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The influence of some sulfur-containing compounds on the toxicity of nitrogen mustards
TOXICOLOGY
The
AND
APPLIED
Influence on
of the
5, 735-749
PHARMACOLOGY
Some
Toxicity
Sulfur-Containing of
BERNARD E. HIETBRINK U. S. Air Force
(1963)
Nitrogen
Mustards’
AND ANN B. RAYMUND
Radiation Laboratory, University Chicago 37, Illinois Received
Compounds
January
of Chicago,
21, 1963
Previous studies in this laboratory demonstrated that sublethal dosesof X-irradiation (DuBois and Petersen, 1954) and nitrogen mustards (DUBois et al., 1956) produce increasesin the adenosine triphosphatase activity of the spleensand thymus glands of rats and mice. The extent of increasein enzyme activity was dependent upon the dose of irradiation or nitrogen mustards. This biochemical effect was sufficiently constant and reproducible with various dosesof radiation to be used as a quantitative measurement of injury to the hematopoietic system and the protective activity of various chemical agents against the injury produced by these agents (Petersen and DuBois, 1955; Hietbrink et al., 1961). Other investigations in this laboratory (Doull and Cummings, 1953) have shown that X-irradiation causesin the cholinesteraseactivity of the intestine a dose-dependentdecreasewhich is maximal at 72 hours after exposure to radiation. The decrease in cholinesterase activity of the intestine has been successfully employed as a quantitative measurement of the influence of protective agents on radiation-induced intestinal injury (Hietbrink et al., 1961). The influence of nitrogen mustards on the cholinesteraseactivity of the intestine has not been studied previously. The present investigation was undertaken to ascertain the effects of two nitrogen mustards, ethyl bis( 2-chloroethyl) amine (HNl ) and methyl bis(2-chloroethyl) amine (HN2 ; mechlorethamine), on the cholinesterase activity of the intestine. Cholinesterasemeasurementson the intestine and adenosine triphosphatase measurementson the spleen were then used to evaluate the efficacy of some sulfur-containing compounds against tissue 1 This work was supported by the AF41(675)-252 monitored by the School Force Base, Texas.
United States Air Force under contract of Aerospace Medicine, USAF, Brooks Air 733
736
BERNARD
E. HIETBRINK
AND
ANN
B. RAYMUND
injury caused by nitrogen mustards as well as the enhancement of protection that results from certain combinations of chemical agents. METHODS
AND
MATERIALS
Young adult, female Sprague-Dawley rats (200-250 g) were used for these experiments. The animals were kept in air-conditioned quarters maintained at 68”-75°F and were fed Rockland Rat Diet and water ad libitum. For the enzyme assays the animals were sacrificed by decapitation, and cold, aqueous homogenates of spleen were prepared. A portion of the small intestine (jejunum) was freed from the mesenteric connective tissue and fat, longitudinally dissected, washed with distilled water, blotted with filter paper, minced, and homogenized in calcium-free Ringer-bicarbonate buffer. The adenosine triphosphatase activity of the spleens was measured by the method of DuBois and Potter (1943) with 0.5% tissue homogenates. Duplicate assays were performed in all cases, using 1 mg and 2 mg of tissue. Inorganic phosphorus was measured by the method of Fiske and Subbarow (1926). The adenosine triphosphatase activity was expressed as micrograms of inorganic phosphorus liberated from adenosine triphosphate per milligram of tissue during a 15-minute incubation period. The manometric method of DuBois and Mangun (1947) was used for measuring the cholinesterase activity of the small intestine. Measurements were conducted in duplicate using 50 mg of tissue in each Warburg vessel. Acetylcholine chloride was used as the substrate. The vessels were gassed with a mixture of 5% carbon dioxide and 95% nitrogen for 5 minutes. Carbon dioxide evolution was recorded at S-minute intervals for 30 minutes following a preliminary lo-minute equilibration period. Cholinesterase activity was expressed as microliters of carbon dioxide evolved per milligram of tissue per 10 minutes. Aqueous solutions of the hydrochlorides of HNI and HN2 were prepared immediately before use and injected by the intraperitoneal route. The sulfur-containing compounds used in this study were administered intraperitoneally as neutral aqueous solutions. Dimethylammonium dimethyldithiocarbamic acid and sodium diethyldithiocarbamic acid were given 5 minutes prior to HNl and HN2. Reduced glutathione and cysteine hydrochloride were injected 15 minutes before the nitrogen mustards, and 2-mercaptoethylamine hydrochloride was injected 10 minutes before the nitrogen mustards. The concentration of the drugs in the solutions was always adjusted so that the total volume injected did not exceed 1.6y0 of the body weight.
