TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
14 (1990)
16305-3
Age-Related Changes in Toxicity and Biotransformation of Potassium Cyanide in Male C57BL/6N Mice TIMOTHY Experimental
F. MCMAHON
AND LINDA
S. BIRNBAUM”~
Tnxicologv Branch, National Institute of Environmental Research Triangle Park, North Carolina -77709
Received
Februar~~
6. 1990; accepted
May
Health
Sciences,
24. I990
Age-Related Changes in Toxicity and Biotransformation of Potassium Cyanide in Male C57BL/6N Mice. MCMAHON, T . F.. AND BIRNBAUM, L. S. (1990). To.uicol. Appl. Pharmacol. 105. 305-3 14. Age-related changes in toxicity and biotransformation of KCN. an ubiquitous environmental toxicant, have not been previously examined. Male C57BL/6N mice aged 2-3 (young), lo- 12 (middle-aged), and 25-30 (old) months were administered KCN at I, 2,4, and 6 mg/kg po, and toxic manifestations were monitored for up to 2 hr. The toxic response to KCN (prostration and labored breathing) was significantly greater in 10-t 2 and 25-30 month vs that in 2-3 month mice at 4 and 6 mg/kg KCN. The basis for this age-related difference in in viva toxicity was examined by studying biotransformation of KCN to thiocyanate by liver and brain rhodanese (RHO), as well as activity of liver and brain cytochrome oxidase (C-OX), inhibition of C-OX by KCN. and activity of /!I-mercaptopyruvate transsulfurase (MT). Tissue and blood levels of CN- following a toxic dose of 6 mg/kg KCN were also measured. No age-related differences were observed in the specific activity of liver and brain RHO, MT, or C-OX. In addition. no differences were observed in the percentage inhibition of C-OX by KCN, or in the K, for inhibition of brain and liver C-OX. However, activity of brain RHO on a per gram tissue basis was significantly lower in lo-12 and 25-30 month vs that in 2-3 month mice. Liver and blood concentrations of CN- were not significantly different in 2-3 vs lo- I2 month mice following treatment with 6 mg/kg KCN; however, significantly greater concentrations of CN- were observed at 4 and 25 min in brains of lo-12 month mice compared to that in 2-3 month mice. These results indicate that increased sensitivity to KCN in older mice may be due in part to a decrease in the amount of brain RHO and altered tissue kinetics of CN- following a toxic dose in older mice. Q 1990 Academic Press. Inc.
The presence of cyanide (CN-) in the environment is widespread. This potent toxicant is found as a component of cigarette and polymer pyrolysis smokes (Way, 1984), tear gas (Frankenberg and Sorbo, 1973), and ver’ To whom correspondence and reprint requests should be addressed at USEPA. Environmental Toxicology Division. MD-66. Research Triangle Park, NC 2771 I. ’ Current address: Environmental Toxicology Division, Health Effects Research Laboratory. USEPA, Research Triangle Park, NC 277 1 I,
305
micidal fumigants (Ballantyne, 1988), while cyanogenic glycosides are present in over 2000 species of plants (Vennesland et al., 1982). In addition, CN- is formed as a primary product from the biotransformation of several aliphatic nitriles which are used in the manufacture of synthetic fibers, resins, plastics, pharmaceuticals, and vitamins (Silver et al., 1982; Willhite and Smith, 1981). Thus, the potential risk of oral or inhalation exposure to CN-, or precursors of CN-, may be very great. Because of the widespread natural occurrence of CN-, it is not surprising that several 0041008X/90
$3.00
Copyright 0 1990 by Academx Ress. Inc Ail rights of reproduction in any fwm rescrvrd
306
MCMAHON
AND BIRNBAUM
mechanisms are operative for CN- detoxication in viva. The primary detoxication reaction is believed to be formation of thiocyanate (SCN-) from CN-, catalyzed by the enzyme rhodanese (RHO; thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1.), which is present mainly in hepatic mitochondria, and to a lesser extent in mitochondria of brain and heart (Rutkowski et al., 1986). Biotransformation by RHO can account for between 6070% of an administered dose of CN- in experimental animals (Fig. 1). Other pathways for CN- detoxication include formation of 2-imino-thiazolidine-4carboxylic acid through the nonenzymatic reaction of CN- with cystine, oxidation to formic acid and COZ through formation of cyanate, and trapping as the cyano group of vitamin Bi2 (Ahmed et al., 1985) (Fig. 1). Formation of 2-imino-thiazolidine-4-carboxylic acid may account for up to 15% of an administered dose of CN- in rats (Wood and Cooley, 1956) while formation of CO* accounts for approximately 2% of an administered dose of KCN in mice (Johnson and Isom, 1985). The toxicologic significance of CN- detoxication by combination with cystine or oxidation to CO2 has not been directly assessed. However, a reduced ability to detoxify cyanide by reaction with cobalamin has been associated with the development of tobacco amblyopia (Freeman, 1988), while Leber’s hereditary optic neuropathy has been linked to deficiency of RHO (Poole and Kind, 1986). Aging has been shown to affect the disposition and toxicity of a number of drugs and xenobiotics (McMahon and Birnbaum, 1990). With regard to CN-, preliminary studies in our laboratory demonstrated that older (10 mo) male C567BL/6N mice were more sensitive than 3 month mice of the same strain to the acute toxic effects of KCN at 4 and 6 mg/kg. In light of the widespread occurrence of CN-, the potential for lifetime exposure to this toxicant, and our preliminary in vivo observations, the present study was
Expired HCN
Cyanocobalamin
2.iminothiazolidineI-carboxylic (15%)
Vitamin
acid
B 2
CNO Cyanate
1 HCOOH 11 Formate (excreted)
One
carbon
pool
I co2 (2%)
FIG. 1. Biotransformation
of cyanide.
