- Email: [email protected]
Antidotal Effect of Dihydroxyacetone against Cyanide Toxicityin Vivo
TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.
138, 186–191 (1996)
0112
Antidotal Effect of Dihydroxyacetone against Cyanide Toxicity in Vivo HOSSEIN NIKNAHAD1
AND
PETER J. O’BRIEN2
Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario, Canada, M5S 2S2 Received November 9, 1995; accepted February 5, 1996
Antidotal Effect of Dihydroxyacetone against Cyanide Toxicity in Vivo. NIKNAHAD, H., AND O’BRIEN, P. J. (1996). Toxicol. Appl. Pharmacol. 138, 186–191. Potassium cyanide (CN) intoxication in mice was found to be effectively antagonized by dihydroxyacetone (DHA), particularly if administered in combination with another CN antidote, sodium thiosulfate. Cyanide-induced convulsions were also prevented by DHA treatment, either alone or in combination with thiosulfate. Injection (ip) of DHA (2 g/kg) 2 min after or 10 min before CN (sc) increased LD50 values of CN (8.7 mg/kg) by factors of 2.1 and 3.0, respectively. Treatment with a combination of DHA and thiosulfate after CN increased the LD50 by a factor of 2.4. Pretreatment with a combination of DHA and thiosulfate (1 g/kg) increased the LD50 of CN to 83 mg/kg. Administration of aketoglutarate (2.0 g/kg), but not pyruvate, 2 min after CN increased the LD50 of CN by a factor of 1.6. Brain, heart, and liver cytochrome oxidase activities were also measured following in vivo CN treatment with and without DHA. Pretreatment with DHA prevented the inhibition of cytochrome oxidase activity by CN and treatment with DHA after CN accelerated the recovery of cytochrome oxidase activity, especially in brain and heart homogenates. DHA is a physiological agent and, therefore, could prove to be a safe and effective antidote for CN, particularly in cases of fire smoke inhalation in which a combination of CN and carbon monoxide is present. In these cases the normally used antidote, sodium nitrite, to induce methemoglobin so as to trap the CN, is contraindicated because some of the oxygen-carrying capacity of the blood will have already been diminished by carbon monoxide. q 1996 Academic Press, Inc.
Because of its rapid lethal effects, cyanide (CN) is used in suicides, homicides, and chemical warfare. Ingestion of CN-containing foods (Osuntokun et al., 1969; Osuntokun 1980; Cook and Coursey, 1981; Rosling, 1989) and occupational exposure to CN in industry have caused serious toxic problems (Blanc et al., 1985; Peden et al., 1986). In medicine, the release of CN from some drugs such as sodium nitroprusside (Davis et al., 1975; Vessy and Cole, 1985) and laetrile (Sadoff et al., 1978; Kalyanaraman et al., 1983) has 1
Current address: Department of Pharmacology & Toxicology, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Fars, Iran. 2 To whom correspondence should be addressed. Fax: (416) 978-8511.
186
0041-008X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AID
TOX 7798
/
6h0c$$$161
also been life threatening. Elevated levels of blood CN have been recorded in victims and survivors of fire smoke inhalation and most of the toxicity and lethality associated with smoke inhalation injury have been attributed to CN (Clark et al., 1981; Cohen and Guzzardi, 1988; Mayes, 1991; Kirk et al., 1993; Barillo et al., 1994). The classical treatment of CN poisoning involves administration of amyl nitrite and sodium nitrite to induce methemoglobin, which reversibly binds the CN (Friedberg, 1968), and administration of thiosulfate which may act by converting CN to thiocyanate with the assistance of the mitochondrial enzyme rhodanese (Himwich and Saunders, 1948). However, nitrites are slow methemoglobin inducers (Kruszyna et al., 1982) and also cause hypotension which can worsen the situation of the patients poisoned with CN (Hall et al., 1989). Furthermore, in the cases of smoke inhalation, induction of methemoglobin formation could be dangerous as a large amount of hemoglobin may already be bound to carbon monoxide (Moore et al., 1987; Hall et al., 1989; Kuling, 1991). The toxic effect of CN has been attributed to its production of histotoxic anoxia by the reversible inhibition of cytochrome c oxidase, the terminal oxidase of the mitochondrial respiratory chain (Solomonson, 1981). Removal of free CN by binding to CN-trapping agents or methemoglobin will result in dissociation of CN from cytochrome oxidase and reactivation of this enzyme (Solomonson, 1981). CN is a nucleophile known to react with various carbonyl groups to form cyanohydrin intermediates. Sodium pyruvate and a-ketoglutarate have therefore been used to trap CN in vivo (Schwartz et al., 1979; Moore et al., 1986; Norris et al., 1990). Previously, we have shown that dihydroxyacetone (DHA), glyceraldehyde, pyruvate, and a-ketoglutarate also rapidly restored CN-inhibited mitochondrial respiration as a result of trapping CN to form cyanohydrins (Niknahad et al., 1994). In the present study, the antidotal effects of DHA against CN poisoning in vivo in mice, alone and in combination with sodium thiosulfate, have been determined and compared. We have found that DHA combined with thiosulfate was the most effective antidotal combination of the agents investigated. DHA could therefore prove an effective antidote for CN poisoning particularly in the cases of smoke
04-09-96 09:20:27
toxa
AP: Tox
187
ANTAGONISM OF CYANIDE LETHALITY BY DIHYDROXYACETONE
inhalation lethality attributed to cyanide when carbon monoxide is present. MATERIALS AND METHODS Animals. Male CD1 mice, weighing 25 to 30 g, were obtained from Charles River Laboratories (Montreal, PQ, Canada). Animals were housed in a temperature-controlled environment with 12-hr light/12-hr dark periods for 2 weeks before experiments and were given feed and tap water ad libitum. Each experimental group was selected randomly from the general mouse population. Chemicals. Potassium cyanide and sodium thiosulfate were purchased from Fisher Scientific Ltd. (Toronto, Canada). Dihydroxyacetone, sodium nitrite, a-ketoglutaric acid, pyruvic acid, and bovine heart cytochrome c were obtained from Sigma Chemical Co. (St. Louis, MO). Other chemicals were of the highest grade commercially available. KCN (2 mg/ml), dihydroxyacetone (500 mg/ml), a-ketoglutarate (500 mg/ml), pyruvate (500 mg/ ml), sodium thiosulfate (500 mg/ml), and sodium nitrite (50 mg/ml) solutions were prepared in normal saline (0.9% NaCl) each day. Lethality studies. For posttreatment studies, KCN at determined doses was given by sc injection and after 2–3 min (as soon as the animal started to convulse), dihydroxyacetone (2 g/kg, ip), a-ketoglutarate (2 g/kg, ip), pyruvate (1 g/kg, ip), sodium thiosulfate (1 g/kg, ip), or sodium nitrite (0.1 g/kg, ip) was injected and the animals were watched for convulsions until 60 min after injection of the antidotes and for 24 hr for survival. For pretreatment studies, sodium nitrite (0.1 g/kg, ip) was given 20 min before CN injection (sc), sodium thiosulfate (1 g/kg, ip) was given 10 min before CN injection, and dihydroxyacetone (2 g/kg, ip), a-ketoglutarate (2 g/kg, ip), and pyruvate (1.0 g/kg, ip) were given 5 min before CN injection and the animals were watched for 60 min for convulsions and for 24 hr for survival. It should be mentioned that pyruvate at the 2 g/kg dose was toxic and caused severe convulsions, stiffness of the tail, and tonic paralysis of limbs in about 5 min and killed all of the animals within 10 min after ip injection. Some of the toxic effects of pyruvate such as partial paralysis of limbs were present even with a dose of 1 g/kg, but none of the animals treated died. The LD50 values are based on 24 hr mortality. Experimental values were obtained for at least four groups of mice with each group containing 6 to 10 mice. Measurement of cytochrome oxidase activity. KCN (7 mg/kg) was injected sc and dihydroxyacetone (1 g/kg, ip), sodium nitrite (0.1 g/kg, ip), or thiosulfate (1 g/kg, ip) was injected 2–3 min after CN. Control animals were receiving normal saline (0.9% NaCl) injections rather than drugs. At specific time points following CN administration, the animals were killed by decapitation and the brain, heart, or liver was removed. Excess blood from the heart or liver was removed by blotting with filter paper. Homogenates (10% w/v) were prepared in chilled (17C) distilled water using a motor drive Teflon tissue homogenizer. Homogenates were subsequently diluted to 1% (w/v) with chilled (17C) 30 mM Tris–HCl buffer, pH 7.4. Cytochrome oxidase activity was determined by the rate of oxidation of reduced cytochrome c, as previously explained (Isom et al., 1982). The assay mixture contained 0.1 ml of homogenate (1% w/v), 2.7 ml of 30 mM Tris–HCl buffer, pH 7.4, and 0.2 ml of 1 mM reduced cytochrome c solution, prepared as previously described by Potter (1964). The absorbance at 550 nm was recorded continuously over a 5-min period using a DW 2000 spectrophotometer. At 5 min, 40 ml of saturated potassium ferricyanide solution was added to completely oxidize the cytochrome c and the cytochrome c oxidase activity was calculated as the first-order rate constant (Smith, 1954) based on the protein concentration of 1% homogenates. The cytochrome c oxidase activities are expressed as percentage of control values in order to compare the enzymatic activities of different treatments. Measurement of protein content of homogenates. Protein content of brain, heart, or liver (1% w/v) homogenates was determined by the Lowry method (Lowry et al., 1951).
