARY AND THKXYANATE AVER INJECTION OF Na’%N INTO THE RAT
CYANIDE,
Radioactivity as % of injected dose” Time after injection (min) 10 20 30 60
Expired air 0.19 0.33 1.21 2.55
+ 0.03 -c 0.04 + 0.05 + 0.29
’ Each value is X + SE for three rats.
Urine
Urinary thiocyanate
Urinary cyanide
0.00 0.49 * 0.21 0.71 * 0.02 3.46 + 2.01
0.00 0.33 f 0.16 0.38 f 0.04 2.57 f 1.67
0.00 0.06 f 0.02 0.09 f 0.02 0.27 + 0.02
339
SHORT COMMUNICATION
activity of which 85% was present in urine, 6.7% in expired air, and 8.1% in feces. This finding agrees with the data reported here for control rats and for rats exposed to chronic dietary cyanide. Furthermore, the results reported here confirm that thiocyanate is the major excretory product in the urine of rats exposed to chronic intake of cyanide. The suggestion that the mode of excretion and metabolism of cyanide carbon is altered when rats are exposed to chronic intake of cyanide is, therefore, unlikely. It is also clear from the results reported here that the respiratory excretion of cyanide carbon is not altered by the chronic intake of cyanide. ACKNOWLEDGEMENT Thanks are expressed to Professor T. W. Goodwin for the opportunity afforded the author to work in his laboratory at the Department of Biochemistry, University of Liverpool, England.
R. G. H.,
SCHIEVELBEIN,
icol. 41, 49-52.
OKE, 0. L., (1969). Role of hydrocyanic acid in nutrition. Wld. Rev. Nutr. Dietetics 11, 170-I 76. OKOH, P. N., (1978). Aspects ofthe Metabolism ofcyanide in the Rat. Ph.D. thesis, University of Liverpool. OKOH, P. N., AND Prrr, G. A. J. ( 1982). The metabolism in the rat of cyanide and the gastrointestinal circulation of the resulting thiocyanate under conditions of chronic cyanide intake. Canad. J. Phsiol. Pharmacol. 60, 38 I 386. SMITH, A. D. M., AND FOULKES, M., (1966). Cyanide excretion in the rat. Nature (London) 209, 919-1000. WILLIAMS, R. T., (1959). Detoxication mechanism. Chapman & Hall, London. WOOD, J. L., ANDCOOLEY, S. L. (1956). Detoxication of cyanide by cystine. .I Biol. Chem. 218, 449-451.
PATRICKN.OKOH
REFERENCES BAUMEISTER,
BOXER, G. E., AND RICKARDS, J. C., (1952). Studies on the metabolism of the carbon of cyanide and thiocyanate. Arch. Biochem Biophys. 39, 7-26. CRAWLEY,F. E. H., AND GODDARD, E. A., (1977). Internal dose from carbon-14 labelled compounds. The metabolism of carbon-14 labelled potassium cyanide in the rat. Health Phys. 32, 135-142. LANG, K., (1933). Thiocyanate formation in the animal body. B&hem. Z. 259,243-256. MEHTA, C. S., AND MCGINITY, J. W., (1977). Chronic administration of cyanide: Urinary excretion of thiocyanate in male and female rats. Acta Pharmacol. TO-Y-
H.,
AND
ZICK-
GRAF-RUDEL, G., (1975). Toxicological and clinical aspects of cyanide metabolism. Arzneim. Forsch. 25, 1056-1064.