undertaken to examine in more detail the basis for this age-related difference in sensitivity to CN-. Differences in CN- toxicity were examined in vivo in male C57BL/6N mice aged 2-3 (young), lo- 12 (middle-aged), and 2530 (old) months over a range of CN- doses (1,2,4, and 6 mg/kg CN- PO). Potential agerelated differences in liver and brain RHO, C-OX (the target enzyme for CN-), and ,& mercaptopyruvate transsulfurase (MT; an enzyme which supplies sulfur for detoxication of CN- by RHO) were examined in vitro, as well as possible differences with age in sensitivity of liver and brain C-OX to inhibition by KCN. In addition, tissue and blood levels of CN- were measured following a toxic dose of 6 mg/kg KCN in young and middle aged mice. METHODS Chemiculs. KCN was obtained from Aldrich Chemical Co. (Milwaukee, WI). Caps buffer, pyridine, sodium
KCN BIOTRANSFORMATION thiosulfate, and barbituric acid were obtained from Sigma Chemical Co. (St Louis, MO). Sucrose and trichloroacetic acid were obtained from J.T. Baker (Phillipsburg, NJ). Sodium mercaptopyruvate was purchased from ICN Biomedical (Irvine, CA). Reagents for preparing phosphate buffers were obtained from Mallinkrodt (Paris, KY). All chemicals were of the highest grade and purity commercially available. Animals and treatments. Male C57BL/6N mice aged 2 months were obtained from Charles River (Raleigh, NC), while retired breeder mice aged IO- I2 months were obtained from Frederick Cancer Research Facility (Frederick, MD). Mice aged 25-30 months were obtained from an aging cohort of retired breeders maintained at NlEHS originally from Frederick. Young mice were housed in groups of five, while older mice were housed singly. All mice were kept under conditions of constant temperature (23 -C2°C) humidity (50 f 5%) and lighting (12-hr light/dark cycle), and were fed Rodent Chow (NIH-31) and water ad libitum. Mice were acclimated for at least 7 days before use in either in vitro or in vivo experiments. For in vivo dosing experiments, 10 mice/age group. singly housed, were administered 1, 2, 4, or 6 mg/kg KCN po in a dosing volume of 10 ml 0.9% NaCl/kg. The same groups of mice were used for each dose of KCN. but were allowed a recovery period of 2 weeks between each dose. Doses of KCN were selected to include both nontoxic ( 1 and 2 mp/kg) and toxic (4 and 6 mg/kg) doses (Rutkowski et al., 1986). Following administration of KCN, mice were observed for the presence or absence of toxic signs (prostration, labored breathing, and tremors) at 5-min intervals up to 30 min, and thereafter every 15 min up to 2 hr. The number of mice in each treatment group manifesting signs of KCN toxicity at each time point was noted. Following the last dose (6 mg/kg) of KCN, mice were allowed to recover and then were used for in vitro enzyme measurements. Enzyme assays.For all enzyme assays,tissues were obtained from mice killed by decapitation under CO* anesthesia. RHO activity in both liver and brain mitochondria was assayed according to a modification of the method of Pettersen and Cohen ( 1985). Organs were removed, weighed, and then homogenized (l/3 and l/4 w/ v, respectively) in ice-cold T&buffered 0.25 M sucrose, pH 7.4. Mitochondria were prepared from tissue homogenates by differential centrifugation according to a modification of the method of Uemura and Chiesara (1976). The washed mitochondrial pellets were resuspended in 1 ml of0.01 M NaPO,/O. 15 M KCL, pH 7.4. Mitochondrial protein (0.1 mg) was then added to prewarmed (37°C) 0.1 M NaP04 buffer. pH 7.4, containing 5 mM sodium thiosulfate in 15-m] test tubes. Concentrations of KCN ranging from 0.46-30 mM were added to initiate the reaction. The reaction volume was 500 ~1. Reactions were carried out at 37°C for 2 min in a shaking water bath, and
AND TOXICITY
IN MICE
307
were terminated by the addition of cold 10% trichloroacetic acid solution containing 5.6 ml formaldehyde/liter. Samples were processed and assayed for thiocyanate as previously described (Pettersen and Cohen, 1985). Apparent K, and V,,,, for hepatic RHO were determined from Lineweaver-Burk transformation of saturation data using PHARM/PCS on an IBM PS/2 computer (Tallarida and Murray, 1987). Reactions were linear from l-5 min and 0.1-0.5 mg protein in liver, and from 2-4 min and 0.2-0.4 mg protein in brain. Increasing the concentration of thiosulfate to 10 mM in either liver or brain incubationsdid not affect theextent ofthe reaction. Blank values were obtained in the absence of thiosulfate. mitochondrial protein, and KCN. and were subtracted from sample values. C-OX activity was determined in liver and brain homogenates according to a modification of the method of Pettersen and Cohen (1985). Tissues were weighed. homogenized with a glass tissue homogenizer using a Teflon pestle, l/10 w/v (liver) or l/2 w/v (brain), in 0.03 M NaPO,, pH 7.4, and diluted l/50 or I/ 100 before use in assays.Incubations were performed at 37°C in a Beckman (Fullerton. CA) DU-8B spectrophotometer using the Kinetics II compuset module. Reaction mixtures contained 2.4 ml 0.03 M NaPO,, pH 7.4. 0.5 ml cytochrome c ( 10v4 M), reduced according to the method of Potter ( 1964), and I5 (liver) or 20 (brain) pg homogenate protein. Reactions were monitored for up to 3 min at 550 nm. The activity ofC-OX was calculated from the initial linear rate using an extinction coefficient of 18.5 mM -’ cm-’ (Hodges and Leonard, 1974). In experiments to determine age-related differences in sensitivity of liver and brain to inhibition of cytochrome oxidase. KCN (0.3130.0 1M) was added prior to the addition of cytochrome c in 100 ~1 0.03 M NaP04. with an appropriate adjustment in reaction volume. MT (EC 2.8.1.2) was determined in homogenates of liver and brain according to a modification of the method of Mimori et al. (1984). The final reaction volume of 500 ~1 contained 300 ~1 Caps buffer. pH 10.0, 100 ~1 diluted homogenate protein (approx 4 mg), 50 11 sodium mercaptopyruvate at a final concentration of 10 mM, and 50 @IKCN at a final concentration of 15 mM. As activity of MT has been previously demonstrated in red blood cell hemolysates (Valentine and Frankenfeld, 1974), MT was also assayed in mouse livers and brains which had been cleared of blood by in situ perfusion with 0.85% NaCl containing 4 mM KPO+ Incubations were carried out for 5 min at 37’C in a shaking water bath (Dubnoff). Reactions were terminated and assayed for thiocyanate production as described above for RHO. Blank values were determined in the absence of homogenate protein and subtracted from sample values. Mitochondrial and tissue homogenate protein was determined according to the method of Bradford ( 1976).
308
MCMAHON
AND BIRNBAUM
a 8
S r
1J
* n 2.3momica n 10.12mo mice n 25.30mo mica
5
b
15
10
20
25
was allowed to proceed for 2 hr. In experiments to determine the recovery of CN- from tissues, no significant interference was found for recovery from tissue or blood controls spiked with CN- in the concentration range of 0.05-4 pg. Statistics.
In vivo toxicity data were analyzed using the Cochran-Armitage x2 test to evaluate the existence of a dose-related response for each age group. In addition, Mantel-Haenzel x2 was used to test the hypothesis of no age effect in the presence of toxicity, and the Wilcoxon signed rank test was used to assesssignificant differences in duration of response for animals in each dose group (SAS Institute, Inc., 1985). In vitro data were analyzed by one-way ANOVA. Means were compared by use of
a 12
l3-
H
t
n
1
2.3mo mice 10.12mo mica
n 2.3mo mice q lo-12momice n 25.30mo mice
5 5
10
20
15
Time
25
(min)
10
15
20
30
'21 l
n q n
FIG.2. Incidence of labored breathing (a) and prostration (b) in male C57BL/6N mice following 4 mg/kg KCN po. *p < 0.05 by x2 analysis vs 2-3 month mice. Bloodand tissue levels ofcyanide. Blood and tissue levels of CN- were determined as described by Blanke (I 976) with minor modifications. For this procedure, mice were dosed po with 6 mg/kg KCN and euthanized under CO1 anesthesia at 2, 4, 6. 8, 10, 20, and 25 min. Blood was obtained by decapitation. Livers and brains were weighed and homogenized either l/l w/v (brain) or l/2 (liver) in ice-cold distilled water using a polytron. Either 0.5 ml whole blood or 1.O ml tissue homogenate was utilized for microdiffusion analysis. Samples of liver, brain, or blood were mixed with 1.O ml of 3.6 N sulfuric acid in the outer well of a Conway microdiffusion dish, to which 0.5 ml of 0.1 N sodium hydroxide had been added in the center well. The contents of the outer well were mixed, the dishes were quickly sealed, and diffusion
25
b
5
10
15
20
Time
(min)
2.3momice lo-12mo mice 25.30mo mice
25
30
FIG. 3. Incidence of labored breathing (a) and prostration (b) in male C57BL/6N mice following 6 mg/kg KCN po. *p < 0.05 by x2 analysis vs 2-3 month mice.