AID
TOX 7798
/
6h0c$$$162
04-09-96 09:20:27
TABLE 1 Antidotal Effect of Dihydroxyacetone, Sodium Thiosulfate, aKetoglutarate, Pyruvate, and Sodium Nitrite on the Lethality of Potassium Cyanide
Treatments (g/kg)a Saline DHAd (1.0) DHA (2.0) DHA (4.0) a-KG (2.0) Pyruvate (1.0) NaNO2 (0.1) Na2S2O3 (1.0) DHA (2.0) / Na2S2O3 (1.0) NaNO2 (0.1) / Na2S2O3 (1.0)
LD50 value (mg/kg)b (95% confidence interval) 8.7 13.5 17.8 22.5 14.2 9.4 12.6 10.9 20.5 13.5
(7.9–9.4) (12.8–14.2) (16.0–19.8) (20.2–25.1) (13.0–15.5) (8.3–10.6) (11.6–13.7) (9.9–12.0) (18.3–23.0) (12.7–14.4)
Potency ratioc
1.55 2.05 2.58 1.63 1.08 1.45 1.25 2.36 1.55
1.00 (1.47–1.63) (1.89–2.22) (2.32–2.87) (1.55–1.71) (0.97–1.19) (1.37–1.54) (1.09–1.43) (2.19–2.54) (1.43–1.68)
a
DHA, NaNO2, and Na2S2O3 were injected (ip) 3 min after cyanide (sc). Each LD50 value was obtained from four to eight graded doses of KCN administered to four groups of mice with six to eight mice per group. c Potency ratio Å LD50 of KCN with antidote(s)/LD50 of KCN without antidote. d DHA, dihydroxyacetone; a-KG, a-ketoglutarate. b
Statistics. The efficacy of the antagonists is shown as a potency ratio, calculated by dividing the LD50 values for CN with antidote by the LD50 value of CN without antidote. The LD50 values were obtained by the method of Litchfield and Wilcoxon (1949) with a 95% confidence level. For cytochrome oxidase activity studies the significant differences between control and experimental groups were calculated using Student’s t test and significantly different groups were chosen when p £ 0.05.
RESULTS
Administration (sc) of cyanide to mice first resulted in a short period (1–2 min) of calmness followed by convulsions which started 2–3 min after CN injection. Death occurred at 4–10 min with an LD50 for CN of 8.7 (7.9–9.4) mg/kg which was similar to that previously reported (Way et al., 1966; Schwartz et al., 1979; McGuinn et al., 1994; Yamamoto, 1990). The effects of DHA, a-ketoglutarate, pyruvate, sodium nitrite, and sodium thiosulfate given intraperitoneally 2–3 min after subcutaneous injection of CN on the LD50 of CN in mice are compared in Table 1. Administration of 1, 2, or 4 g/kg of DHA increased the LD50 value about 1.6-, 2.1-, and 2.6-fold, respectively (Table 1). aKetoglutarate (2 g/kg) increased the LD50 of CN only 1.6fold (equal to 1 g/kg of DHA) and pyruvate (1 g/kg) did not affect the LD50 value of CN. Sodium nitrite, but not sodium thiosulfate, also slightly increased the LD50 value for CN but was less effective than DHA. However, in most of the cases sodium nitrite but not sodium thiosulfate prolonged the survival time. Sodium thiosulfate slightly increased the antidotal effectiveness of
toxa
AP: Tox
188
NIKNAHAD AND O’BRIEN
TABLE 2 Prophylactic Effects of Dihydroxyacetone, Sodium Thiosulfate, a-Ketoglutarate, Pyruvate, and Sodium Nitrite on the Lethality of Potassium Cyanide
Treatments (g/kg)a Saline DHAd (2.0) a-KG (2.0) Pyruvate (1.0) NaNO2 (0.1) Na2S2O3 (1.0) DHA (2.0) / NaNO2 (0.1) DHA (2.0) / Na2S2O3 (1.0) NaNO2 (0.1) / Na2S2O3 (1.0)
LD50 value (mg/kg, ip)b (95% confidence interval)
Potency ratioc (95% confidence)
8.7 26.0 28.4 14.2 23.5 21.3 47.8 83.6 47.3
2.99 3.26 1.63 2.70 2.44 5.49 9.61 5.44
(7.9–9.4) (24.5–27.6) (25.3–31.9) (12.1–16.7) (20.6–26.8) (19.3–23.3) (42.8–53.2) (78.9–88.6) (45.1–49.7)
1.00 (2.76–3.24) (3.01–3.53) (1.52–1.75) (2.38–2.91) (2.27–2.62) (5.03–5.99) (8.71–10.60) (5.19–5.71)
a DHA (ip), NaNO2 (ip), and Na2S2O3 (ip) were injected 10, 20, and 15 min respectively before the administration of cyanide (sc). b Each LD50 value was obtained from four to eight graded doses of KCN administered to four to six groups of mice with six to eight mice per group. c Potency ratio Å LD50 of KCN with antidote(s)/LD50 of KCN without antidote. d DHA, dihydroxyacetone; a-KG, a-ketoglutarate.
DHA (Table 1). The combination of DHA with a-ketoglutarate or pyruvate had additive antidotal effects against CN (data not shown). Table 2 shows the effect of pretreatment with different antidotes on the lethality of CN in mice. Pretreatment with sodium nitrite or sodium thiosulfate increased the LD50 for CN 2.7 and 2.4 times, respectively, values similar to those previously reported (Yamamoto, 1990; Moore et al., 1986; McGuinn et al., 1994). However, pretreatment with 2 g/kg DHA or a-ketoglutarate was more effective than that with pyruvate, sodium nitrite, or sodium thiosulfate in antagonizing CN lethality. DHA and thiosulfate were synergistic and were the most effective antidotes found by which the LD50 for CN was increased more than ninefold. In all cases that DHA was administered, animals did not convulse. The time course for the effect of CN, with and without DHA treatment, on cytochrome oxidase activity in mouse brain, heart, and liver homogenates was investigated. Brain, heart, or liver cytochrome oxidase was inactivated very rapidly after sc administration of 7 mg/kg CN. The maximum inhibition of cytochrome oxidase occurred around 5 min after CN injection. The cytochrome oxidase activity of brain and liver declined to approximately 37% of control at 5 min following CN treatment (Figs. 1A and 1C), whereas that of heart declined to about 25% of control at 5 min (Fig. 1B). Cytochrome oxidase activities started to recover around 10 min and returned to 70, 64, and 92% of control at 30 min in brain, heart, and liver, respectively. Cytochrome oxidase activity in all these organs recovered 60 min after CN injection. If DHA was administered 2 min after
AID
TOX 7798
/
6h0c$$$162
04-09-96 09:20:27
CN injection the cytochrome oxidase activity in brain and heart started to recover at 5 min after CN injection (Figs. 1A and 1B). However, the recovery in the liver was less pronounced (Fig. 1C). If DHA was injected 5 min before CN, the inhibition of cytochrome oxidase activity in brain, heart, and liver homogenates was partially prevented (Figs. 1A–1C). Maximal inhibition occurred at 10 min following CN injection. Cytochrome oxidase activities at 10 min were about 65, 61, and 59% of control in brain, heart, and liver, respectively. Administration of thiosulfate after CN caused a rapid recovery of cytochrome oxidase activity in brain, heart, and liver (Table 3). Sodium nitrite was much less effective than thiosulfate or DHA in restoring cytochrome oxidase activity. Pretreatment with thiosulfate was as effective as or better than DHA in preventing the inactivation of cytochrome oxidase of brain, heart, and liver (Table 4). Pretreatment with sodium nitrite, however, effectively prevented the inhibition of cytochrome oxidase in brain and liver but not heart (Table 4). DISCUSSION
We have previously shown that DHA binds CN and restores mitochondrial respiration inhibited by CN in isolated hepatocytes (Niknahad et al., 1994). In the present study, we have shown that DHA provided a better antidotal effect against the lethal effects of CN than sodium nitrite or sodium thiosulfate alone. The combination of DHA and thiosulfate had a strong synergistic effect and was much more effective than the sodium nitrite and thiosulfate combination. DHA could have a potential therapeutic advantage over thiosulfate and sodium nitrite as a CN antidote as it is a physiological component of the cell whose phosphorylated form is a part of the glycolysis pathway. Previously, we showed that the administration of DHA to hepatocytes increased ATP and lactate formation in hypoxic or antimycin A-treated hepatocytes (Niknahad et al., 1995; Khan and O’Brien, 1995). Since at least one of the antidotal mechanisms of DHA involves trapping the CN to form a cyanohydrin, it could have a very rapid onset of action compared to those agents which induce methemoglobin to trap CN (Friedberg, 1968). DHA had a stronger antidotal effect than pyruvate or aketoglutarate on CN lethality if administered after CN. However, although much better than pyruvate, DHA had a similar effect to that of a-ketoglutarate, if administered before CN injection. This may be due to a faster absorption and distribution of DHA to the site of CN localization. All of the CN antidotes examined were much more effective when given prophylactically than when given after CN. One reason for this is possibly more complete absorption from the site of injection and better distribution of the antidotes to the site of detoxification or to the target organs, when given prophylactically. Sodium thiosulfate is known
toxa
AP: Tox
ANTAGONISM OF CYANIDE LETHALITY BY DIHYDROXYACETONE
189
FIG. 1. The effects of treatment with dihydroxyacetone on cyanide-induced inhibition of cytochrome oxidase in brain (A), heart (B), and liver (C) homogenates. Dihydroxyacetone (1 g/kg) was injected ip 10 min before (l), or 2 min after (s), sc injection of 7 mg/kg cyanide (j). Cytochrome oxidase activity was measured as explained under Materials and Methods. Results shown are mean { SD of three to five experiments. The first-order rate constants for cytochrome oxidase activity of control brain, heart, and liver were 0.058 { 0.003, 0.107 { 0.015, and 0,072 { 0.005 mg01 sec01, respectively.