Department of Animal Science Faculty of Agriculture Ahmadu Belle University Zaria, Nigeria Received November 20, 1982; accepted
March
3. 1983
AND
APPLIED
PHARMACOLOGY
70, 335-339 (1983)
SHORT COMMUNICATION Excretion of “C-labeled Cyanide in Rats Exposed to Chronic Intake of Potassium Cyanide Excretion of “C-labeled Cyanide in Rats Exposed to Chronic Intake of Potassium Cyanide. P. N. (1983). Toxicol. Appl. Pharmocol. 70, 335-339. The excretion of an acute dose of “C-labeled cyanide in urine, feces,and expired air was studied in rats exposed to daily intake of unlabeled KCN in the diet for 6 weeks. Urinary excretion was the main route of elimination of cyanide carbon in these rats, accounting for 83% of the total excreted radioactivity in 12 hr and 89% of the total excreted radioactivity in 24 hr. The major excretion metabolite of cyanide in urine was thiocyanate, and this metabolite accounted for 71 and 79% of the total urinary activity in 12 hr and 24 hr, respectively. The mean total activity excreted in expired air after 12 hr was only 4%, and this value did not change after 24 hr. Of the total activity in expired air in 24 hr, 90% was present as carbon dioxide and 9% as cyanide. When these results were compared with those observed for control rats, it was clear that the mode of elimination of cyanide carbon in both urine and breath was not altered by the chronic intake of cyanide. OKOH,
Several routes of metabolism of cyanide have been described; these routes have been reviewed by Williams (1959) Oke (1969), and Baumeister et al. (1975). The best known mechanism for detoxification of cyanide is the conversion of cyanide to the relatively nontoxic thiocyanate which is excreted largely in the urine (Boxer and Rickards, 1952). Another route is the reaction with cystine to form 2iminothiazolidine-4-carboxylic acid (Wood and Cooley, 1956). Following injection of Na14CN in dogs and rats, r4C appears in urine as free cyanide, as a constituent of cyanocobalamin, and in thiocyanate. A part of the cyanide and thiocyanate is oxidized directly to form carbon dioxide (Boxer and Rickards, 1952). The chronic intake of cyanide has been implicated in the pathogenesis of a number of diseases. Although it has been suggested that a diminution of cyanide metabolism is probably responsible (Baumeister et al., 1975), the exact cause of the clinical state has not been established. Smith and Foulkes ( 1966) claimed that urinary excretion of thiocyanate is decreased in the rat following chronic administration of cyanide. These workers suggested that the main excretory pathway for cyanide excretion was affected by chronic but unphys335
iologic cyanide stimulation. They concluded that some unknown degradation product of cyanide contributed to the observed clinical state in chronic cyanide intoxication. Mehta and McGinity (1977) failed to confirm this observation of altered cyanide metabolism made by Smith and Foulkes (1966) by the same chemical technique. Confirmation of any change in the metabolism of cyanide as a result of chronic intake will be of value in understanding the nature of chronic cyanide toxicity in humans. By studying the pattern of excretion of 14Clabeled cyanide in rats chronically exposed to cyanide, it was hoped that any effect of chronic intake of cyanide on the urinary elimination of cyanide carbon would be readily observed by the more sensitive radiochemical technique. The effect of chronic intake of cyanide on the respiratory excretion of cyanide carbon was also studied. METHODS Twenty male albino rats (from the stock colony of the Department of Biochemistry, University of Liverpool, England) weighing about 100 g each were randomly assigned to individual cages and divided into two groups of 10 rats each. They were maintained on a semisynthetic diet containing extracted soyabean meal as the main protein source (Okoh, 1978; Okoh and Pitt, 1982). Potassium 0041-008X/83
$3.00
Copyright 0 1983 by Academic Press Inc. All rights of reproductmn in any form reserved.