KCN BIOTRANSFORMATION
AND TOXICITY
309
IN MICE
TABLE 1 AGE-RELATED
CHANGES
Specific activity (nmol/min/mg protein) Liver Brain Organ activity (nmol/min/g tissue) Liver Brain
IN LIVER AND BRAIN RHODANESE
IN MALE
C57BL/6N
MICE
2-3 Months
IO- 12 Months
25-30 Months
1239.7 f 74.1” 23.6 zk 0.6
1744.6 f 150.1* 31.1 + 3.0
1358.6 r 44.7 24.6 t 1.9
2805.6 k 420.7 248.6 f 21.6
4611.2 f 315.0* 178.8 ~fr 5.7*
3091.8 t 452.3 155.3 t 5.9*
-
a Mean rt_SEM for four mice per age group. * Significantly different vs 2-3 month mice (p < 0.05).
Fisher’s LSD test. Differences were considered significant at p c 0.05.
RESULTS The effect of age on toxicity of 4 mg KCN/ kg in male C57BL/6N mice is shown in Figs. 2a and 2b. In Fig. 2a, the incidence of labored breathing was significantly increased in lo12 and 25-30 month mice at 5 min following KCN administration. As compared to toxicity at 2 mg KCN/kg (data not shown), a significant effect of dose was also noted at 4 mg KCN/kg on the incidence of labored breathing in lo- 12 and 25 month old mice. The duration of the toxic response was also significantly longer in lo- 12 and 25 month old mice vs that in 3 month mice (Figs. 2a and 2b). No significant effects of age were noted on prostration following 4 mg KCN/kg (Fig. 2b). The effect of age on toxicity of 6 mg KCN/ kg in male C57BL/6N mice is shown in Figs. 3a and 3b. At this dose, lo- 12 and 25-30 month mice demonstrated a significant increase in the incidence of labored breathing at 15, 20,25, and 30 min following KCN administration (Fig. 3a). In addition, the incidence of prostration was significantly greater in lo- 12 and 25-30 month mice at 10 min after KCN administration (Fig. 3b).
Activity of hepatic and brain RHO as a function of age is shown in Table 1. Activity in liver on a per milligram protein and per gram tissue basis was equivalent in 2-3 and 25-30 month mice, but was significantly increased in lo-12 month mice. This increase could be attributed to a significant increase in V,,, for RHO in 12 month mice (Table 2). No significant age-related changes were observed in activity of brain RHO on a per milligram protein basis; however, in both lo- 12 and 25-30 month mice, brain RHO was significantly decreased vs that in 3 month mice when expressed on a per gram tissue basis (Table 1). This alteration occurred due to a significant decrease in the brain mitochondrial protein content of older mice (7.4 and 6.9 mg protein/g tissue in 10-l 2 and 25-30 TABLE 2 AGE-RELATED CHANGES IN KINETICS OF HEPATIC RHODANESE IN MALE C57BL/6N MICE f-ma,
Months 2-3 LO-12 25-30
(nmol/min) 120.51 k 6.9” 166.81 f 14.4* 134.70 k 20.8
Kn (mM) 0.86 t 0. I 0.70 +- 0.04 0.57 * 0.06
a Mean f SEM of four mice per age group. * Significantly different vs 2-3 month mice (p < 0.05).
310
MCMAHON
AND TABLE
AGE-RELATED
ALTERATIONS
’ Mean
3
IN ACTIVITY OF LIVER AND BRAIN CYTOCHROME IN MALE C57BLi6N MICE 2-3 months
Specific activity (nmoI/min/rg Liver Brain Organ activity (pmol/min/g Liver Brain
BIRNBAUM
IO-12
months
OXIDASE
25-30
months
protein) 0.75 * 0.02” 1.13 kO.02
0.79+ 1.33 f
0.01 0.02
0.90 + 1.28 +
0.03 0.02
tissue) 100.6 53.2
f 7.0 f 2.3
152.4 52.4
f 20.4 + 5.3
129.5 51.6
zk 17.3 k 3.5
k SEM of four mice per age group.
month mice, respectively, vs 10.4 mg protein/g tissue in 3 month mice). Brain RHO activity in all age groups of mice was approximately 1/50th that of liver (data not shown). As shown in Table 3, no significant alterations were observed with age in activity of C-OX in either liver or brain. It should be noted, however, that brain activity on a per milligram protein basis was equivalent or greater than that of the liver. Inhibition of C-OX by KCN in liver and brain is illustrated in Figs. 4a and 4b. Within the range of KCN concentrations used for inhibition of brain and liver C-OX, there were no significant differences in inhibition occurring with age in either organ. Estimation of inhibition constants from Dixon plot transformation of these data also showed no significant changes with age (Table 4). Activity of MT in both liver and brain, as shown in Table 5, was significantly increased in both organs in 12 and 25 month mice vs that in 3 month mice. Removal of blood by perfusion had no effect on activity of MT in either liver or brain (data not shown). Tissue and blood levels of CN- following a dose of 6 mg KCN/kg in 2-4 and lo-12 month mice are shown in Figs. 5a-5c. No significant age-related differences were observed in either blood (Fig. 5a) or liver (Fig. 5b) CNconcentrations over the time course of CN-
measurements; however, in the brain of middle-aged mice, CN- concentrations were significantly greater vs those in young mice at 4 and 25 min after KCN administration (Fig. 5~). In liver, the mean area under the curve (AUC) was not significantly different in lo12 month (62.6 f 2.4) vs that in 2-3 month (70.9 t- 6.7) mice; a similar result was observed for blood AUC (147.6 f 13.9 vs 124.9 + 17.3) in lo-12 vs that in 2-3 month mice, respectively. However, in brain, mean AUC was significantly higher in lo-12 mo (8.0 + 0.4) vs that in 2-3 month (4.5 f 0.5) mice. DISCUSSION Although several reports have appeared concerning age-related changes in activity of C-OX, the target enzyme in CN- toxicity (Wilson et al., 1975; Chiu and Richardson, 1980; Muller-Hacker, 1989), the relationship of age to biotransformation and toxicity of CN- has not been previously investigated. In this study, the mechanistic basis for age-related differences in the in vivo toxicity of CNwas investigated through examination of enzymes involved in biotransformation of CNas well as tissue and blood levels of CN- following a toxic dose. Biotransformation of CN- to SCN- by hepatic RHO has been considered as the pri-
KCN BIOTRANSFORMATION lo
a a
0.0 ! 0
10
I 20
I
,
30
40
10
20
30
40
o.o0
KCN W)
4. Inhibition of C-OX by KCN in liver (a) and brain (b) homogenates of male C57BL/6N mice of various ages. Each value represents the mean + SEM of four animals per age group. FIG.