to penetrate the cell membrane very slowly and also needs to cross the mitochondrial membrane as rhodanese is located in the mitochondrial matrix (Westly et al., 1983). Sodium nitrite, which is believed to antagonize CN lethality largely by inducing methemoglobin formation, is, however, a slow methemoglobin inducer with maximum methemoglobin formation at 30 min following its injection to mice (Moore et al., 1987). Cyanide-trapping agents DHA, a-ketoglutarate, and pyruvate were also more effective prophylactically possibly as a result of a better distribution to the target organs such as brain or heart. Alternatively, these agents may bind more readily to CN when given prophylactically as they compete for CN with cytochrome oxidase. The preventive and antidotal effects of DHA against CN
AID
TOX 7798
/
6h0c$$$163
04-09-96 09:20:27
were accompanied by the prevention or faster restoration of cytochrome oxidase activity in brain, heart, and liver. The restoration of the oxidase activity after CN treatment was faster in liver than in heart or brain. The faster restoration of cytochrome oxidase activity in the liver is not surprising since liver has a high level of rhodanese activity (Devlin et al., 1989). Skeletal muscle is also known to possess high rhodanese activity (Devlin et al., 1989) but restoration of cytochrome oxidase activity of heart was not faster than that of brain and was much slower than that of liver. It is possible that rhodanese activity of the heart cells is lower than that of liver or maybe more CN is localized in the heart. A more pronounced inhibition by CN of heart cytochrome oxidase activity than brain or liver cytochrome oxidase activity is probably because of the greater
toxa
AP: Tox
190
NIKNAHAD AND O’BRIEN
concentration of mitochondria in the heart cells. Heart cytochrome oxidase activity was also restored better following administration of DHA than following administration of sodium nitrite or thiosulfate. This may be one of the reasons for the better antidotal effectiveness of DHA compared to sodium nitrite or thiosulfate. Although the antidotal effectiveness of DHA was greater than that of thiosulfate at preventing CN lethality, its effectiveness at restoring cytochrome oxidase activity was similar. On the other hand, pretreatment with sodium nitrite prevented CN lethality to a similar degree as DHA, although the heart cytochrome oxidase activity was still inactivated. These suggest that cytochrome oxidase inhibition is not the only toxic mechanism involved in the lethality of CN. Lethality by CN was also reported without liver or brain cytochrome oxidase inhibition (Isom and Way, 1976; Tadic, 1992). It is not clear why cytochrome oxidase activity in the brain, heart, or liver of CN-treated animals reaches levels higher than control values 60 min after treatment. However, the lower activity of the enzyme at this time point in animals treated with DHA, before or after CN treatment, probably indicates that the DHA–CN cyanohydrin slowly releases CN as DHA is metabolized in the body. Previously, we showed that glycolytic substrates can prevent CN or antimycin A toxicity in isolated cells by averting reductive stress and/or supplying ATP (Niknahad et al., 1995). DHA can also supply ATP via glycolysis, and it has been used as a dietary regimen in combination with pyruvate in human studies (Stanko et al., 1992). As a medicine, DHA is already in use in dermatological tests (Pierard and Pierard-
TABLE 4 Prophylactic Effects of Dihydroxyacetone, Sodium Thiosulfate, a-Ketoglutarate, and Sodium Nitrite on the Inhibition of Cytochrome Oxidase Activity by Cyanide Cytochrome oxidase activity (% of control) Treatments (g/kg) Cyanide (0.007) / DHAa (2.0) /NaNO2 (0.1) /Na2S2O3 (1.0) / a-KG (2.0)
Brain 36.7 71.2 84.7 82.5 74.4
{ { { { {
3.6* 5.0** 5.8** 5.7** 4.6**
Heart 24.7 63.0 38.3 62.2 56.8
{ { { { {
4.1* 4.5** 3.5 3.8** 4.3**
Liver 38.1 60.7 77.5 85.6 63.7
{ { { { {
3.8* 3.5** 5.2** 6.2** 4.4**
Note. DHA (ip), NaNO2 (ip), and Na2S2O3 (ip) were injected 10, 20, and 15 min respectively before the administration of cyanide (sc) to mice. Cytochrome oxidase activity was measured 7 min after Cn injection as explained under Materials and Methods. Each value is the mean ({SD) of three to five different experiments. a DHA, dihydroxyacetone; a-KG, a-ketoglutarate. * Significantly lower than control (p £ 0.001). ** Significantly higher than cyanide-treated (p £ 0.005).
Franchimont, 1993). The results of the present study suggest that DHA may be a promising safe CN antidote which can be used in combination with other CN antidotes, especially thiosulfate. In particular, DHA may be considered as a substitute for sodium nitrite in the cases of CN poisoning associated with fire smoke inhalation in which the oxygen-carrying capacity of the blood has already been compromised by carbon monoxide as a result of carbon monoxy hemoglobin complex formation. ACKNOWLEDGMENT
TABLE 3 Antidotal Effect of Dihydroxyacetone, Sodium Thiosulfate, aKetoglutarate, and Sodium Nitrite on the Inhibition of Cytochrome Oxidase Activity by Cyanide
Hossein Niknahad was financially supported by a scholarship from Iran’s Ministry of Health and Medical Education.
REFERENCES Cytochrome oxidase activity (% of control) Treatments (g/kg) Cyanide (0.007) / DHAa (2.0) / NaNO2 (0.1) / Na2S2O3 (1.0) / a-KG (2.0)
Brain 37.5 57.2 41.8 51.6 46.3
{ { { { {
4.0* 3.2** 6.3 5.7** 3.6**
Heart 26.4 47.3 28.0 44.2 38.6
{ { { { {
2.8* 3.6** 3.7 3.8** 3.5**
Liver 37.4 49.2 40.3 52.5 41.7
{ { { { {
3.5* 2.9** 4.4 6.2** 4.4
Note. DHA (ip), NaNO2 (ip), and Na2S2O3 (ip) were injected 2 min after the administration of cyanide (sc) to mice. Cytochrome oxidase activity was measured 7 min after CN injection as explained under Materials and Methods. Each value is the mean ({SD) of three to five different experiments. a DHA, dihydroxyacetone; a-KG, a-ketoglutarate. * Significantly lower than control (p £ 0.05). ** Significantly higher than cyanide-treated or cyanide / sodium nitritetreated (p £ 0.05).