ANTAGONISM
OF NITROGEN
737
MUSTARDS
RESULTS
of Nitrogen testines of Rats
ZnfEuence
Mustards
on the Cholinesterase
Activity
of the In-
To ascertain whether the nitrogen mustards resemble X-irradiation (Burn et al., 1952; Doull and Cummings, 1953) in their effects on the cholinesterase activity of the small intestine, groups each containing 5 animals were given 0.5 mg/kg of HNI or HN2 and the small intestines were
FIG. 1. Effect of ethyl bis(2-chloroethyl)amine bis(2-chloroethyl)amine (HN2) hydrochloride small intestine of rats. Curve A, HN2; curve
(HNl) hydrochloride on the choline&erase B, HNI.
and activity
methyl of the
assayed for cholinesterase activity at various intervals. The results of these measurements are shown in Fig. 1. The data in Fig. 1 show that a sublethal dose of 0.5 mg/kg of HN2 causes a decrease in the cholinesterase activity of the small intestine. The decrease was evident at 1 day, reached a maximum in 2 to 3 days, and the activity then returned to normal within 7 days. A similar but more pronounced decrease in enzyme activity resulted from intraperitoneal administration of 0.5 mg/kg of HNl. A marked decrease in cholinesterase activity was evident at 2 days after this dose. It reached a maximum in 3
738
BERNARD
E. HIETBRINK
AND
ANN
B. RAYMUND
days and the activity returned to normal within 10 days, Another experiment was conducted to ascertain whether the decrease in cholinesterase activity caused by HNI and HN2 was dose dependent as it is in the case of X-irradiation (Doull and Cummings, 1953). For this experiment groups each containing 5 rats were given various doses of HN2 ranging from 0.25 mg/kg to 0.75 mg/kg intraperitoneally. Since the decrease in enzyme activity is maximal at approximately 3 days after nitrogen mustard administration (Fig. l), the animals were sacrificed at this time interval and the choline&erase activity of the intestine was measured. The results of these measurements demonstrated that doses ranging from 0.25 mg/kg to 1.0 mg/kg of HN2 or 0.25 mg/kg to 0.5 mg/kg of HNl produce a dose-dependent decrease in cholinesterase activity and that higher doses of these agents do not cause further decreases in enzyme activity. Thus it is evident that HNl and HN2 produce a decrease in the cholinesterase activity of the intestine that is dose dependent within a relatively narrow range. Influence of Various Chemical Agents on the Nitrogen Changes in Enzyme Activity of Rat Tissues
Mustard-Induced
Several sulfur-containing compounds have been found to protect rodents against the lethal effect of X-irradiation and nitrogen mustards (Goldenthal et ai., 1959). Our previous studies demonstrated that these compounds reduce the amount of radiation-induced changes in the adenosine triphosphatase activity of the spleens and the cholinesterase activity of the intestines of rats. Experiments were, therefore, undertaken to determine the ability of several sulfur-containing compounds to reduce the changes caused by nitrogen mustards in the adenosine triphosphatase activity of the spleens and the cholinesterase activity of the small intestines of rats. The effects of these compounds on the enzyme activities of tissues from rats treated with HNl and HN2 are shown in Tables 1 and 2. The enzyme assays were performed at 3 days after administration of the nitrogen mustards and drug treatment and each value represents the average obtained on tissues from at least 4 animals. The data in Table 1 indicate that certain sulfur-containing compounds have the ability to counteract the changes in enzyme activity caused by 1.5 mg/kg of HN2 which is the LDjo of this compound for rats under the conditions of our experiments. In the untreated control animals this dose of HN2 caused an increase to 188% of normal in the adenosine triphosphatase activity of the spleen, and it reduced the cholinesterase activity of the intestine to 41% of normal, Mercaptoethylamine and reduced glutathione
600
400
Sodium diethyldithiocarhamate
Dimethylammonium dimethyldithiocarbamate
the mean.