mar-y detoxication reaction for CN-. However, it has been observed that experimental manipulation of hepatic RHO does not always result in the expected change in toxicity of CN-. For example, Rutkowski et al. (1985) observed an increase in acute CN- lethality in mice fed a protein-free diet, despite a significant increase in hepatic RHO activity. In addition, the acute lethal effect of CN- in mice was not altered by pretreatment with CCL+ or two-thirds partial hepatectomy (Rutkowski et al., 1986). Thus, the role of hepatic
AND TOXICITY
311
IN MICE
RHO as a key enzyme in detoxication of CNmay be open to question (Rutkowski et al., 1986). The observation in the present study of increased toxicity of KCN in older mice despite no decrease in hepatic RHO lends support to this idea. Inhibition of C-OX by CN- is followed by restoration of C-OX activity through competition for CN- by RHO (Isom and Way, 1984). Thus, tissues with high activities of RHO would be expected to display minimal inhibition of C-OX (Isom and Way, 1976). In this respect, Isom and Way (1976) demonstrated minimal inhibition of liver vs brain C-OX after a lethal dose of CN- in the presence of the CN- antidotes sodium nitrite and sodium thiosulfate. While tissue distribution of CN- and CN- antidotes could play a role in this differential inhibition, reactivation of C-OX through biotransformation of CN- by RHO would occur more rapidly in liver relative to that in brain. due to the significantly greater activity of liver RHO as compared to that of brain RHO (Table 1). The increased sensitivity of older mice in the present study to the acute toxic effects of KCN could thus be explained in part by slower reactivation of C-OX in the brains of these mice as opposed to that in young mice, due to the significantly decreased amount of brain RHO in these mice (Table 1). Support for this hypothesis is derived from the observations that no changes were observed in the activity of MT (Table 5), which supplies sulfur for detoxication of CN- by RHO (DeBruin, 1976), or an
TABLE 4 AGE-RELATED CHANGES IN INHIBITION CYT~CHROME OXIDASE BY KCN
OF
K (PM )
2-3 Months
IO-12 Months
25-30 Months
Liver Brain
6.22 -c 1.3” 3.70 k 0.7
8.22 f 1.4 5.82 + I .5
8.36 k 1.3 5.32 rt 1.4
’ Mean + SEM of four mice per age group.
312
MCMAHON + +
a
AND BIRNBAUM
blood 2.3mo blood 10.12mo
b 81 -:+
6
ltver 2.3mo liver 10.12mo
4
2
0 0
10
20
30
C 1.0
l
-S--D-
T
3
0.6
brain 2.3mo bran 10.12mo
0.6 0.4 0.2 0.0 0
10
20
30
Time (min)
FIG.5. Concentration ofcyanide in blood (a), liver(b), and brain (c) of male C57BL/6N mice following 6 mg/ kg KCN po. Each value represents the mean rt SEM of three to four animals per age group. *p < 0.05 by oneway ANOVA vs 2-3 month mice.
serum albumin (data not shown), which has been proposed as a carrier of sulfane sulfur in vivo for detoxication of CN-. The age-related increase in acute toxicity of CN- may also be expected based upon the observations that (1) significantly greater CN- concentrations were found in the brains of 10-l 2 month mice at 4 and 25 min in
comparison to those in 2-4 month mice; (2) the uptake of CN- in the brains of lo-12 month mice appeared to be more rapid, as the peak concentration of CN- occurred sooner; (3) disappearance of CN- from the brain was prolonged in lo-12 month mice relative to that in 2-4 month mice (Fig. 5~); and (4) the tissue AUC for brain CN- was significantly greater in lo-12 month vs that in 2-3 month mice. While a significant age-related decrease in brain RHO and significantly greater brain concentrations of CN- in older mice would offer a plausible explanation for the age-related increase in in vivo toxicity of KCN, this does not exclude other mechanisms which may contribute to this phenomenon. For example, it is known that enzymes involved in antioxidant defense, such as superoxide dismutase, catalase, and glutathione peroxidase, are more sensitive to inhibition by CN- than is C-OX. In a study by Ardelt et al. (1989) sublethal doses of CN- in mice resulted in rapid peroxidation of brain lipids which correlated with the temporal profile of acute CN- toxicity. While a temporal correlation can be observed in the disappearance of CNfrom the brain and recovery from acute toxicity in our study, age-related decreases in antioxidant defense capacity (McMahon and Birnbaum, 1990) if inhibited by CN- to a similar degree with age, could also contribute to alterations in acute CN- toxicity. Additionally, there is evidence that CN- disposition includes reversible binding to unknown tissue constituents (Devlin et al., 1989) and that this represents a “sink” of metabolizable CN-. Although not investigated in the present study, potential age-related alterations in this “sink” of CN- may also result in altered tissue levels and thus influence toxicity of CN- in older subjects. In conclusion, this study has demonstrated that the significant age-related increase in acute toxicity of CN- observed in male C57BL/6N mice may be related to a significant decrease in the amount of brain RHO,
KCN BIOTRANSFORMATION
AND TOXICITY
313
IN MICE
TABLE 5 AGE-RELATED
CHANGES
Activity (nmol/min/mg Liver Brain
IN ACTIVITY
OF &MERCAPTOPYRUVATE
TRANSSULFERASE
2-3 Months
IO-12 Months
IN MALE
C57BLi6N MICE 25-30 Months
protein) 5.33 *0.7* 1.51 + 0.2*
3.26 kO.3" 0.92 kO.07
5.37 +0.7*
1.27 f 0.09*
a Mean * SEM of four mice per age group. * Significantly different vs 2-3 month mice (p < 0.05).
as well as altered tissue kinetics of CN- in the brains of older mice. These observations suggest that it is the disposition of CN- within the brain that may be most important in assessing acute toxicity of this compound, as well as alterations in susceptibility to CN- occurring with age. ACKNOWLEDGMENTS The authors thank Ann-Marie Clark for statistical analysis of the data, and Janet J. Diliberto for technical assistance.
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A. ( 1976). Biochemical To.xicology qf Environmental Agents. Elsevier/North-Holland. New York. DEVLIN. D. J.. SMITH, R. P., AND THRON, C. D. ( 1989). Cyanide metabolism in the isolated. perfused. bloodless hindlimbs or liver of the rat. Toxicol. .4ppl. Pharmacol. 98,338-349. FRANKENBERG, L., AND SORBO, B. ( 1973). Formation of cyanide from a-chloro-benylidene malonitrile and its toxicologic significance. Arch. Toxikol. 31,99-108. FREEMAN. A. G. (1988). Optic neuropathy and chronic cyanide intoxication: A review. J. Royal Sot. Med. 81, DEBRUIN,
103-106.
HODGES, T. K., AND LEONARD, R. T. (1974). Purification of a plasma membrane bound adenosine triphosphatase from plant roots. In Meth0d.y in Enzymology (S. P. Colowick and N. 0. Kaplan, Eds.). Vol. 32. pp. 397-398. Academic Press, New York. ISOM, G. E.. AND WAY, J. L. (1976). Lethality ofcyanide in the absence of inhibition of liver cytochrome oxidase. Biochem. Phurmacol. 25,605-608. ISOM. G. E., AND WAY, J. L. ( 1984). Effects of oxygen on the antagonism of cyanide intoxication:cytochrome oxidase. in vitro. Toxicol. .4ppl. Pharmucol. 74, 57-62. JOHNSON, J. D., AND ISOM. G. E. (1985). The oxidative disposition of potassium cyanide in mice. Touicrkg~ 37,215-224.
MCMAHON, T. F.. AND BIRNBAUM, L. S. (1990). Agerelated changes in the disposition and metabolism of xenobiotics. In Aging and Environmental To.uicologJ (Cooper and Goldwin, Eds.). Johns Hopkins Univ. Press, Maryland, in press. MIMORI. Y.. NAKAMURA. S.. AND KAMEYAMA. M. (1984). Regional and subcellular distribution of cyanide metabolizing enzymes in the central nervous system. J. Neurochem. 43, 540-545. MULLER-HOCKER, J. (1989). Cytochrome-c-oxidase deficient cardiomyocytes in the human heart-an age-related phenomenon. Amer. J. Pathol. 134, 1167-I 173. PETTERSEN, J. C.. AND COHEN, S. D. (1985). Antagonism of cyanide poisoning by chlorpromazine and so-
314
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AND BIRNBAUM
dium thiosulfate. Toxicol. Appl. Pharmacol. 81, 265213.