AID
TOX 7798
/
6h0c$$$163
04-09-96 09:20:27
Barillo, D. J., Goode, R., and Esh, V. (1994). Cyanide poisoning in victims of fire: Analysis of 364 cases and review of the literature. J. Burn Care Rehabil. 15, 46–57. Blanc, P., Hogan, M., Malin, K., Hryhorczuk, D., Hessel, S., and Bernard, B. (1985). Cyanide intoxication among silver reclaiming workers. J. Am. Med. Assoc. 253, 367–371. Clark, C. J., Campbell, D., and Reid, W. H. (1981). Blood carboxyhaemoglobin and cyanide levels in fire survivors. Lancet 1, 1332–1335. Cohen, M. A., and Guzzardi, L. J. (1988). The smoke inhalation and cyanide poisoning. Am. J. Emerg. Med. 6, 203–204. Cook, R. D., and Coursey, D. B. (1981). Cassava: A major cyanide-containing food crop. In Cyanide in Biology (B. Vennesland, E. E. Conn, C. J. Knowles, J. Westley, and F. Wissing, Eds.), pp. 93–114. Academic Press, New York. Davis, D. W., Griess, L., Kadar, D., and Stewart, D. J. (1975). A sudden death associated with the use of sodium nitroprusside for induction of hypotension during anaesthesia. Can. Anaesth. Soc. J. 22, 547–552.
toxa
AP: Tox
ANTAGONISM OF CYANIDE LETHALITY BY DIHYDROXYACETONE Devlin, D. J., Mills, J. W., and Smith, R. P. (1989). Histochemical localization of rhodanese activity in rat liver and skeletal muscle. Toxicol. Appl. Pharmacol. 97, 247–255. Friedberg, K. D. (1968). Antidotes by cyanide poisoning. Arch. Toxikol. 24, 41–48. Hall, A. H., Kuling, K. W., and Rumack, B. H. (1989). Suspected cyanide poisoning in smoke inhalation: Complications of sodium nitrite therapy. J. Toxicol. Clin. Exp. 9, 3–9. Himwich, W. A., and Saunders, J. P. (1948). Enzymatic conversion of cyanide to thiocyanate. Am. J. Physiol. 153, 348–354. Isom, G. E., and Way, J. L. (1976). Lethality of cyanide in the absence of inhibition of liver cytochrome oxidase. Biochem. Pharmacol. 25, 605– 608. Isom, G. E., Burrows, G. E., and Way, J. L. (1982). Effect of oxygen on the antagonism of cyanide intoxication-cytochrome oxidase, in vivo. Toxicol. Appl. Pharmacol. 65, 250–256. Kalyanaraman, U. P., Kalyanaraman, K., and Cullinan, S. A. (1983). Neuropathy of cyanide intoxication due to laetrile (amygdalin). Cancer 51, 2126–2133. Khan, S., and O’Brien, P. J. (1995). Modulating hypoxia-induced hepatocyte injury by affecting intracellular redox state. Biochim. Biophys. Acta 1269, 153–161. Kirk, M. A., Gerace, R., and Kuling, K. W. (1993). Cyanide and methemoglobin kinetics in smoke inhalation victims treated with the cyanide antidote kit. Ann. Emerg. Med. 22, 1413–1418. Kruszyna, R., Kruszyna, H., and Smith, R. P. (1982). Comparison of hydroxylamine, 4-dimethylaminophenol and nitrite protection against cyanide poisoning in mice. Arch. Toxicol. 49, 191–202. Kuling, K. W. (1991). Cyanide antidotes and fire toxicology. N. Engl. J. Med. 325, 1801–1802. Litchfield, J. T., Jr., and Wilcoxon, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96, 99– 113. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Mayes, R. W. (1991). The toxicological examination of the victims of the British Air Tours Boeing 737 accident at Manchester in 1985. J. Forens. Sci. 36, 179–184. McGuinn, W. D., Baxter, L., Pei, L., Petrikovics, I., Cannon, E. P., and Way, J. L. (1994). Antagonism of the lethal effects of cyanide by a synthetic water-soluble cobalt(III) porphyrin compound. Fundam. Appl. Toxicol. 23, 76–80. Moore, S. J., Norris, J. C., Ho, I. K., and Hume, A. S. (1986). The efficacy of a-ketoglutaric acid in the antagonism of cyanide intoxication. Toxicol. Appl. Pharmacol. 82, 40–44. Moore, S. J., Norris, J. C., Walsh, D. A., and Hume, A. S. (1987). Antidotal use of methemoglobin forming cyanide antagonists in concurrent carbon monoxide/cyanide intoxication. J. Pharmacol. Exp. Ther. 242, 70–73. Niknahad, H., Khan, S., and O’Brien, P. J. (1995). Hepatocyte injury resulting from the inhibition of mitochondrial respiration at low oxygen
AID
TOX 7798
/
6h0c$$$163
04-09-96 09:20:27
191
concentrations involves reductive stress and oxygen activation. Chem.Biol. Interact. 98, 1–18. Niknahad, H., Khan, S., Sood, C., and O’Brien, P. J. (1994). Prevention of cyanide-induced cytotoxicity by nutrients in isolated rat hepatocytes. Toxicol. Appl. Pharmacol. 128, 271–279. Norris, J. C., Utley, W. A., and Hume, A. S. (1990). Mechanism of antagonizing cyanide-induced lethality by a-ketoglutaric acid. Toxicology 62, 275–288. Osuntokun, B. O., Monekosso, G. L., and Wilson, J. (1969). Relationship of a degenerative tropic neuropathy to diet: A report of a field study. Br. Med. J. 1, 547–550. Osuntokun, B. O. (1980). A degenerative neuropathy with blindness and chronic cyanide intoxication of dietary origin. The evidence in Nigerians. In Toxicology in the Tropics (R. L. Smith and E. A. Bababunmi, Eds.), pp. 16–79. Taylor and Francis, London. Peden, N. R., Taha, A., McSorley, P. D., Bryden, G. T., Murdoch, I. B., and Anderson, J. M. (1986). Industrial exposure to hydrogen cyanide: Implications for treatment. Br. Med. J. 293, 538. Pierard, G. E., and Pierard-Franchimont, C. (1993). Dihydroxyacetone test as a substitute for the dansyl chloride test. Dermatology 186, 133–137. Potter, V. R. (1964). Preparation and standardization of cytochrome c. In Manometric Techniques (W. W. Umbreit, R. H. Burris, and J. F. Stouffer, Eds.), pp. 270–273. Burgess, Minneapolis. Rosling, H. (1989). Cassava associated neurotoxicity in Africa. In Proceedings of the 5th International Congress of Toxicology (G. N. Volans, J. Sims, F. M. Sullivan, and P. Turner, Eds.), pp. 605–614. Taylor and Francis, Birghton. Sadoff, L., Fuchs, J., and Hollander, J. (1978). Rapid death associated with laetrile ingestion. J. Am. Med. Assoc. 239, 1532. Schwartz, C., Morgan, R. L., Way, L. M., and Way, L. J. (1979). Antagonism of cyanide intoxication with sodium pyruvate. Toxicol. Appl. Pharmacol. 50, 437–441. Smith, L. (1954). Spectrophotometric assay of cytochrome c oxidase. In Methods of Biochemical Analysis (D. Glick, Ed.), pp. 427–434. Academic Press, New York. Solomonson, L. P. (1981). Cyanide as a metabolic inhibitor. In Cyanide in Biology (B. Vennesland, E. E. Conn, C. J. Knowles, J. Westley, and F. Wissing, Eds.), pp. 11–28. Academic Press, New York. Stanko, R. T., Tietze, D. L., and Arch, J. E. (1992). Body composition, energy utilization, and nitrogen metabolism with a severely restricted diet supplemented with dihydroxyacetone and pyruvate. Am. J. Clin. Nutr. 55, 771–776. Tadic, V. (1992). The in vivo effects of cyanide and its antidotes on rat brain cytochrome oxidase activity. Toxicology 76, 59–67. Vessy, C. J., and Cole, P. V. (1985). Blood cyanide and thiocyanate concentrations produced by long-term therapy with sodium nitroprusside. Br. J. Anaesth. 57, 148–155. Way, J. L., Gibbon, S. L., and Sheehy, M. (1966). Effect of oxygen on cyanide intoxication. I. Prophylactic protection. J. Pharmacol. Exp. Ther. 153, 381–385. Westley, J. L., Adler, H., Westley, L., and Nishida, C. (1983). The sulfurtransferases. Fundam. Appl. Toxicol. 3, 377–382. Yamamoto, H.-A. (1990). Protection against cyanide-induced convulsion with a-ketoglutarate. Toxicology 61, 221–228.