336
SHORT COMMUNICATION
cyanide (unlabeled) was added to the diet (mixed with 5% arachis oil) of each rat in one group (cyanide rats) to provide 77 pmole cyanide per rat per day for six weeks. The cyanide was added to 10 g of diet which was given to each rat in the evening. Ten g was about the amount of diet each rat consumed overnight. By the following morning, each rat was given the soyabean diet without cyanide for the rest of the day. Potassium cyanide is stable in the soyabean meal diet when mixed with 5% arachis oil (Okoh, 1978; Okoh and Pitt, 1982). Also the mixing of the diet with arachis oil reduced spilling by the rats. The rats in the second group (control rats) were fed similarly except that no potassium cyanide was added to their diet. At the end of 6 weeks, each rat was given two doses of 8.3 rmol ‘%N- with a 30-min interval by sc injection. The specific activity of the injected material was 1.15 rCi/ rmol. Following injection, each rat was placed in a metabolism unit which allowed for collection of respiratory activity in NaOH absorbers, feces an a fine screen, and urine in the bottom of a dessicator (Boxer and Rickards, 1952). At the end of each study period, any urine in the bladder was forced out of each rat into a stainless-steel tray by gripping the rat firmly at the back of the neck with the thumb and forefingers and then stretching the animal by a slight pull at the tail. The used NaOH absorbers and the collected feces and urine were stored separately at -20°C. To achieve quantitative separation of carbon dioxide and cyanide in the urine and the NaOH absorbers, the following procedure described by Boxer and Rickards (1952) was used. A known volume of the sample was put into a tube with a rubber stopper equipped with gas inlet and outlet tubes. Silicon antifoaming agent (1 ml 8% solution) was added, and the mixture was acidified to pH 3 to 4 by addition of 0.5 M H$O,. Four traps were connected in series with the first and the second containing 6 ml 0.02 M Ag2S04 in 0.05 M HzS04 each, while the third and fourth contained 5 ml 0.1 M NaOH each. Cyanide and carbon dioxide were transferred over a period of 45 min by aerating with oxygen-free nitrogen at a flow rate of between 700 and 800 ml/min. The silver sulfate traps removed all the cyanide while the carbon dioxide liberated was trapped in the NaOH. In the urine samples, thiocyanate left in the mixture after removal of cyanide and carbon dioxide was oxidized to cyanide by the addition of I ml 0.01 M potassium permanganate; oxidation was usually completed in 3 min. Stannous chloride (1 ml 2% solution) was added to reduce excess permanganate, and the cyanide formed was aerated into two tubes containing 5 ml 0.1 M NaOH each by aerating with nitrogen as previously described. Samples were prepared for counting in a toluene scintillator fluid mixed with NCS solubilizer (New England Nuclear, Boston). The amount of radioactivity in each sample was determined in a liquid scintillation counter (Model SL 40 Intertechnique Ltd., Bringhton, England). Appropriate corrections were made for quenching by the
external standard method. Data were subjected to analysis of variance by the least square method with a digital computer.
RESULTS
AND
DISCUSSION
Rats on the KCN diet ate adequate diet and grew like the control rats fed a similar diet without KCN. Furthermore, the addition of KCN (77 pmol/rat/day) to the diet of the rats resulted in increased thiocyanate excretion in the urine. The mean thiocyanate content of 24-hr urine collected from four rats just before the addition of KCN to the diet was 1.69 -+ 0.19 pmol SE. By contrast, the first 24-hr urine collection from the same rats following the addition of KCN to the basal diet was 30.80 + 2.57 pmol SE. Table 1 shows the result of the elimination of 14C activity in urine, expired air, and feces after injection of the Na14CN to normal rats and those exposed to chronic intake of cyanide in the diet (cyanide rats). Urine was the main route of excretion of radioactivity in all the rats, accounting for over 80% of the total excreted radioactivity in 12 hr and over 85% of the total excreted radioactivity in 24 hr. Moreover, the cumulative 24-hr urinary excretion of radioactivity in both control and cyanide rats was significantly higher (p c 0.05) than the total excretion in 12 hr. By contrast, the small increase in the radioactivity excreted in expired air in 24 hr compared with the 12hr period was not statistically significant in either control or cyanide rats. The fecal radioactivity was highly variable (C.V. 94.0% Table 1). This result was due to the large difference in the amount of feces collected from individual rats. However, the radioactivity excreted in feces by the cyanide rats was higher than that excreted by control rats at the same period, but this finding was not statistically significant. Okoh and Pitt (Okoh, 1978; Okoh and Pitt, 1982) have shown that a large amount of radioactive cyanide injected into rats was largely secreted into the stomach contents as thiocyanate but reabsorbed into the body fluid in the lower part of the gut. The higher amount of radioactivity in the feces of
337
SHORT COMMUNICATION TABLE CUMULATIVE
AMOUNT
OF 14C IN URINE,
KCN in diet (woVrac/day)
Time after injection (W 12 12 24 24
0.0
77.0 0.0
77.0 SE cv
EXPIRED
(%)
1
AIR, AND FECES AITER INJECTION
OF Na’%ZN
TO RATS
Radioactivity as % of dose injected”’ Urine
Expired air
24.31' f 1.32 23.91' + 1.48
4.16=-c 0.18 4.32' + 0.49 4.53=+ 0.45 4.90' -+ 0.43 0.41 20.0
57.28*
f 3.40
51.72* f 2.45 2.32 12.0
FeOeS 0.37' 0.48' 1.15"* 1.70* 0.39 94.0
* f -+ f
0.25 0.45 0.24 0.54
a Each value is X k SE for five rats. ’ Means in the same column followed by different letters differ significantly at p G 0.05.