POOLE, C. J. M., AND KIND, P. R. N. (1986). Deficiency of thiosulfate sulphurtransferase (rhodanese) in Leber’s hereditary optic neuropathy. Brit. Med. J. Clin. Res. 292,1229- 1230. 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. RUTKOWSKI, J. V., ROEBUCK, B. D., AND SMITH, R. P. (1985). Effects of protein-free diet and food deprivation on hepatic rhodanese activity, serum proteins and acute cyanide lethality in mice. J. Nutr. 115, 132- 137. RUTKOWSKI, J. V., ROEBUCK, B. D., AND SMITH, R. P. (1986). Liver damage does not increase the sensitivity of mice to cyanide given acutely. Toxicology 38,305314.
SAS Institute, Inc. (1985). SAS User’s Guide: Statistics, Version 5 ed., p. 2 1. SAS Institute, Cary, NC. SILVER, E. H., KUTTAB, S. H., HASAN, T., AND HASSAN, M. (1982). Structural considerations in the metabolism of nitriles to cyanide in vivo. Drug Metab. Dispos. 10(5),
495-498.
TALLARIDA, R. J., AND MURRAY, R. B. (1987). Manual of Pharmacologic Calculations with Computer Programs, 2nd ed. Springer-Verlag, New York. UEMURA, AND CHIESARA, (1976). NADH-dependent aryl hydrocarbon hydroxylase in rat liver mitochondrial outer membrane. Eur. J. Biochem. 66,293307.
VALENTINE, W. N., AND FRANKENFELD, J. K. (1974). 3-Mercaptopyruvate sulfurtransferase (EC 2.8.1.2): A simple assay adapted to human red blood cells. Clin. Chim. Acta 51,205-210. VENNESLAND, B., GASTRIC, P. A., CONN, E. E., SOLOMONSON, L. P., VOLINI, M., AND WESTLEY, J. (1982). Cyanide metabolism. Fed. Proc. 41,2639-2648. WAY, J. L. (1984). Cyanide intoxication and its mechanism of antagonism. Ann. Rev. Pharmacol. Toxicol. 24,451-481. WILLHITE, C. C., AND SMITH, R. P. (198 1). The role of cyanide liberation in the acute toxicity of aliphatic nitriles. Toxicol. Appl. Pharmacol. 59,589-602. WILSON, P. D., HILL, B. T., AND FRANKS, L. M. (1975). The effect of age on mitochondrial enzymes and respiration. Gerontologia 21,95-101. WOOD, J. L., AND COOLEY, S. L. (1956). Detoxication of cyanide by cystine. J. Biol. Chem. 218,449-451.
AND
APPLIED
PHARMACOLOGY
14 (1990)
16305-3
Age-Related Changes in Toxicity and Biotransformation of Potassium Cyanide in Male C57BL/6N Mice TIMOTHY Experimental
F. MCMAHON
AND LINDA
S. BIRNBAUM”~
Tnxicologv Branch, National Institute of Environmental Research Triangle Park, North Carolina -77709
Received
Februar~~
6. 1990; accepted
May
Health
Sciences,
24. I990
Age-Related Changes in Toxicity and Biotransformation of Potassium Cyanide in Male C57BL/6N Mice. MCMAHON, T . F.. AND BIRNBAUM, L. S. (1990). To.uicol. Appl. Pharmacol. 105. 305-3 14. Age-related changes in toxicity and biotransformation of KCN. an ubiquitous environmental toxicant, have not been previously examined. Male C57BL/6N mice aged 2-3 (young), lo- 12 (middle-aged), and 25-30 (old) months were administered KCN at I, 2,4, and 6 mg/kg po, and toxic manifestations were monitored for up to 2 hr. The toxic response to KCN (prostration and labored breathing) was significantly greater in 10-t 2 and 25-30 month vs that in 2-3 month mice at 4 and 6 mg/kg KCN. The basis for this age-related difference in in viva toxicity was examined by studying biotransformation of KCN to thiocyanate by liver and brain rhodanese (RHO), as well as activity of liver and brain cytochrome oxidase (C-OX), inhibition of C-OX by KCN. and activity of /!I-mercaptopyruvate transsulfurase (MT). Tissue and blood levels of CN- following a toxic dose of 6 mg/kg KCN were also measured. No age-related differences were observed in the specific activity of liver and brain RHO, MT, or C-OX. In addition. no differences were observed in the percentage inhibition of C-OX by KCN, or in the K, for inhibition of brain and liver C-OX. However, activity of brain RHO on a per gram tissue basis was significantly lower in lo-12 and 25-30 month vs that in 2-3 month mice. Liver and blood concentrations of CN- were not significantly different in 2-3 vs lo- I2 month mice following treatment with 6 mg/kg KCN; however, significantly greater concentrations of CN- were observed at 4 and 25 min in brains of lo-12 month mice compared to that in 2-3 month mice. These results indicate that increased sensitivity to KCN in older mice may be due in part to a decrease in the amount of brain RHO and altered tissue kinetics of CN- following a toxic dose in older mice. Q 1990 Academic Press. Inc.
The presence of cyanide (CN-) in the environment is widespread. This potent toxicant is found as a component of cigarette and polymer pyrolysis smokes (Way, 1984), tear gas (Frankenberg and Sorbo, 1973), and ver’ To whom correspondence and reprint requests should be addressed at USEPA. Environmental Toxicology Division. MD-66. Research Triangle Park, NC 2771 I. ’ Current address: Environmental Toxicology Division, Health Effects Research Laboratory. USEPA, Research Triangle Park, NC 277 1 I,
305
micidal fumigants (Ballantyne, 1988), while cyanogenic glycosides are present in over 2000 species of plants (Vennesland et al., 1982). In addition, CN- is formed as a primary product from the biotransformation of several aliphatic nitriles which are used in the manufacture of synthetic fibers, resins, plastics, pharmaceuticals, and vitamins (Silver et al., 1982; Willhite and Smith, 1981). Thus, the potential risk of oral or inhalation exposure to CN-, or precursors of CN-, may be very great. Because of the widespread natural occurrence of CN-, it is not surprising that several 0041008X/90
$3.00
Copyright 0 1990 by Academx Ress. Inc Ail rights of reproduction in any fwm rescrvrd
306
MCMAHON
AND BIRNBAUM
mechanisms are operative for CN- detoxication in viva. The primary detoxication reaction is believed to be formation of thiocyanate (SCN-) from CN-, catalyzed by the enzyme rhodanese (RHO; thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1.), which is present mainly in hepatic mitochondria, and to a lesser extent in mitochondria of brain and heart (Rutkowski et al., 1986). Biotransformation by RHO can account for between 6070% of an administered dose of CN- in experimental animals (Fig. 1). Other pathways for CN- detoxication include formation of 2-imino-thiazolidine-4carboxylic acid through the nonenzymatic reaction of CN- with cystine, oxidation to formic acid and COZ through formation of cyanate, and trapping as the cyano group of vitamin Bi2 (Ahmed et al., 1985) (Fig. 1). Formation of 2-imino-thiazolidine-4-carboxylic acid may account for up to 15% of an administered dose of CN- in rats (Wood and Cooley, 1956) while formation of CO* accounts for approximately 2% of an administered dose of KCN in mice (Johnson and Isom, 1985). The toxicologic significance of CN- detoxication by combination with cystine or oxidation to CO2 has not been directly assessed. However, a reduced ability to detoxify cyanide by reaction with cobalamin has been associated with the development of tobacco amblyopia (Freeman, 1988), while Leber’s hereditary optic neuropathy has been linked to deficiency of RHO (Poole and Kind, 1986). Aging has been shown to affect the disposition and toxicity of a number of drugs and xenobiotics (McMahon and Birnbaum, 1990). With regard to CN-, preliminary studies in our laboratory demonstrated that older (10 mo) male C567BL/6N mice were more sensitive than 3 month mice of the same strain to the acute toxic effects of KCN at 4 and 6 mg/kg. In light of the widespread occurrence of CN-, the potential for lifetime exposure to this toxicant, and our preliminary in vivo observations, the present study was
Expired HCN
Cyanocobalamin
2.iminothiazolidineI-carboxylic (15%)
Vitamin
acid
B 2
CNO Cyanate
1 HCOOH 11 Formate (excreted)
One
carbon
pool
I co2 (2%)
FIG. 1. Biotransformation
of cyanide.