toxa
AP: Tox
138, 186–191 (1996)
0112
Antidotal Effect of Dihydroxyacetone against Cyanide Toxicity in Vivo HOSSEIN NIKNAHAD1
AND
PETER J. O’BRIEN2
Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario, Canada, M5S 2S2 Received November 9, 1995; accepted February 5, 1996
Antidotal Effect of Dihydroxyacetone against Cyanide Toxicity in Vivo. NIKNAHAD, H., AND O’BRIEN, P. J. (1996). Toxicol. Appl. Pharmacol. 138, 186–191. Potassium cyanide (CN) intoxication in mice was found to be effectively antagonized by dihydroxyacetone (DHA), particularly if administered in combination with another CN antidote, sodium thiosulfate. Cyanide-induced convulsions were also prevented by DHA treatment, either alone or in combination with thiosulfate. Injection (ip) of DHA (2 g/kg) 2 min after or 10 min before CN (sc) increased LD50 values of CN (8.7 mg/kg) by factors of 2.1 and 3.0, respectively. Treatment with a combination of DHA and thiosulfate after CN increased the LD50 by a factor of 2.4. Pretreatment with a combination of DHA and thiosulfate (1 g/kg) increased the LD50 of CN to 83 mg/kg. Administration of aketoglutarate (2.0 g/kg), but not pyruvate, 2 min after CN increased the LD50 of CN by a factor of 1.6. Brain, heart, and liver cytochrome oxidase activities were also measured following in vivo CN treatment with and without DHA. Pretreatment with DHA prevented the inhibition of cytochrome oxidase activity by CN and treatment with DHA after CN accelerated the recovery of cytochrome oxidase activity, especially in brain and heart homogenates. DHA is a physiological agent and, therefore, could prove to be a safe and effective antidote for CN, particularly in cases of fire smoke inhalation in which a combination of CN and carbon monoxide is present. In these cases the normally used antidote, sodium nitrite, to induce methemoglobin so as to trap the CN, is contraindicated because some of the oxygen-carrying capacity of the blood will have already been diminished by carbon monoxide. q 1996 Academic Press, Inc.
Because of its rapid lethal effects, cyanide (CN) is used in suicides, homicides, and chemical warfare. Ingestion of CN-containing foods (Osuntokun et al., 1969; Osuntokun 1980; Cook and Coursey, 1981; Rosling, 1989) and occupational exposure to CN in industry have caused serious toxic problems (Blanc et al., 1985; Peden et al., 1986). In medicine, the release of CN from some drugs such as sodium nitroprusside (Davis et al., 1975; Vessy and Cole, 1985) and laetrile (Sadoff et al., 1978; Kalyanaraman et al., 1983) has 1
Current address: Department of Pharmacology & Toxicology, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Fars, Iran. 2 To whom correspondence should be addressed. Fax: (416) 978-8511.
186
0041-008X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AID
TOX 7798
/
6h0c$$$161
also been life threatening. Elevated levels of blood CN have been recorded in victims and survivors of fire smoke inhalation and most of the toxicity and lethality associated with smoke inhalation injury have been attributed to CN (Clark et al., 1981; Cohen and Guzzardi, 1988; Mayes, 1991; Kirk et al., 1993; Barillo et al., 1994). The classical treatment of CN poisoning involves administration of amyl nitrite and sodium nitrite to induce methemoglobin, which reversibly binds the CN (Friedberg, 1968), and administration of thiosulfate which may act by converting CN to thiocyanate with the assistance of the mitochondrial enzyme rhodanese (Himwich and Saunders, 1948). However, nitrites are slow methemoglobin inducers (Kruszyna et al., 1982) and also cause hypotension which can worsen the situation of the patients poisoned with CN (Hall et al., 1989). Furthermore, in the cases of smoke inhalation, induction of methemoglobin formation could be dangerous as a large amount of hemoglobin may already be bound to carbon monoxide (Moore et al., 1987; Hall et al., 1989; Kuling, 1991). The toxic effect of CN has been attributed to its production of histotoxic anoxia by the reversible inhibition of cytochrome c oxidase, the terminal oxidase of the mitochondrial respiratory chain (Solomonson, 1981). Removal of free CN by binding to CN-trapping agents or methemoglobin will result in dissociation of CN from cytochrome oxidase and reactivation of this enzyme (Solomonson, 1981). CN is a nucleophile known to react with various carbonyl groups to form cyanohydrin intermediates. Sodium pyruvate and a-ketoglutarate have therefore been used to trap CN in vivo (Schwartz et al., 1979; Moore et al., 1986; Norris et al., 1990). Previously, we have shown that dihydroxyacetone (DHA), glyceraldehyde, pyruvate, and a-ketoglutarate also rapidly restored CN-inhibited mitochondrial respiration as a result of trapping CN to form cyanohydrins (Niknahad et al., 1994). In the present study, the antidotal effects of DHA against CN poisoning in vivo in mice, alone and in combination with sodium thiosulfate, have been determined and compared. We have found that DHA combined with thiosulfate was the most effective antidotal combination of the agents investigated. DHA could therefore prove an effective antidote for CN poisoning particularly in the cases of smoke
04-09-96 09:20:27
toxa
AP: Tox
187
ANTAGONISM OF CYANIDE LETHALITY BY DIHYDROXYACETONE
inhalation lethality attributed to cyanide when carbon monoxide is present. MATERIALS AND METHODS Animals. Male CD1 mice, weighing 25 to 30 g, were obtained from Charles River Laboratories (Montreal, PQ, Canada). Animals were housed in a temperature-controlled environment with 12-hr light/12-hr dark periods for 2 weeks before experiments and were given feed and tap water ad libitum. Each experimental group was selected randomly from the general mouse population. Chemicals. Potassium cyanide and sodium thiosulfate were purchased from Fisher Scientific Ltd. (Toronto, Canada). Dihydroxyacetone, sodium nitrite, a-ketoglutaric acid, pyruvic acid, and bovine heart cytochrome c were obtained from Sigma Chemical Co. (St. Louis, MO). Other chemicals were of the highest grade commercially available. KCN (2 mg/ml), dihydroxyacetone (500 mg/ml), a-ketoglutarate (500 mg/ml), pyruvate (500 mg/ ml), sodium thiosulfate (500 mg/ml), and sodium nitrite (50 mg/ml) solutions were prepared in normal saline (0.9% NaCl) each day. Lethality studies. For posttreatment studies, KCN at determined doses was given by sc injection and after 2–3 min (as soon as the animal started to convulse), dihydroxyacetone (2 g/kg, ip), a-ketoglutarate (2 g/kg, ip), pyruvate (1 g/kg, ip), sodium thiosulfate (1 g/kg, ip), or sodium nitrite (0.1 g/kg, ip) was injected and the animals were watched for convulsions until 60 min after injection of the antidotes and for 24 hr for survival. For pretreatment studies, sodium nitrite (0.1 g/kg, ip) was given 20 min before CN injection (sc), sodium thiosulfate (1 g/kg, ip) was given 10 min before CN injection, and dihydroxyacetone (2 g/kg, ip), a-ketoglutarate (2 g/kg, ip), and pyruvate (1.0 g/kg, ip) were given 5 min before CN injection and the animals were watched for 60 min for convulsions and for 24 hr for survival. It should be mentioned that pyruvate at the 2 g/kg dose was toxic and caused severe convulsions, stiffness of the tail, and tonic paralysis of limbs in about 5 min and killed all of the animals within 10 min after ip injection. Some of the toxic effects of pyruvate such as partial paralysis of limbs were present even with a dose of 1 g/kg, but none of the animals treated died. The LD50 values are based on 24 hr mortality. Experimental values were obtained for at least four groups of mice with each group containing 6 to 10 mice. Measurement of cytochrome oxidase activity. KCN (7 mg/kg) was injected sc and dihydroxyacetone (1 g/kg, ip), sodium nitrite (0.1 g/kg, ip), or thiosulfate (1 g/kg, ip) was injected 2–3 min after CN. Control animals were receiving normal saline (0.9% NaCl) injections rather than drugs. At specific time points following CN administration, the animals were killed by decapitation and the brain, heart, or liver was removed. Excess blood from the heart or liver was removed by blotting with filter paper. Homogenates (10% w/v) were prepared in chilled (17C) distilled water using a motor drive Teflon tissue homogenizer. Homogenates were subsequently diluted to 1% (w/v) with chilled (17C) 30 mM Tris–HCl buffer, pH 7.4. Cytochrome oxidase activity was determined by the rate of oxidation of reduced cytochrome c, as previously explained (Isom et al., 1982). The assay mixture contained 0.1 ml of homogenate (1% w/v), 2.7 ml of 30 mM Tris–HCl buffer, pH 7.4, and 0.2 ml of 1 mM reduced cytochrome c solution, prepared as previously described by Potter (1964). The absorbance at 550 nm was recorded continuously over a 5-min period using a DW 2000 spectrophotometer. At 5 min, 40 ml of saturated potassium ferricyanide solution was added to completely oxidize the cytochrome c and the cytochrome c oxidase activity was calculated as the first-order rate constant (Smith, 1954) based on the protein concentration of 1% homogenates. The cytochrome c oxidase activities are expressed as percentage of control values in order to compare the enzymatic activities of different treatments. Measurement of protein content of homogenates. Protein content of brain, heart, or liver (1% w/v) homogenates was determined by the Lowry method (Lowry et al., 1951).