1000
Cysteine
from
900
Glutathione
deviation
200
Mercaptoethylamine
a Average
-
Control
1.5
1.5
1.5
1.5
1.5
1.5
Dose of HN2 (w/kg) -
AGENTS ON ENZYME
Dose of dwz (w/k)
OF CHEMICAL
HN2
Treatment
EFFECTS
1
32.0 -r- 3.4
27.5 k 0.8
29.6 -+ 1.5
36.9 -c 3.4
36.6 k 1.6
41.9 ‘- 3.4
22.1 -c 2.0”
HN2-TREATED
145
124
134
167
166
12
4
116e4
112 IL 16
105 f
42 f
57 _c 12
48 ?I 13
117+6”
188
Activity (units)
Intestine
RATS
$7 of Control
triphosphatase
OF TISSUES FROM adenosine
Activity (units)
Spleen
ACTIWTIES
TABLS
99
96
90
36
49
41
-
yo of Control
cholinesterase
5 2 2 z1
E E
8 z z
$ Et
n9
740
BERNARD
E. HIETBRINK
AND
ANN
B. RAYMUND
caused a moderate reduction in the amount of nitrogen mustard-induced change in the adenosine triphosphatase activity of the spleen. Mercaptoethylamine also afforded someprotection to the intestine. Very substantial protection was provided to the spleen by cysteine, sodium diethyldithiocarbamate, and dimethylammonium dimethyldithiocarbamate, and these agents almost completely prevented the HN2-induced decrease in the cholinesteraseactivity of the intestine. Similar experiments on the influence of sulfur-containing compounds on the injury to the spleenand intestine causedby HNl demonstrated that most of the compounds almost completely prevented the changes in enzyme activity produced by the LDsO (0.75 mg/kg) of this nitrogen mustard. Additional experiments were, therefore, conducted to measure the ability of someof these compounds to reduce changesin enzyme activity caused by lethal dosesof HNl. The results of these experiments are summarized in Table 2, in which average values for groups of at least 4 animals are presented.These data indicate that 0.75 mg/kg of HNI causedan increase in the adenosinetriphosphatase activity of the spleensto 188% of normal and a decreasein the cholinesteraseactivity of the intestine to 31% of normal. All the compoundsthat were tested provided substantial protection against the changescausedby HNl in enzyme activities in the spleenand intestine. Glutathione was the least effective and sodium diethyldithiocarbamate was the most effective of the agents tested. The latter compound substantially reduced the changes in enzyme activity of the spleen and intestines after dosesof HNl as high as 5 mg/kg, which is more than 6 times the LDjo. Effect
of Combinations of Chemical Agents on the Enzyme Activities of
Tissuesof Nitrogen Mustard-Treated Rats After the ability of various sulfur-containing compounds to reduce the nitrogen mustard-induced changes in enzyme activities of the spleen and intestine was demonstrated, studies were undertaken to ascertain the protective influence of several combinations of these agents. In preliminary experiments it was found that most of the combinations tested almost completely eliminated the changes in enzyme activity in the spleen and intestine caused by 3.0 mg/kg of HNl or HN2. Thus, in order to obtain information on the relative efficacy of the various combinations, the protective compounds were administered before injection of 5 mg/kg of the nitrogen mustards. Groups each containing 4 animalswere sacrificed 3 days later for adenosinetriphosphatase assayson the spleensand cholinesterase
Treatment
1.25 1.25 3.5
5.0 4.0
200
900 1000
600
400
5 Average deviation from the mean.
Control HNl Mercaptoethylamine Glutathione Cysteine Sodium diethyldithiocarbamate Dimethylammonium dimethyldithiocarbamate
Dose of HNl bdkd 0.75
Dose of drug (w/W -
32.4
29.5
135 147
2 1.5
137
132
129
188
% of ControI -
2.7
2
22.1 IL 2.0a 41.9 * 1.0 28.5 f 2.5 29.2 ? 2.6 29.8 2~ 4.6
Activity (units)
Spleen adenosine triphosphatase
73
-c
I4
77 f 18
62
66
Intestine cholinesterase Activity % of (units) ControI 117 t 6” 38 ‘- 6 31 77 k 6 66 65 2 4 56 85 2 15 73
TABLE 2 EFFECTS OF CHEMICAL AGENTS ON ENZYME ACTIVITIES OF TISSUES PROM HNI-TREATED RATS
z % $ i?l
2T 2 ;: z % 2 z i2 i2
1000
Cysteine plus mercaptoethylamine
a Average
deviation
Glutathione plus dimethylammonium dithiocarbamate
from
the mean.
dimethyl400
900
900 600
Glutathione sodium
plus diethyldithiocarbamate
400
dimethyl-
loo0
Cysteine plus dimethylammonium dithiocarbamate
600
loo0
plus diethyldithiocarbamate
Cysteine sodium
900
loo0
Cysteine plus glutathione
5.0
5.0
5 .o
5.0
s .o
5.0
1.5
200
-
(mg/W
HN2
Dose
AWNTS
-
Dose of drug (mg/kd
OF CHEMICAL
HNZ
OF COMBINATIONS
Control
Treatment
EFFECTS
TABLE
of
3
29.2
31.4
35.3
34.9
36.6
36.2
41.9
22.1
2.4
2.9
-r- 1.7
zk 1.1
& 2.0
-t
zk 3.0
f
+ 3.4
z!z 2.0a
Activity (unit.s)
adenosine
ACTIVITIES
Spleen
ON ENZYME
FROM
132
142
160
158
166
164
188
-
% of Control
triphosphatase
OF TISSUES
90 f
75 -I
57212
16
18
65 k 9
77
64
49
56
35
33
39 k 2
41k4
41
-
% of Control
cholinesterase
RATS
117&6= 48-c13
Activity (units)
Intestine
HNZ-TREATED
2
$
s m
5
z z z ?t
$
E M
3 T?*
a Average
deviation
from
the mean.