undertaken to examine in more detail the basis for this age-related difference in sensitivity to CN-. Differences in CN- toxicity were examined in vivo in male C57BL/6N mice aged 2-3 (young), lo- 12 (middle-aged), and 2530 (old) months over a range of CN- doses (1,2,4, and 6 mg/kg CN- PO). Potential agerelated differences in liver and brain RHO, C-OX (the target enzyme for CN-), and ,& mercaptopyruvate transsulfurase (MT; an enzyme which supplies sulfur for detoxication of CN- by RHO) were examined in vitro, as well as possible differences with age in sensitivity of liver and brain C-OX to inhibition by KCN. In addition, tissue and blood levels of CN- were measured following a toxic dose of 6 mg/kg KCN in young and middle aged mice. METHODS Chemiculs. KCN was obtained from Aldrich Chemical Co. (Milwaukee, WI). Caps buffer, pyridine, sodium
KCN BIOTRANSFORMATION thiosulfate, and barbituric acid were obtained from Sigma Chemical Co. (St Louis, MO). Sucrose and trichloroacetic acid were obtained from J.T. Baker (Phillipsburg, NJ). Sodium mercaptopyruvate was purchased from ICN Biomedical (Irvine, CA). Reagents for preparing phosphate buffers were obtained from Mallinkrodt (Paris, KY). All chemicals were of the highest grade and purity commercially available. Animals and treatments. Male C57BL/6N mice aged 2 months were obtained from Charles River (Raleigh, NC), while retired breeder mice aged IO- I2 months were obtained from Frederick Cancer Research Facility (Frederick, MD). Mice aged 25-30 months were obtained from an aging cohort of retired breeders maintained at NlEHS originally from Frederick. Young mice were housed in groups of five, while older mice were housed singly. All mice were kept under conditions of constant temperature (23 -C2°C) humidity (50 f 5%) and lighting (12-hr light/dark cycle), and were fed Rodent Chow (NIH-31) and water ad libitum. Mice were acclimated for at least 7 days before use in either in vitro or in vivo experiments. For in vivo dosing experiments, 10 mice/age group. singly housed, were administered 1, 2, 4, or 6 mg/kg KCN po in a dosing volume of 10 ml 0.9% NaCl/kg. The same groups of mice were used for each dose of KCN. but were allowed a recovery period of 2 weeks between each dose. Doses of KCN were selected to include both nontoxic ( 1 and 2 mp/kg) and toxic (4 and 6 mg/kg) doses (Rutkowski et al., 1986). Following administration of KCN, mice were observed for the presence or absence of toxic signs (prostration, labored breathing, and tremors) at 5-min intervals up to 30 min, and thereafter every 15 min up to 2 hr. The number of mice in each treatment group manifesting signs of KCN toxicity at each time point was noted. Following the last dose (6 mg/kg) of KCN, mice were allowed to recover and then were used for in vitro enzyme measurements. Enzyme assays.For all enzyme assays,tissues were obtained from mice killed by decapitation under CO* anesthesia. RHO activity in both liver and brain mitochondria was assayed according to a modification of the method of Pettersen and Cohen ( 1985). Organs were removed, weighed, and then homogenized (l/3 and l/4 w/ v, respectively) in ice-cold T&buffered 0.25 M sucrose, pH 7.4. Mitochondria were prepared from tissue homogenates by differential centrifugation according to a modification of the method of Uemura and Chiesara (1976). The washed mitochondrial pellets were resuspended in 1 ml of0.01 M NaPO,/O. 15 M KCL, pH 7.4. Mitochondrial protein (0.1 mg) was then added to prewarmed (37°C) 0.1 M NaP04 buffer. pH 7.4, containing 5 mM sodium thiosulfate in 15-m] test tubes. Concentrations of KCN ranging from 0.46-30 mM were added to initiate the reaction. The reaction volume was 500 ~1. Reactions were carried out at 37°C for 2 min in a shaking water bath, and
AND TOXICITY
IN MICE
307
were terminated by the addition of cold 10% trichloroacetic acid solution containing 5.6 ml formaldehyde/liter. Samples were processed and assayed for thiocyanate as previously described (Pettersen and Cohen, 1985). Apparent K, and V,,,, for hepatic RHO were determined from Lineweaver-Burk transformation of saturation data using PHARM/PCS on an IBM PS/2 computer (Tallarida and Murray, 1987). Reactions were linear from l-5 min and 0.1-0.5 mg protein in liver, and from 2-4 min and 0.2-0.4 mg protein in brain. Increasing the concentration of thiosulfate to 10 mM in either liver or brain incubationsdid not affect theextent ofthe reaction. Blank values were obtained in the absence of thiosulfate. mitochondrial protein, and KCN. and were subtracted from sample values. C-OX activity was determined in liver and brain homogenates according to a modification of the method of Pettersen and Cohen (1985). Tissues were weighed. homogenized with a glass tissue homogenizer using a Teflon pestle, l/10 w/v (liver) or l/2 w/v (brain), in 0.03 M NaPO,, pH 7.4, and diluted l/50 or I/ 100 before use in assays.Incubations were performed at 37°C in a Beckman (Fullerton. CA) DU-8B spectrophotometer using the Kinetics II compuset module. Reaction mixtures contained 2.4 ml 0.03 M NaPO,, pH 7.4. 0.5 ml cytochrome c ( 10v4 M), reduced according to the method of Potter ( 1964), and I5 (liver) or 20 (brain) pg homogenate protein. Reactions were monitored for up to 3 min at 550 nm. The activity ofC-OX was calculated from the initial linear rate using an extinction coefficient of 18.5 mM -’ cm-’ (Hodges and Leonard, 1974). In experiments to determine age-related differences in sensitivity of liver and brain to inhibition of cytochrome oxidase. KCN (0.3130.0 1M) was added prior to the addition of cytochrome c in 100 ~1 0.03 M NaP04. with an appropriate adjustment in reaction volume. MT (EC 2.8.1.2) was determined in homogenates of liver and brain according to a modification of the method of Mimori et al. (1984). The final reaction volume of 500 ~1 contained 300 ~1 Caps buffer. pH 10.0, 100 ~1 diluted homogenate protein (approx 4 mg), 50 11 sodium mercaptopyruvate at a final concentration of 10 mM, and 50 @IKCN at a final concentration of 15 mM. As activity of MT has been previously demonstrated in red blood cell hemolysates (Valentine and Frankenfeld, 1974), MT was also assayed in mouse livers and brains which had been cleared of blood by in situ perfusion with 0.85% NaCl containing 4 mM KPO+ Incubations were carried out for 5 min at 37’C in a shaking water bath (Dubnoff). Reactions were terminated and assayed for thiocyanate production as described above for RHO. Blank values were determined in the absence of homogenate protein and subtracted from sample values. Mitochondrial and tissue homogenate protein was determined according to the method of Bradford ( 1976).