AID
TOX 7798
/
6h0c$$$162
04-09-96 09:20:27
TABLE 1 Antidotal Effect of Dihydroxyacetone, Sodium Thiosulfate, aKetoglutarate, Pyruvate, and Sodium Nitrite on the Lethality of Potassium Cyanide
Treatments (g/kg)a Saline DHAd (1.0) DHA (2.0) DHA (4.0) a-KG (2.0) Pyruvate (1.0) NaNO2 (0.1) Na2S2O3 (1.0) DHA (2.0) / Na2S2O3 (1.0) NaNO2 (0.1) / Na2S2O3 (1.0)
LD50 value (mg/kg)b (95% confidence interval) 8.7 13.5 17.8 22.5 14.2 9.4 12.6 10.9 20.5 13.5
(7.9–9.4) (12.8–14.2) (16.0–19.8) (20.2–25.1) (13.0–15.5) (8.3–10.6) (11.6–13.7) (9.9–12.0) (18.3–23.0) (12.7–14.4)
Potency ratioc
1.55 2.05 2.58 1.63 1.08 1.45 1.25 2.36 1.55
1.00 (1.47–1.63) (1.89–2.22) (2.32–2.87) (1.55–1.71) (0.97–1.19) (1.37–1.54) (1.09–1.43) (2.19–2.54) (1.43–1.68)
a
DHA, NaNO2, and Na2S2O3 were injected (ip) 3 min after cyanide (sc). Each LD50 value was obtained from four to eight graded doses of KCN administered to four groups of mice with six to eight mice per group. c Potency ratio Å LD50 of KCN with antidote(s)/LD50 of KCN without antidote. d DHA, dihydroxyacetone; a-KG, a-ketoglutarate. b
Statistics. The efficacy of the antagonists is shown as a potency ratio, calculated by dividing the LD50 values for CN with antidote by the LD50 value of CN without antidote. The LD50 values were obtained by the method of Litchfield and Wilcoxon (1949) with a 95% confidence level. For cytochrome oxidase activity studies the significant differences between control and experimental groups were calculated using Student’s t test and significantly different groups were chosen when p £ 0.05.
RESULTS
Administration (sc) of cyanide to mice first resulted in a short period (1–2 min) of calmness followed by convulsions which started 2–3 min after CN injection. Death occurred at 4–10 min with an LD50 for CN of 8.7 (7.9–9.4) mg/kg which was similar to that previously reported (Way et al., 1966; Schwartz et al., 1979; McGuinn et al., 1994; Yamamoto, 1990). The effects of DHA, a-ketoglutarate, pyruvate, sodium nitrite, and sodium thiosulfate given intraperitoneally 2–3 min after subcutaneous injection of CN on the LD50 of CN in mice are compared in Table 1. Administration of 1, 2, or 4 g/kg of DHA increased the LD50 value about 1.6-, 2.1-, and 2.6-fold, respectively (Table 1). aKetoglutarate (2 g/kg) increased the LD50 of CN only 1.6fold (equal to 1 g/kg of DHA) and pyruvate (1 g/kg) did not affect the LD50 value of CN. Sodium nitrite, but not sodium thiosulfate, also slightly increased the LD50 value for CN but was less effective than DHA. However, in most of the cases sodium nitrite but not sodium thiosulfate prolonged the survival time. Sodium thiosulfate slightly increased the antidotal effectiveness of
toxa
AP: Tox
188
NIKNAHAD AND O’BRIEN
TABLE 2 Prophylactic Effects of Dihydroxyacetone, Sodium Thiosulfate, a-Ketoglutarate, Pyruvate, and Sodium Nitrite on the Lethality of Potassium Cyanide
Treatments (g/kg)a Saline DHAd (2.0) a-KG (2.0) Pyruvate (1.0) NaNO2 (0.1) Na2S2O3 (1.0) DHA (2.0) / NaNO2 (0.1) DHA (2.0) / Na2S2O3 (1.0) NaNO2 (0.1) / Na2S2O3 (1.0)
LD50 value (mg/kg, ip)b (95% confidence interval)
Potency ratioc (95% confidence)
8.7 26.0 28.4 14.2 23.5 21.3 47.8 83.6 47.3
2.99 3.26 1.63 2.70 2.44 5.49 9.61 5.44
(7.9–9.4) (24.5–27.6) (25.3–31.9) (12.1–16.7) (20.6–26.8) (19.3–23.3) (42.8–53.2) (78.9–88.6) (45.1–49.7)
1.00 (2.76–3.24) (3.01–3.53) (1.52–1.75) (2.38–2.91) (2.27–2.62) (5.03–5.99) (8.71–10.60) (5.19–5.71)
a DHA (ip), NaNO2 (ip), and Na2S2O3 (ip) were injected 10, 20, and 15 min respectively before the administration of cyanide (sc). b Each LD50 value was obtained from four to eight graded doses of KCN administered to four to six groups of mice with six to eight mice per group. c Potency ratio Å LD50 of KCN with antidote(s)/LD50 of KCN without antidote. d DHA, dihydroxyacetone; a-KG, a-ketoglutarate.
DHA (Table 1). The combination of DHA with a-ketoglutarate or pyruvate had additive antidotal effects against CN (data not shown). Table 2 shows the effect of pretreatment with different antidotes on the lethality of CN in mice. Pretreatment with sodium nitrite or sodium thiosulfate increased the LD50 for CN 2.7 and 2.4 times, respectively, values similar to those previously reported (Yamamoto, 1990; Moore et al., 1986; McGuinn et al., 1994). However, pretreatment with 2 g/kg DHA or a-ketoglutarate was more effective than that with pyruvate, sodium nitrite, or sodium thiosulfate in antagonizing CN lethality. DHA and thiosulfate were synergistic and were the most effective antidotes found by which the LD50 for CN was increased more than ninefold. In all cases that DHA was administered, animals did not convulse. The time course for the effect of CN, with and without DHA treatment, on cytochrome oxidase activity in mouse brain, heart, and liver homogenates was investigated. Brain, heart, or liver cytochrome oxidase was inactivated very rapidly after sc administration of 7 mg/kg CN. The maximum inhibition of cytochrome oxidase occurred around 5 min after CN injection. The cytochrome oxidase activity of brain and liver declined to approximately 37% of control at 5 min following CN treatment (Figs. 1A and 1C), whereas that of heart declined to about 25% of control at 5 min (Fig. 1B). Cytochrome oxidase activities started to recover around 10 min and returned to 70, 64, and 92% of control at 30 min in brain, heart, and liver, respectively. Cytochrome oxidase activity in all these organs recovered 60 min after CN injection. If DHA was administered 2 min after
AID
TOX 7798
/
6h0c$$$162
04-09-96 09:20:27
CN injection the cytochrome oxidase activity in brain and heart started to recover at 5 min after CN injection (Figs. 1A and 1B). However, the recovery in the liver was less pronounced (Fig. 1C). If DHA was injected 5 min before CN, the inhibition of cytochrome oxidase activity in brain, heart, and liver homogenates was partially prevented (Figs. 1A–1C). Maximal inhibition occurred at 10 min following CN injection. Cytochrome oxidase activities at 10 min were about 65, 61, and 59% of control in brain, heart, and liver, respectively. Administration of thiosulfate after CN caused a rapid recovery of cytochrome oxidase activity in brain, heart, and liver (Table 3). Sodium nitrite was much less effective than thiosulfate or DHA in restoring cytochrome oxidase activity. Pretreatment with thiosulfate was as effective as or better than DHA in preventing the inactivation of cytochrome oxidase of brain, heart, and liver (Table 4). Pretreatment with sodium nitrite, however, effectively prevented the inhibition of cytochrome oxidase in brain and liver but not heart (Table 4). DISCUSSION
We have previously shown that DHA binds CN and restores mitochondrial respiration inhibited by CN in isolated hepatocytes (Niknahad et al., 1994). In the present study, we have shown that DHA provided a better antidotal effect against the lethal effects of CN than sodium nitrite or sodium thiosulfate alone. The combination of DHA and thiosulfate had a strong synergistic effect and was much more effective than the sodium nitrite and thiosulfate combination. DHA could have a potential therapeutic advantage over thiosulfate and sodium nitrite as a CN antidote as it is a physiological component of the cell whose phosphorylated form is a part of the glycolysis pathway. Previously, we showed that the administration of DHA to hepatocytes increased ATP and lactate formation in hypoxic or antimycin A-treated hepatocytes (Niknahad et al., 1995; Khan and O’Brien, 1995). Since at least one of the antidotal mechanisms of DHA involves trapping the CN to form a cyanohydrin, it could have a very rapid onset of action compared to those agents which induce methemoglobin to trap CN (Friedberg, 1968). DHA had a stronger antidotal effect than pyruvate or aketoglutarate on CN lethality if administered after CN. However, although much better than pyruvate, DHA had a similar effect to that of a-ketoglutarate, if administered before CN injection. This may be due to a faster absorption and distribution of DHA to the site of CN localization. All of the CN antidotes examined were much more effective when given prophylactically than when given after CN. One reason for this is possibly more complete absorption from the site of injection and better distribution of the antidotes to the site of detoxification or to the target organs, when given prophylactically. Sodium thiosulfate is known
toxa
AP: Tox
ANTAGONISM OF CYANIDE LETHALITY BY DIHYDROXYACETONE
189
FIG. 1. The effects of treatment with dihydroxyacetone on cyanide-induced inhibition of cytochrome oxidase in brain (A), heart (B), and liver (C) homogenates. Dihydroxyacetone (1 g/kg) was injected ip 10 min before (l), or 2 min after (s), sc injection of 7 mg/kg cyanide (j). Cytochrome oxidase activity was measured as explained under Materials and Methods. Results shown are mean { SD of three to five experiments. The first-order rate constants for cytochrome oxidase activity of control brain, heart, and liver were 0.058 { 0.003, 0.107 { 0.015, and 0,072 { 0.005 mg01 sec01, respectively.