dimethyl-
plus diethyldithiocarbamate
dimethyldimethyldithio-
Glutathione plus dimethylammonium dithiocarbamate
Glutathione sodium
Cysteine plus ammonium carbamate
400
600 900
900
400
1000
600
1000
Cysteine sodium
plus diethyldithiocarhamate
-
Dose of drug (w/kg)
OF CHEMICAL
-
COMBINATIONS
Control
OF
HNl
Treatment
EFFECTS
TAB’LE
4
5.0
5.0
5.0
5.0
0.75
Dose of HNI bdkd -
adenosine
ACTIVITJES
1.0
24.8 k 1.5
24.8 2 2.0
27.3 ?z 0.7
25.0 2 0.5
41.9 *
22.1 k 2.oa
Activity (units)
Spleen
AGENTS ON ENZYME
112
112
123
113
188
-
76 of Control
triphosphatase
OF TISSUES FROM
94 f 12
108 c 4
114 * 14
92 -c- 13
38 f 6
117 k 65
Activity (units)
Intestine
HNl-TREATED
80
92
97
79
31
-
7%of Control
cholinesterase
RATS
744
BERNARD
E. HIETBRINK
AND
ANN
B. RAYMUND
assays on the intestines. The results of these measurements are shown in Tables. 3 and 4. The data in Table 3 show that all the combinations of sulfur-containing compounds tested were effective in reducing the degree of change in the enzyme activity of the spleen caused by 5 mg/kg of HN2. The combinations of glutathione with sodium diethyldithiocarbamate and glutathione with dimethylammonium dimethyldithiocarbamate were the most effective in preventing the HN2-induced changes in the tissues studied. The data in Table 4 show the influence of combinations of compounds on the changes in enzyme activity caused by HNl. The combination of cysteine or glutathione with sodium diethyldithiocarbamate or with dimethylammonium dimethyldithiocarbamate provided marked protection against HNl-induced changes in adenosine triphosphatase activity of the spleen and cholinesterase activity of the intestine. Eflect of Various Sulfur-Containing Nitrogen Mustards
Compounds on the Acute Toxicity
of
Acute toxicity studies were performed to ascertain whether a relationship exists between the effectiveness of the sulfur-containing chemicals in reducing changes in enzyme activity (Tables 1-4) and in preventing mortality caused by nitrogen mustards. For these measurements groups each containing 10 rats were treated with the same doses of the sulfur-containing protective agents that were used for the enzyme assays described above and were then given normally lethal doses of nitrogen mustards. The results of these measurements are summarized in Tables 5 and 6. The data in Table 5 indicate that cysteine was the most effective agent administered singly in preventing mortality from otherwise lethal doses of HN2. Only one of the animals given mercaptoethylamine (MEA) or glutathione prior to 4 mg/kg of HN2 survived whereas most of the rats injected with sodium diethyldithiocarbamate or dimethylammonium dimethyldithiocarabamate were able to tolerate this dose of nitrogen mustard. The combination of cysteine with MEA permitted survival of 60% of the animals given 7.5 mg/kg of HN2, but the animals given cysteine in combination with glutathione, sodium diethyldithiocarbamate or dimethylammonium dimethyldithiocarbamate were not able to tolerate 5 mg/kg of this nitrogen mustard as well as those which had received cysteine alone. Combination of glutathione with sodium diethyldithiocarbamate provided protection similar to that obtained with sodium diethyldithiocarabamate given singly. The data in Table 3 indicate that the combination of gluta-
ANTAGONISM
OF NITROGEN
745
MUSTARDS
thione and dimethylammonium dimethyldithiocarbamate is the most effective combination of those tested in preventing the changes in enzyme activity caused by 5 mg/kg of HN2. This combination prevented mortality of 90% of the animals given 7.5 mg/kg of HN2 and 40% of those given TABLE EFFECT
OF CHEMICAL GNEN
5
AGENTS
ON THE SUWIVAL
Doss
LETHAL
OF
OF RATS
HN2
Dose of drug tmdkd
Dose of HN2 (m&d
Mortality
% Mortality
Control
-
2.5
lO/lO
100
Mercaptoethylamine
200
4.0
9110
90
Glutathione
900
4.0
g/10
90
Cysteine
1000
5.0
o/10
Cysteine
1000
7.5
7/10
70
600
4.0
2/10
20
400
4.0
l/10
10
7.5
4/10
40
Drug
Sodium
treatment
diethyldithiocarhamate
Dimethylammonium dimethyldithiocarbamate
0
Cysteine plus mercaptoethylamine
1000
Cysteine plus glutathione
1000
900
5.0
2/lO
20
Cysteine plus sodium diethyldithiocarbamate