308
MCMAHON
AND BIRNBAUM
a 8
S r
1J
* n 2.3momica n 10.12mo mice n 25.30mo mica
5
b
15
10
20
25
was allowed to proceed for 2 hr. In experiments to determine the recovery of CN- from tissues, no significant interference was found for recovery from tissue or blood controls spiked with CN- in the concentration range of 0.05-4 pg. Statistics.
In vivo toxicity data were analyzed using the Cochran-Armitage x2 test to evaluate the existence of a dose-related response for each age group. In addition, Mantel-Haenzel x2 was used to test the hypothesis of no age effect in the presence of toxicity, and the Wilcoxon signed rank test was used to assesssignificant differences in duration of response for animals in each dose group (SAS Institute, Inc., 1985). In vitro data were analyzed by one-way ANOVA. Means were compared by use of
a 12
l3-
H
t
n
1
2.3mo mice 10.12mo mica
n 2.3mo mice q lo-12momice n 25.30mo mice
5 5
10
20
15
Time
25
(min)
10
15
20
30
'21 l
n q n
FIG.2. Incidence of labored breathing (a) and prostration (b) in male C57BL/6N mice following 4 mg/kg KCN po. *p < 0.05 by x2 analysis vs 2-3 month mice. Bloodand tissue levels ofcyanide. Blood and tissue levels of CN- were determined as described by Blanke (I 976) with minor modifications. For this procedure, mice were dosed po with 6 mg/kg KCN and euthanized under CO1 anesthesia at 2, 4, 6. 8, 10, 20, and 25 min. Blood was obtained by decapitation. Livers and brains were weighed and homogenized either l/l w/v (brain) or l/2 (liver) in ice-cold distilled water using a polytron. Either 0.5 ml whole blood or 1.O ml tissue homogenate was utilized for microdiffusion analysis. Samples of liver, brain, or blood were mixed with 1.O ml of 3.6 N sulfuric acid in the outer well of a Conway microdiffusion dish, to which 0.5 ml of 0.1 N sodium hydroxide had been added in the center well. The contents of the outer well were mixed, the dishes were quickly sealed, and diffusion
25
b
5
10
15
20
Time
(min)
2.3momice lo-12mo mice 25.30mo mice
25
30
FIG. 3. Incidence of labored breathing (a) and prostration (b) in male C57BL/6N mice following 6 mg/kg KCN po. *p < 0.05 by x2 analysis vs 2-3 month mice.
KCN BIOTRANSFORMATION
AND TOXICITY
309
IN MICE
TABLE 1 AGE-RELATED
CHANGES
Specific activity (nmol/min/mg protein) Liver Brain Organ activity (nmol/min/g tissue) Liver Brain
IN LIVER AND BRAIN RHODANESE
IN MALE
C57BL/6N
MICE
2-3 Months
IO- 12 Months
25-30 Months
1239.7 f 74.1” 23.6 zk 0.6
1744.6 f 150.1* 31.1 + 3.0
1358.6 r 44.7 24.6 t 1.9
2805.6 k 420.7 248.6 f 21.6
4611.2 f 315.0* 178.8 ~fr 5.7*
3091.8 t 452.3 155.3 t 5.9*
-
a Mean rt_SEM for four mice per age group. * Significantly different vs 2-3 month mice (p < 0.05).
Fisher’s LSD test. Differences were considered significant at p c 0.05.
RESULTS The effect of age on toxicity of 4 mg KCN/ kg in male C57BL/6N mice is shown in Figs. 2a and 2b. In Fig. 2a, the incidence of labored breathing was significantly increased in lo12 and 25-30 month mice at 5 min following KCN administration. As compared to toxicity at 2 mg KCN/kg (data not shown), a significant effect of dose was also noted at 4 mg KCN/kg on the incidence of labored breathing in lo- 12 and 25 month old mice. The duration of the toxic response was also significantly longer in lo- 12 and 25 month old mice vs that in 3 month mice (Figs. 2a and 2b). No significant effects of age were noted on prostration following 4 mg KCN/kg (Fig. 2b). The effect of age on toxicity of 6 mg KCN/ kg in male C57BL/6N mice is shown in Figs. 3a and 3b. At this dose, lo- 12 and 25-30 month mice demonstrated a significant increase in the incidence of labored breathing at 15, 20,25, and 30 min following KCN administration (Fig. 3a). In addition, the incidence of prostration was significantly greater in lo- 12 and 25-30 month mice at 10 min after KCN administration (Fig. 3b).
Activity of hepatic and brain RHO as a function of age is shown in Table 1. Activity in liver on a per milligram protein and per gram tissue basis was equivalent in 2-3 and 25-30 month mice, but was significantly increased in lo-12 month mice. This increase could be attributed to a significant increase in V,,, for RHO in 12 month mice (Table 2). No significant age-related changes were observed in activity of brain RHO on a per milligram protein basis; however, in both lo- 12 and 25-30 month mice, brain RHO was significantly decreased vs that in 3 month mice when expressed on a per gram tissue basis (Table 1). This alteration occurred due to a significant decrease in the brain mitochondrial protein content of older mice (7.4 and 6.9 mg protein/g tissue in 10-l 2 and 25-30 TABLE 2 AGE-RELATED CHANGES IN KINETICS OF HEPATIC RHODANESE IN MALE C57BL/6N MICE f-ma,
Months 2-3 LO-12 25-30
(nmol/min) 120.51 k 6.9” 166.81 f 14.4* 134.70 k 20.8
Kn (mM) 0.86 t 0. I 0.70 +- 0.04 0.57 * 0.06
a Mean f SEM of four mice per age group. * Significantly different vs 2-3 month mice (p < 0.05).
310
MCMAHON
AND TABLE
AGE-RELATED
ALTERATIONS
’ Mean
3
IN ACTIVITY OF LIVER AND BRAIN CYTOCHROME IN MALE C57BLi6N MICE 2-3 months
Specific activity (nmoI/min/rg Liver Brain Organ activity (pmol/min/g Liver Brain
BIRNBAUM
IO-12
months
OXIDASE
25-30
months
protein) 0.75 * 0.02” 1.13 kO.02
0.79+ 1.33 f
0.01 0.02
0.90 + 1.28 +
0.03 0.02
tissue) 100.6 53.2
f 7.0 f 2.3
152.4 52.4
f 20.4 + 5.3
129.5 51.6
zk 17.3 k 3.5
k SEM of four mice per age group.