to penetrate the cell membrane very slowly and also needs to cross the mitochondrial membrane as rhodanese is located in the mitochondrial matrix (Westly et al., 1983). Sodium nitrite, which is believed to antagonize CN lethality largely by inducing methemoglobin formation, is, however, a slow methemoglobin inducer with maximum methemoglobin formation at 30 min following its injection to mice (Moore et al., 1987). Cyanide-trapping agents DHA, a-ketoglutarate, and pyruvate were also more effective prophylactically possibly as a result of a better distribution to the target organs such as brain or heart. Alternatively, these agents may bind more readily to CN when given prophylactically as they compete for CN with cytochrome oxidase. The preventive and antidotal effects of DHA against CN
AID
TOX 7798
/
6h0c$$$163
04-09-96 09:20:27
were accompanied by the prevention or faster restoration of cytochrome oxidase activity in brain, heart, and liver. The restoration of the oxidase activity after CN treatment was faster in liver than in heart or brain. The faster restoration of cytochrome oxidase activity in the liver is not surprising since liver has a high level of rhodanese activity (Devlin et al., 1989). Skeletal muscle is also known to possess high rhodanese activity (Devlin et al., 1989) but restoration of cytochrome oxidase activity of heart was not faster than that of brain and was much slower than that of liver. It is possible that rhodanese activity of the heart cells is lower than that of liver or maybe more CN is localized in the heart. A more pronounced inhibition by CN of heart cytochrome oxidase activity than brain or liver cytochrome oxidase activity is probably because of the greater
toxa
AP: Tox
190
NIKNAHAD AND O’BRIEN
concentration of mitochondria in the heart cells. Heart cytochrome oxidase activity was also restored better following administration of DHA than following administration of sodium nitrite or thiosulfate. This may be one of the reasons for the better antidotal effectiveness of DHA compared to sodium nitrite or thiosulfate. Although the antidotal effectiveness of DHA was greater than that of thiosulfate at preventing CN lethality, its effectiveness at restoring cytochrome oxidase activity was similar. On the other hand, pretreatment with sodium nitrite prevented CN lethality to a similar degree as DHA, although the heart cytochrome oxidase activity was still inactivated. These suggest that cytochrome oxidase inhibition is not the only toxic mechanism involved in the lethality of CN. Lethality by CN was also reported without liver or brain cytochrome oxidase inhibition (Isom and Way, 1976; Tadic, 1992). It is not clear why cytochrome oxidase activity in the brain, heart, or liver of CN-treated animals reaches levels higher than control values 60 min after treatment. However, the lower activity of the enzyme at this time point in animals treated with DHA, before or after CN treatment, probably indicates that the DHA–CN cyanohydrin slowly releases CN as DHA is metabolized in the body. Previously, we showed that glycolytic substrates can prevent CN or antimycin A toxicity in isolated cells by averting reductive stress and/or supplying ATP (Niknahad et al., 1995). DHA can also supply ATP via glycolysis, and it has been used as a dietary regimen in combination with pyruvate in human studies (Stanko et al., 1992). As a medicine, DHA is already in use in dermatological tests (Pierard and Pierard-
TABLE 4 Prophylactic Effects of Dihydroxyacetone, Sodium Thiosulfate, a-Ketoglutarate, and Sodium Nitrite on the Inhibition of Cytochrome Oxidase Activity by Cyanide Cytochrome oxidase activity (% of control) Treatments (g/kg) Cyanide (0.007) / DHAa (2.0) /NaNO2 (0.1) /Na2S2O3 (1.0) / a-KG (2.0)
Brain 36.7 71.2 84.7 82.5 74.4
{ { { { {
3.6* 5.0** 5.8** 5.7** 4.6**
Heart 24.7 63.0 38.3 62.2 56.8
{ { { { {
4.1* 4.5** 3.5 3.8** 4.3**
Liver 38.1 60.7 77.5 85.6 63.7
{ { { { {
3.8* 3.5** 5.2** 6.2** 4.4**
Note. DHA (ip), NaNO2 (ip), and Na2S2O3 (ip) were injected 10, 20, and 15 min respectively before the administration of cyanide (sc) to mice. Cytochrome oxidase activity was measured 7 min after Cn injection as explained under Materials and Methods. Each value is the mean ({SD) of three to five different experiments. a DHA, dihydroxyacetone; a-KG, a-ketoglutarate. * Significantly lower than control (p £ 0.001). ** Significantly higher than cyanide-treated (p £ 0.005).
Franchimont, 1993). The results of the present study suggest that DHA may be a promising safe CN antidote which can be used in combination with other CN antidotes, especially thiosulfate. In particular, DHA may be considered as a substitute for sodium nitrite in the cases of CN poisoning associated with fire smoke inhalation in which the oxygen-carrying capacity of the blood has already been compromised by carbon monoxide as a result of carbon monoxy hemoglobin complex formation. ACKNOWLEDGMENT
TABLE 3 Antidotal Effect of Dihydroxyacetone, Sodium Thiosulfate, aKetoglutarate, and Sodium Nitrite on the Inhibition of Cytochrome Oxidase Activity by Cyanide
Hossein Niknahad was financially supported by a scholarship from Iran’s Ministry of Health and Medical Education.
REFERENCES Cytochrome oxidase activity (% of control) Treatments (g/kg) Cyanide (0.007) / DHAa (2.0) / NaNO2 (0.1) / Na2S2O3 (1.0) / a-KG (2.0)
Brain 37.5 57.2 41.8 51.6 46.3
{ { { { {
4.0* 3.2** 6.3 5.7** 3.6**
Heart 26.4 47.3 28.0 44.2 38.6
{ { { { {
2.8* 3.6** 3.7 3.8** 3.5**
Liver 37.4 49.2 40.3 52.5 41.7
{ { { { {
3.5* 2.9** 4.4 6.2** 4.4
Note. DHA (ip), NaNO2 (ip), and Na2S2O3 (ip) were injected 2 min after the administration of cyanide (sc) to mice. Cytochrome oxidase activity was measured 7 min after CN injection as explained under Materials and Methods. Each value is the mean ({SD) of three to five different experiments. a DHA, dihydroxyacetone; a-KG, a-ketoglutarate. * Significantly lower than control (p £ 0.05). ** Significantly higher than cyanide-treated or cyanide / sodium nitritetreated (p £ 0.05).