1000 600
5.0
2/10
20
Cysteine plus dimethylammonium dithiocarbamate
1000 5.0
4/10
40
4.0
2/10
20
7.5
l/10
10
6/10
60
Glutathione sodium
200
dimethyl400
plus diethyldithiocarbamate
Glutathione plus dimethylammonium dithiocarbamate
dimethyl-
Ghrtakhione plus dimethylammonium dithiocarbamate
dimethyl-
900 600 900 400 900
10.0
400
10 mg/kg of this nitrogen mustard. Thus, the combination of glutathione with dimethylammonium dimethyldithiocarbamate increased the LDrio of HN2 at least sixfold. The data presented in Table 6 show the effect of sulfur-containing compounds alone and in combination on the mortality of rats given lethal
746
BERNARD
E. HIETBRINK
AND
ANN
B. RAYMUND
doses of HNl. Mercaptoethylamine and glutathione were the least effective of the compounds tested in preventing HNl-induced mortality. Cysteine and the salts of dithiocarbamic acid were essentially equal in protective activity. All the combinations that were tested produced additive protective TABLE EFFECT
OF CHEMICAL GIVEN
6
AGENTS LETHAL
ON THE SURVIVAL DOSES
Dose of drug Drug
treatment
b-&k)
Control
OF RATS
OF HNl
%
Dose of HNl b-&kg)
Mortality
Mortality
1.5
10/10
100
Mercaptoethylamine
200
2.5
8/10
80
Glutathione
900
3.0
9/10
90
1000
5.0
2/10
20
600
5.0
2/10
20
400
5.0
4/10
40
7.5
2/10
20
7.5
l/l0
10
7.5
o/10
0
7.5
3/10
30
Cysteine Sodium
diethyldithiocarbamate
Dimethylammonium dithiocarbamate
dimethyl-
Cysteine plus sodium diethyldithiocarbamate
1000
Cysteine plus dimethylammonium dithiocarbamate
1000
Glutathione sodium
dimethyl400
plus diethyldithiocarbamate
Glutathione plus dimethylammonium dithiocarbamate
600
1000 600 900
dimethyl400
effects as measured by enzyme assays (Table 4), and the mortality studies illustrate that almost all the animals treated with these combinations were able to tolerate 7.5 mg/kg of HNI, which is 10 times the LDbO. DISCUSSION
A qualitative similarity between the nitrogen mustards and X-irradiation in their ability to inhibit citrate synthesis (DuBois et al., 1951, 1952) and to increase the adenosine triphosphatase activity of the hematopoietic tissues (DuBois et al., 1956) has been demonstrated previously in this laboratory. The results of studies described in this communication have shown that the nitrogen mustards, like X-irradiation (Burn et al., 1952 ; Doull and Cummings, 1953) cause a substantial decrease in the cholinesterase ac-
ANTAGONISM
OF NITROGEN
MUSTARDS
747
tivity of the small intestine of rats. The ability of chemical compounds to modify the radiation-induced changes in the adenosine triphosphatase activity of the spleens and cholinesterase activity of the small intestine of rats has been employed with satisfactory results for quantitating the protective activity of chemical agents against radiation damage to these tissues (Hietbrink et al., 1961). In view of the marked similarity in the biochemical effects of nitrogen mustards and X-irradiation, it was reasoned that the sameenzyme assaysshould constitute a suitable method of searching for agents capable of protecting against the toxicity of nitrogen mustards. The results of the present study demonstrated that several sulfur-containing compounds reduce the degree of nitrogen mustard-induced changes in the enzyme activities of the spleen and intestine. The protective action of cysteine, mercaptoethylamine (Goldenthal et al., 1959)) and glutathione (Therkelsen, 1958) on the toxicity of HN2 has been well established. In the present study the use of enzyme assays to test a number of other chemicals for protective activity resulted in the finding that derivatives of dithiocarbamic acid substantially reduce the injurious effects of the nitrogen mustards on the spleensand intestines of rats. A comparison of dimethylammonium dimethyldithiocarbamate and sodium diethyldithiocarbamate with cysteine, mercaptoethylamine, and glutathione indicated that these derivatives of dithiocarbamic acid exceed glutathione and mercaptoethylamine in protective activity to the spleen and intestine and they are similar to cysteine in protective activity against damage to these tissues. The results of acute toxicity