cyanide rats may therefore be due to a decrease in the efficiency of reabsorption of cyanide carbon in the lower region of the gut. Regardless of this small increase in the fecal r4C of cyanide rats, it is clear from Table 1 that feces are not a major route of excretion of cyanide carbon by either group of rats. The quantitative distribution of the injected cyanide carbon in the various fractions of the urine is illustrated in Table 2. The urinary cyanide includes Bi2 cyanide, while the CO* fraction includes carbonate and bicarbonate. Neither cyanide nor carbon dioxide plays a major role as an excretory pathway. In each group of rats, the largest urinary activity was the thiocyanate fraction, and the amount in 24 hr was significantly higher (p -L 0.05) than
in the 12-hr collection. This increase in the proportion of thiocyanate in urine with time is likely associated with increase in the amount of this radical formed from cyanide by the tissue rhodanese enzyme (Lang, 1933). The proportion of urinary radioactivity in cyanide and carbon dioxide fractions did not significantly change with time in either control or cyanide rats. The distribution of respiratory activity between cyanide and carbon dioxide fraction is given in Table 3. Of the total activity in expired air in 24 hr, 90% Was present as carbon dioxide and only 9% as cyanide. This distribution is not significantly different from that observed for control rats, not on chronic cyanide intake. However, in both groups of rats, the propor-
TABLE 2 CUMULATIVE
DISTRIBUTION
KCN in diet (rmol/rat/day) 0.0 77.0 0.0 77.0 SE cv (o/o)
OF RADIOACTIVITY
Time after injection (hd 12 12 24 24
IN VARIOUS
URINE
FRACTIONS
AFTER INJECTION
OF Na’%N
Radioactivity as % of activity in Urinea~b SCN-
CN-
69.50' +- 1.74 70.86' k 1.85 80.28*~ 2.87 78.80* zk 1.97 2.16 6.0
1.08' -+ 0.20 1.13c + 0.15 1.41'? 0.10 1.31'2 0.13 0.14 26.0
u Each value is X _t SE for five rats. b Means in the same column followed by different letters differ significantly at p < 0.05.
co2 5.10' + 5.34' f 5.51=_+ 6.15'+ 0.37 15.0
0.54 0.34 0.31 0.27
338
SHORT COMMUNICATION TABLE 3 CUMULATIVE
DISTRIBUTION OF RADIOA~TIVW IN VARIOUS FFMXIONS OF THE EXPIRED AIR AFTER INJECI-ION OF Na?ZN
Radioactivity as % of total activity in expired aiFb
Time after injection (hd
KCN in diet bmobat/W) 0.0 77.0 0.0 77.0 SE cv (%b)
CN-
co2
12 12 24 24
85.59’ + 0.12 85.69’ f 1.52 91.15’f 0.93 89.55’ + 1.88 1.34 3.0
13.05’ f 14.12’-c 9.58d f 9.44d f 0.96 18.0
0.41 1.71 0.64 0.41
’ Each value X f SE for 5 rats. b Means in the same column followed by different letters differ significantly at p G 0.05.