month mice, respectively, vs 10.4 mg protein/g tissue in 3 month mice). Brain RHO activity in all age groups of mice was approximately 1/50th that of liver (data not shown). As shown in Table 3, no significant alterations were observed with age in activity of C-OX in either liver or brain. It should be noted, however, that brain activity on a per milligram protein basis was equivalent or greater than that of the liver. Inhibition of C-OX by KCN in liver and brain is illustrated in Figs. 4a and 4b. Within the range of KCN concentrations used for inhibition of brain and liver C-OX, there were no significant differences in inhibition occurring with age in either organ. Estimation of inhibition constants from Dixon plot transformation of these data also showed no significant changes with age (Table 4). Activity of MT in both liver and brain, as shown in Table 5, was significantly increased in both organs in 12 and 25 month mice vs that in 3 month mice. Removal of blood by perfusion had no effect on activity of MT in either liver or brain (data not shown). Tissue and blood levels of CN- following a dose of 6 mg KCN/kg in 2-4 and lo-12 month mice are shown in Figs. 5a-5c. No significant age-related differences were observed in either blood (Fig. 5a) or liver (Fig. 5b) CNconcentrations over the time course of CN-
measurements; however, in the brain of middle-aged mice, CN- concentrations were significantly greater vs those in young mice at 4 and 25 min after KCN administration (Fig. 5~). In liver, the mean area under the curve (AUC) was not significantly different in lo12 month (62.6 f 2.4) vs that in 2-3 month (70.9 t- 6.7) mice; a similar result was observed for blood AUC (147.6 f 13.9 vs 124.9 + 17.3) in lo-12 vs that in 2-3 month mice, respectively. However, in brain, mean AUC was significantly higher in lo-12 mo (8.0 + 0.4) vs that in 2-3 month (4.5 f 0.5) mice. DISCUSSION Although several reports have appeared concerning age-related changes in activity of C-OX, the target enzyme in CN- toxicity (Wilson et al., 1975; Chiu and Richardson, 1980; Muller-Hacker, 1989), the relationship of age to biotransformation and toxicity of CN- has not been previously investigated. In this study, the mechanistic basis for age-related differences in the in vivo toxicity of CNwas investigated through examination of enzymes involved in biotransformation of CNas well as tissue and blood levels of CN- following a toxic dose. Biotransformation of CN- to SCN- by hepatic RHO has been considered as the pri-
KCN BIOTRANSFORMATION lo
a a
0.0 ! 0
10
I 20
I
,
30
40
10
20
30
40
o.o0
KCN W)
4. Inhibition of C-OX by KCN in liver (a) and brain (b) homogenates of male C57BL/6N mice of various ages. Each value represents the mean + SEM of four animals per age group. FIG.
mar-y detoxication reaction for CN-. However, it has been observed that experimental manipulation of hepatic RHO does not always result in the expected change in toxicity of CN-. For example, Rutkowski et al. (1985) observed an increase in acute CN- lethality in mice fed a protein-free diet, despite a significant increase in hepatic RHO activity. In addition, the acute lethal effect of CN- in mice was not altered by pretreatment with CCL+ or two-thirds partial hepatectomy (Rutkowski et al., 1986). Thus, the role of hepatic
AND TOXICITY
311
IN MICE
RHO as a key enzyme in detoxication of CNmay be open to question (Rutkowski et al., 1986). The observation in the present study of increased toxicity of KCN in older mice despite no decrease in hepatic RHO lends support to this idea. Inhibition of C-OX by CN- is followed by restoration of C-OX activity through competition for CN- by RHO (Isom and Way, 1984). Thus, tissues with high activities of RHO would be expected to display minimal inhibition of C-OX (Isom and Way, 1976). In this respect, Isom and Way (1976) demonstrated minimal inhibition of liver vs brain C-OX after a lethal dose of CN- in the presence of the CN- antidotes sodium nitrite and sodium thiosulfate. While tissue distribution of CN- and CN- antidotes could play a role in this differential inhibition, reactivation of C-OX through biotransformation of CN- by RHO would occur more rapidly in liver relative to that in brain. due to the significantly greater activity of liver RHO as compared to that of brain RHO (Table 1). The increased sensitivity of older mice in the present study to the acute toxic effects of KCN could thus be explained in part by slower reactivation of C-OX in the brains of these mice as opposed to that in young mice, due to the significantly decreased amount of brain RHO in these mice (Table 1). Support for this hypothesis is derived from the observations that no changes were observed in the activity of MT (Table 5), which supplies sulfur for detoxication of CN- by RHO (DeBruin, 1976), or an
TABLE 4 AGE-RELATED CHANGES IN INHIBITION CYT~CHROME OXIDASE BY KCN
OF
K (PM )
2-3 Months
IO-12 Months
25-30 Months
Liver Brain
6.22 -c 1.3” 3.70 k 0.7
8.22 f 1.4 5.82 + I .5
8.36 k 1.3 5.32 rt 1.4
’ Mean + SEM of four mice per age group.
312
MCMAHON + +
a
AND BIRNBAUM
blood 2.3mo blood 10.12mo
b 81 -:+
6
ltver 2.3mo liver 10.12mo
4
2
0 0
10
20
30
C 1.0
l
-S--D-
T
3
0.6
brain 2.3mo bran 10.12mo
0.6 0.4 0.2 0.0 0
10
20
30
Time (min)
FIG.5. Concentration ofcyanide in blood (a), liver(b), and brain (c) of male C57BL/6N mice following 6 mg/ kg KCN po. Each value represents the mean rt SEM of three to four animals per age group. *p < 0.05 by oneway ANOVA vs 2-3 month mice.
serum albumin (data not shown), which has been proposed as a carrier of sulfane sulfur in vivo for detoxication of CN-. The age-related increase in acute toxicity of CN- may also be expected based upon the observations that (1) significantly greater CN- concentrations were found in the brains of 10-l 2 month mice at 4 and 25 min in
comparison to those in 2-4 month mice; (2) the uptake of CN- in the brains of lo-12 month mice appeared to be more rapid, as the peak concentration of CN- occurred sooner; (3) disappearance of CN- from the brain was prolonged in lo-12 month mice relative to that in 2-4 month mice (Fig. 5~); and (4) the tissue AUC for brain CN- was significantly greater in lo-12 month vs that in 2-3 month mice. While a significant age-related decrease in brain RHO and significantly greater brain concentrations of CN- in older mice would offer a plausible explanation for the age-related increase in in vivo toxicity of KCN, this does not exclude other mechanisms which may contribute to this phenomenon. For example, it is known that enzymes involved in antioxidant defense, such as superoxide dismutase, catalase, and glutathione peroxidase, are more sensitive to inhibition by CN- than is C-OX. In a study by Ardelt et al. (1989) sublethal doses of CN- in mice resulted in rapid peroxidation of brain lipids which correlated with the temporal profile of acute CN- toxicity. While a temporal correlation can be observed in the disappearance of CNfrom the brain and recovery from acute toxicity in our study, age-related decreases in antioxidant defense capacity (McMahon and Birnbaum, 1990) if inhibited by CN- to a similar degree with age, could also contribute to alterations in acute CN- toxicity. Additionally, there is evidence that CN- disposition includes reversible binding to unknown tissue constituents (Devlin et al., 1989) and that this represents a “sink” of metabolizable CN-. Although not investigated in the present study, potential age-related alterations in this “sink” of CN- may also result in altered tissue levels and thus influence toxicity of CN- in older subjects. In conclusion, this study has demonstrated that the significant age-related increase in acute toxicity of CN- observed in male C57BL/6N mice may be related to a significant decrease in the amount of brain RHO,
KCN BIOTRANSFORMATION
AND TOXICITY
313
IN MICE
TABLE 5 AGE-RELATED
CHANGES
Activity (nmol/min/mg Liver Brain
IN ACTIVITY
OF &MERCAPTOPYRUVATE
TRANSSULFERASE
2-3 Months
IO-12 Months
IN MALE
C57BLi6N MICE 25-30 Months
protein) 5.33 *0.7* 1.51 + 0.2*
3.26 kO.3" 0.92 kO.07
5.37 +0.7*
1.27 f 0.09*
a Mean * SEM of four mice per age group. * Significantly different vs 2-3 month mice (p < 0.05).
as well as altered tissue kinetics of CN- in the brains of older mice. These observations suggest that it is the disposition of CN- within the brain that may be most important in assessing acute toxicity of this compound, as well as alterations in susceptibility to CN- occurring with age. ACKNOWLEDGMENTS The authors thank Ann-Marie Clark for statistical analysis of the data, and Janet J. Diliberto for technical assistance.
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