measurementsdemonstrated that the enzyme assaysprovide a reliable indication of the protective activity of sulfur-containing compounds against nitrogen mustard lethality. The administration of certain combinations of sulfur-containing compounds before injection of HNl or HN2 provided more protection to the spleen and intestine than could be achieved by one compound alone. Enzyme assaysindicated that several of the combinations provided substantial reductions in the biological effects of high dosesof nitrogen mustards. The data indicated that in most instances combinations of cysteine or glutathione with derivatives of dithiocarbamic acid were considerably more effective in reducing the acute toxicity of HNl than the acute toxicity of HN2. In this connection most of the animals given these combinations were able to tolerate dosesof HN1 equivalent to about 10 times the normal LDjo while this treatment permitted survival of a majority of the rats given 3 to 5 times the LDjo of HN2. The exact mechanismsresponsible for the protective activity of sulfur-containing compounds are not com-
748
BERNARD
E. HIETBRINK
AND
ANN
B. RAYMUND
pletely understood, but Goldenthal et al. (1959) have demonstrated that cysteine combines with HN2 in vivo and that the protection provided by this compound is at least partly due to this chemical inactivation. Thus the ability of these agents to protect against the toxicity of HNl to a greater degree than against HN2 may be due to a difference in the rate at which these toxic agents react with sulfur-containing compounds. SUMMARY Measurements of the effect of nitrogen mustards on the cholinesterase activity of the small intestine of rats indicated that HNI and HN2 cause a decrease in enzyme activity of the intestine that is maximal at 2-4 days after administration and is reversible after sublethal doses of the nitrogen mustards. Modification of nitrogen mustard-induced changes in the adenosine triphosphatase activity of the spleens and the cholinesterase activity of the small intestines of rats was employed as a means of evaluating protective activity provided by sulfur-containing compounds against injury by nitrogen mustards to these tissues. Cysteine, dimethylammonium dimethyldithiocarbamate, and sodium diethyldithiocarbamate, when given before the nitrogen mustards, were more effective than mercaptoethylamine or reduced glutathione in reducing the biological effects of HNl and HN2 on the spleen and intestine of rats. Acute toxicity studies showed that the enzyme assays were valid indicators of the degree of protection provided by these compounds. Combinations of cysteine with mercaptoethylamine or glutathione were more effective in preventing HNZ-induced mortality than when these sulfur-containing compounds were administered alone. Most of the animals given combinations of cysteine or glutathione with the derivatives of dithiocarbamic acid were able to survive after doses of HNl as great as 10 times the normal LD,,. The results of this study demonstrated the applicability of enzyme assays on tissues known to be damaged by a particular toxic agent as valuable tools in the search for chemical protective agents. REFERENCES J. H., KORDICK, P., and MOLE, R. H. (1952). The effect of x-irradiation on the response of the intestine to acetylcholine and on its content of “pseudo” cholinesterase. Brit. J. Pharmacol. 7, 58-66. DOULL, J., and CUMMINGS, 0. K. (1953). Studies on the effects of x-irradiation on the neurohumoral mechanisms of the gastro-intestinal tract, hematopoietic tissues and various other tissues of rats. USAF Radiation Lab. Quart. Progr. Kept. NO.
BURN,
8, 28-49. DUBOIS, K.
P., and MANGUN, G. H. (1947). Effect of hexaethyl tetraphosphate on choline esterase in vitro and in vivo. PYOC. So&. Exptl. Biol. Med. %, 137-139. DIJBOIS, K. P., and PETERSEN, D. F. (1954). Adenosine triphosphatase and 5nucleotidase activity of hematopoietic tissues of irradiated animals. Am. J. Pkysiol. 176, 282-286. DuRors, K. P., and POTTER, V. R. (1943). The assay of animal tissues for respiratory enzymes. III. Adenosinetriphosphatase. J. Biol. Chem. 160, 185-195.