tion of cyanide in expired air significantly (p < 0.05) decreased in 24 hr compared with the 12-hr collection. This result is because the increase in activity in the expired air at 24 hr compared with the 12-hr period (Table 1) is largely associated with an increase in the carbon dioxide fraction and not in cyanide. The rate of excretion of radioactivity in the urine and expired air was studied in four groups of rats previously exposed to daily cyanide intake in the diet for 3 weeks. The elimination of the 14C activity in the urine and expired air up to 1 hr after sc injection of Na14CN is summarized in Table 4. No radioactivity was detected in the urine of rats 10 min after the injection of the Na14CN. However, urine collected by 20 min contained de-
tectable activity in all rats. In contrast, excretion of radioactivity by the expired air was detectable 10 min after injection of the dose (Table 4). The urinary excretion of radioactivity was more variable than respiratory excretion. When urinary cyanide and thiocyanate were separated from the total urinary excretion, it was observed that the latter radical was the major excretory component at each period (20, 30 and 60 min). Excretion of cyanide carbon has been examined in a number of animal species not exposed to chronic cyanide intake (Boxer and Rickards, 1952; Crawley and Goddard, 1977). One such study in rats (Crawley and Goddard, 1977) showed that the amount of cyanide carbon excreted in 24 hr was 54% of the injected
TABLE 4 CUMULATIVE
RATE OF APPEARANCE OF RADIOACWITY IN EXPIRED AIR, URINARY AND THKXYANATE AVER INJECTION OF Na’%N INTO THE RAT
CYANIDE,
Radioactivity as % of injected dose” Time after injection (min) 10 20 30 60
Expired air 0.19 0.33 1.21 2.55
+ 0.03 -c 0.04 + 0.05 + 0.29
’ Each value is X + SE for three rats.
Urine
Urinary thiocyanate
Urinary cyanide
0.00 0.49 * 0.21 0.71 * 0.02 3.46 + 2.01
0.00 0.33 f 0.16 0.38 f 0.04 2.57 f 1.67
0.00 0.06 f 0.02 0.09 f 0.02 0.27 + 0.02
339
SHORT COMMUNICATION
activity of which 85% was present in urine, 6.7% in expired air, and 8.1% in feces. This finding agrees with the data reported here for control rats and for rats exposed to chronic dietary cyanide. Furthermore, the results reported here confirm that thiocyanate is the major excretory product in the urine of rats exposed to chronic intake of cyanide. The suggestion that the mode of excretion and metabolism of cyanide carbon is altered when rats are exposed to chronic intake of cyanide is, therefore, unlikely. It is also clear from the results reported here that the respiratory excretion of cyanide carbon is not altered by the chronic intake of cyanide. ACKNOWLEDGEMENT Thanks are expressed to Professor T. W. Goodwin for the opportunity afforded the author to work in his laboratory at the Department of Biochemistry, University of Liverpool, England.
R. G. H.,
SCHIEVELBEIN,
icol. 41, 49-52.
OKE, 0. L., (1969). Role of hydrocyanic acid in nutrition. Wld. Rev. Nutr. Dietetics 11, 170-I 76. OKOH, P. N., (1978). Aspects ofthe Metabolism ofcyanide in the Rat. Ph.D. thesis, University of Liverpool. OKOH, P. N., AND Prrr, G. A. J. ( 1982). The metabolism in the rat of cyanide and the gastrointestinal circulation of the resulting thiocyanate under conditions of chronic cyanide intake. Canad. J. Phsiol. Pharmacol. 60, 38 I 386. SMITH, A. D. M., AND FOULKES, M., (1966). Cyanide excretion in the rat. Nature (London) 209, 919-1000. WILLIAMS, R. T., (1959). Detoxication mechanism. Chapman & Hall, London. WOOD, J. L., ANDCOOLEY, S. L. (1956). Detoxication of cyanide by cystine. .I Biol. Chem. 218, 449-451.
PATRICKN.OKOH
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BOXER, G. E., AND RICKARDS, J. C., (1952). Studies on the metabolism of the carbon of cyanide and thiocyanate. Arch. Biochem Biophys. 39, 7-26. CRAWLEY,F. E. H., AND GODDARD, E. A., (1977). Internal dose from carbon-14 labelled compounds. The metabolism of carbon-14 labelled potassium cyanide in the rat. Health Phys. 32, 135-142. LANG, K., (1933). Thiocyanate formation in the animal body. B&hem. Z. 259,243-256. MEHTA, C. S., AND MCGINITY, J. W., (1977). Chronic administration of cyanide: Urinary excretion of thiocyanate in male and female rats. Acta Pharmacol. TO-Y-
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Department of Animal Science Faculty of Agriculture Ahmadu Belle University Zaria, Nigeria Received November 20, 1982; accepted
March
3. 1983