AID
TOX 7798
/
6h0c$$$163
04-09-96 09:20:27
Barillo, D. J., Goode, R., and Esh, V. (1994). Cyanide poisoning in victims of fire: Analysis of 364 cases and review of the literature. J. Burn Care Rehabil. 15, 46–57. Blanc, P., Hogan, M., Malin, K., Hryhorczuk, D., Hessel, S., and Bernard, B. (1985). Cyanide intoxication among silver reclaiming workers. J. Am. Med. Assoc. 253, 367–371. Clark, C. J., Campbell, D., and Reid, W. H. (1981). Blood carboxyhaemoglobin and cyanide levels in fire survivors. Lancet 1, 1332–1335. Cohen, M. A., and Guzzardi, L. J. (1988). The smoke inhalation and cyanide poisoning. Am. J. Emerg. Med. 6, 203–204. Cook, R. D., and Coursey, D. B. (1981). Cassava: A major cyanide-containing food crop. In Cyanide in Biology (B. Vennesland, E. E. Conn, C. J. Knowles, J. Westley, and F. Wissing, Eds.), pp. 93–114. Academic Press, New York. Davis, D. W., Griess, L., Kadar, D., and Stewart, D. J. (1975). A sudden death associated with the use of sodium nitroprusside for induction of hypotension during anaesthesia. Can. Anaesth. Soc. J. 22, 547–552.
toxa
AP: Tox
ANTAGONISM OF CYANIDE LETHALITY BY DIHYDROXYACETONE Devlin, D. J., Mills, J. W., and Smith, R. P. (1989). Histochemical localization of rhodanese activity in rat liver and skeletal muscle. Toxicol. Appl. Pharmacol. 97, 247–255. Friedberg, K. D. (1968). Antidotes by cyanide poisoning. Arch. Toxikol. 24, 41–48. Hall, A. H., Kuling, K. W., and Rumack, B. H. (1989). Suspected cyanide poisoning in smoke inhalation: Complications of sodium nitrite therapy. J. Toxicol. Clin. Exp. 9, 3–9. Himwich, W. A., and Saunders, J. P. (1948). Enzymatic conversion of cyanide to thiocyanate. Am. J. Physiol. 153, 348–354. Isom, G. E., and Way, J. L. (1976). Lethality of cyanide in the absence of inhibition of liver cytochrome oxidase. Biochem. Pharmacol. 25, 605– 608. Isom, G. E., Burrows, G. E., and Way, J. L. (1982). Effect of oxygen on the antagonism of cyanide intoxication-cytochrome oxidase, in vivo. Toxicol. Appl. Pharmacol. 65, 250–256. Kalyanaraman, U. P., Kalyanaraman, K., and Cullinan, S. A. (1983). Neuropathy of cyanide intoxication due to laetrile (amygdalin). Cancer 51, 2126–2133. Khan, S., and O’Brien, P. J. (1995). Modulating hypoxia-induced hepatocyte injury by affecting intracellular redox state. Biochim. Biophys. Acta 1269, 153–161. Kirk, M. A., Gerace, R., and Kuling, K. W. (1993). Cyanide and methemoglobin kinetics in smoke inhalation victims treated with the cyanide antidote kit. Ann. Emerg. Med. 22, 1413–1418. Kruszyna, R., Kruszyna, H., and Smith, R. P. (1982). Comparison of hydroxylamine, 4-dimethylaminophenol and nitrite protection against cyanide poisoning in mice. Arch. Toxicol. 49, 191–202. Kuling, K. W. (1991). Cyanide antidotes and fire toxicology. N. Engl. J. Med. 325, 1801–1802. Litchfield, J. T., Jr., and Wilcoxon, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96, 99– 113. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Mayes, R. W. (1991). The toxicological examination of the victims of the British Air Tours Boeing 737 accident at Manchester in 1985. J. Forens. Sci. 36, 179–184. McGuinn, W. D., Baxter, L., Pei, L., Petrikovics, I., Cannon, E. P., and Way, J. L. (1994). Antagonism of the lethal effects of cyanide by a synthetic water-soluble cobalt(III) porphyrin compound. Fundam. Appl. Toxicol. 23, 76–80. Moore, S. J., Norris, J. C., Ho, I. K., and Hume, A. S. (1986). The efficacy of a-ketoglutaric acid in the antagonism of cyanide intoxication. Toxicol. Appl. Pharmacol. 82, 40–44. Moore, S. J., Norris, J. C., Walsh, D. A., and Hume, A. S. (1987). Antidotal use of methemoglobin forming cyanide antagonists in concurrent carbon monoxide/cyanide intoxication. J. Pharmacol. Exp. Ther. 242, 70–73. Niknahad, H., Khan, S., and O’Brien, P. J. (1995). Hepatocyte injury resulting from the inhibition of mitochondrial respiration at low oxygen
AID
TOX 7798
/
6h0c$$$163
04-09-96 09:20:27
191
concentrations involves reductive stress and oxygen activation. Chem.Biol. Interact. 98, 1–18. Niknahad, H., Khan, S., Sood, C., and O’Brien, P. J. (1994). Prevention of cyanide-induced cytotoxicity by nutrients in isolated rat hepatocytes. Toxicol. Appl. Pharmacol. 128, 271–279. Norris, J. C., Utley, W. A., and Hume, A. S. (1990). Mechanism of antagonizing cyanide-induced lethality by a-ketoglutaric acid. Toxicology 62, 275–288. Osuntokun, B. O., Monekosso, G. L., and Wilson, J. (1969). Relationship of a degenerative tropic neuropathy to diet: A report of a field study. Br. Med. J. 1, 547–550. Osuntokun, B. O. (1980). A degenerative neuropathy with blindness and chronic cyanide intoxication of dietary origin. The evidence in Nigerians. In Toxicology in the Tropics (R. L. Smith and E. A. Bababunmi, Eds.), pp. 16–79. Taylor and Francis, London. Peden, N. R., Taha, A., McSorley, P. D., Bryden, G. T., Murdoch, I. B., and Anderson, J. M. (1986). Industrial exposure to hydrogen cyanide: Implications for treatment. Br. Med. J. 293, 538. Pierard, G. E., and Pierard-Franchimont, C. (1993). Dihydroxyacetone test as a substitute for the dansyl chloride test. Dermatology 186, 133–137. Potter, V. R. (1964). Preparation and standardization of cytochrome c. In Manometric Techniques (W. W. Umbreit, R. H. Burris, and J. F. Stouffer, Eds.), pp. 270–273. Burgess, Minneapolis. Rosling, H. (1989). Cassava associated neurotoxicity in Africa. In Proceedings of the 5th International Congress of Toxicology (G. N. Volans, J. Sims, F. M. Sullivan, and P. Turner, Eds.), pp. 605–614. Taylor and Francis, Birghton. Sadoff, L., Fuchs, J., and Hollander, J. (1978). Rapid death associated with laetrile ingestion. J. Am. Med. Assoc. 239, 1532. Schwartz, C., Morgan, R. L., Way, L. M., and Way, L. J. (1979). Antagonism of cyanide intoxication with sodium pyruvate. Toxicol. Appl. Pharmacol. 50, 437–441. Smith, L. (1954). Spectrophotometric assay of cytochrome c oxidase. In Methods of Biochemical Analysis (D. Glick, Ed.), pp. 427–434. Academic Press, New York. Solomonson, L. P. (1981). Cyanide as a metabolic inhibitor. In Cyanide in Biology (B. Vennesland, E. E. Conn, C. J. Knowles, J. Westley, and F. Wissing, Eds.), pp. 11–28. Academic Press, New York. Stanko, R. T., Tietze, D. L., and Arch, J. E. (1992). Body composition, energy utilization, and nitrogen metabolism with a severely restricted diet supplemented with dihydroxyacetone and pyruvate. Am. J. Clin. Nutr. 55, 771–776. Tadic, V. (1992). The in vivo effects of cyanide and its antidotes on rat brain cytochrome oxidase activity. Toxicology 76, 59–67. Vessy, C. J., and Cole, P. V. (1985). Blood cyanide and thiocyanate concentrations produced by long-term therapy with sodium nitroprusside. Br. J. Anaesth. 57, 148–155. Way, J. L., Gibbon, S. L., and Sheehy, M. (1966). Effect of oxygen on cyanide intoxication. I. Prophylactic protection. J. Pharmacol. Exp. Ther. 153, 381–385. Westley, J. L., Adler, H., Westley, L., and Nishida, C. (1983). The sulfurtransferases. Fundam. Appl. Toxicol. 3, 377–382. Yamamoto, H.-A. (1990). Protection against cyanide-induced convulsion with a-ketoglutarate. Toxicology 61, 221–228.
toxa
AP: Tox