ANTAGONISM
OF NITROGEN
MUSTARDS
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DUBOIS, K. P., COCHRAN, K. W., and DOULL, J. (1951). Inhibition of citric acid synthesis in vivo by x-irradiation. Proc. Sot. Exptl. Biol. Med. 76, 422-427. DUBOIS, K. P., DEROIN, J., and COCHRAN, K. W. (1952). Influence of nitrogen mustards on citric acid synthesis in vivo. Proc. Sot. Exptl. Biol. Med. 81, 230-234. DUBOIS, K. P., PETERSEN, D. F., and ZINS, G. R. (1956). Phosphatase activity of hematopoietic tissues of nitrogen mustard-treated animals. Proc. SOL Exptl. Biol. Med. 91, 244-248. FISKE, C. H., and SUBBAROW, Y. (1926). The calorimetric determination of phosphorus. J. Biol. Chem. 66, 375-400. GOLDENTHAL, E. J., NADKARNI, M. V., and SMITH, P. K. (1959). A study of comparative protection against lethality of triethylenemelamine, nitrogen mustard and x-irradiation in mice. Radiation Res. 10, 571-583. HIETBRINK, B. E., RAYMUND, A. B., ZINS, G. R., and DUBOIS, K. P. (1961). Enzymatic measurement of the radioprotective activity of chemical agents. Toxicol. Appl. Pharmacol. 3, 267-277. PETERSEN, D. F., and DUBOIS, K. P. (1955). Modification of the radiation-induced increases in phosphatase activity of hematopoietic tissues by chemical agents. Am. J. Physiol. 181, 513-518. THERKELSEN, A. J. (1958). Studies of the mechanism of the protective action of sulfhydryl compounds and amines against nitrogen mustard (HN2) and roentgen irradiation in mice. Biochem. Pharmacol. 1. 258-266.
The
AND
APPLIED
Influence on
of the
5, 735-749
PHARMACOLOGY
Some
Toxicity
Sulfur-Containing of
BERNARD E. HIETBRINK U. S. Air Force
(1963)
Nitrogen
Mustards’
AND ANN B. RAYMUND
Radiation Laboratory, University Chicago 37, Illinois Received
Compounds
January
of Chicago,
21, 1963
Previous studies in this laboratory demonstrated that sublethal dosesof X-irradiation (DuBois and Petersen, 1954) and nitrogen mustards (DUBois et al., 1956) produce increasesin the adenosine triphosphatase activity of the spleensand thymus glands of rats and mice. The extent of increasein enzyme activity was dependent upon the dose of irradiation or nitrogen mustards. This biochemical effect was sufficiently constant and reproducible with various dosesof radiation to be used as a quantitative measurement of injury to the hematopoietic system and the protective activity of various chemical agents against the injury produced by these agents (Petersen and DuBois, 1955; Hietbrink et al., 1961). Other investigations in this laboratory (Doull and Cummings, 1953) have shown that X-irradiation causesin the cholinesteraseactivity of the intestine a dose-dependentdecreasewhich is maximal at 72 hours after exposure to radiation. The decrease in cholinesterase activity of the intestine has been successfully employed as a quantitative measurement of the influence of protective agents on radiation-induced intestinal injury (Hietbrink et al., 1961). The influence of nitrogen mustards on the cholinesteraseactivity of the intestine has not been studied previously. The present investigation was undertaken to ascertain the effects of two nitrogen mustards, ethyl bis( 2-chloroethyl) amine (HNl ) and methyl bis(2-chloroethyl) amine (HN2 ; mechlorethamine), on the cholinesterase activity of the intestine. Cholinesterasemeasurementson the intestine and adenosine triphosphatase measurementson the spleen were then used to evaluate the efficacy of some sulfur-containing compounds against tissue 1 This work was supported by the AF41(675)-252 monitored by the School Force Base, Texas.
United States Air Force under contract of Aerospace Medicine, USAF, Brooks Air 733
736
BERNARD
E. HIETBRINK
AND
ANN
B. RAYMUND
injury caused by nitrogen mustards as well as the enhancement of protection that results from certain combinations of chemical agents. METHODS
AND
MATERIALS
Young adult, female Sprague-Dawley rats (200-250 g) were used for these experiments. The animals were kept in air-conditioned quarters maintained at 68”-75°F and were fed Rockland Rat Diet and water ad libitum. For the enzyme assays the animals were sacrificed by decapitation, and cold, aqueous homogenates of spleen were prepared. A portion of the small intestine (jejunum) was freed from the mesenteric connective tissue and fat, longitudinally dissected, washed with distilled water, blotted with filter paper, minced, and homogenized in calcium-free Ringer-bicarbonate buffer. The adenosine triphosphatase activity of the spleens was measured by the method of DuBois and Potter (1943) with 0.5% tissue homogenates. Duplicate assays were performed in all cases, using 1 mg and 2 mg of tissue. Inorganic phosphorus was measured by the method of Fiske and Subbarow (1926). The adenosine triphosphatase activity was expressed as micrograms of inorganic phosphorus liberated from adenosine triphosphate per milligram of tissue during a 15-minute incubation period. The manometric method of DuBois and Mangun (1947) was used for measuring the cholinesterase activity of the small intestine. Measurements were conducted in duplicate using 50 mg of tissue in each Warburg vessel. Acetylcholine chloride was used as the substrate. The vessels were gassed with a mixture of 5% carbon dioxide and 95% nitrogen for 5 minutes. Carbon dioxide evolution was recorded at S-minute intervals for 30 minutes following a preliminary lo-minute equilibration period. Cholinesterase activity was expressed as microliters of carbon dioxide evolved per milligram of tissue per 10 minutes. Aqueous solutions of the hydrochlorides of HNI and HN2 were prepared immediately before use and injected by the intraperitoneal route. The sulfur-containing compounds used in this study were administered intraperitoneally as neutral aqueous solutions. Dimethylammonium dimethyldithiocarbamic acid and sodium diethyldithiocarbamic acid were given 5 minutes prior to HNl and HN2. Reduced glutathione and cysteine hydrochloride were injected 15 minutes before the nitrogen mustards, and 2-mercaptoethylamine hydrochloride was injected 10 minutes before the nitrogen mustards. The concentration of the drugs in the solutions was always adjusted so that the total volume injected did not exceed 1.6y0 of the body weight.
ANTAGONISM
OF NITROGEN
737
MUSTARDS
RESULTS
of Nitrogen testines of Rats
ZnfEuence
Mustards
on the Cholinesterase
Activity
of the In-
To ascertain whether the nitrogen mustards resemble X-irradiation (Burn et al., 1952; Doull and Cummings, 1953) in their effects on the cholinesterase activity of the small intestine, groups each containing 5 animals were given 0.5 mg/kg of HNI or HN2 and the small intestines were
FIG. 1. Effect of ethyl bis(2-chloroethyl)amine bis(2-chloroethyl)amine (HN2) hydrochloride small intestine of rats. Curve A, HN2; curve
(HNl) hydrochloride on the choline&erase B, HNI.
and activity
methyl of the
assayed for cholinesterase activity at various intervals. The results of these measurements are shown in Fig. 1. The data in Fig. 1 show that a sublethal dose of 0.5 mg/kg of HN2 causes a decrease in the cholinesterase activity of the small intestine. The decrease was evident at 1 day, reached a maximum in 2 to 3 days, and the activity then returned to normal within 7 days. A similar but more pronounced decrease in enzyme activity resulted from intraperitoneal administration of 0.5 mg/kg of HNl. A marked decrease in cholinesterase activity was evident at 2 days after this dose. It reached a maximum in 3
738
BERNARD
E. HIETBRINK
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ANN
B. RAYMUND
days and the activity returned to normal within 10 days, Another experiment was conducted to ascertain whether the decrease in cholinesterase activity caused by HNI and HN2 was dose dependent as it is in the case of X-irradiation (Doull and Cummings, 1953). For this experiment groups each containing 5 rats were given various doses of HN2 ranging from 0.25 mg/kg to 0.75 mg/kg intraperitoneally. Since the decrease in enzyme activity is maximal at approximately 3 days after nitrogen mustard administration (Fig. l), the animals were sacrificed at this time interval and the choline&erase activity of the intestine was measured. The results of these measurements demonstrated that doses ranging from 0.25 mg/kg to 1.0 mg/kg of HN2 or 0.25 mg/kg to 0.5 mg/kg of HNl produce a dose-dependent decrease in cholinesterase activity and that higher doses of these agents do not cause further decreases in enzyme activity. Thus it is evident that HNl and HN2 produce a decrease in the cholinesterase activity of the intestine that is dose dependent within a relatively narrow range. Influence of Various Chemical Agents on the Nitrogen Changes in Enzyme Activity of Rat Tissues
Mustard-Induced
Several sulfur-containing compounds have been found to protect rodents against the lethal effect of X-irradiation and nitrogen mustards (Goldenthal et ai., 1959). Our previous studies demonstrated that these compounds reduce the amount of radiation-induced changes in the adenosine triphosphatase activity of the spleens and the cholinesterase activity of the intestines of rats. Experiments were, therefore, undertaken to determine the ability of several sulfur-containing compounds to reduce the changes caused by nitrogen mustards in the adenosine triphosphatase activity of the spleens and the cholinesterase activity of the small intestines of rats. The effects of these compounds on the enzyme activities of tissues from rats treated with HNl and HN2 are shown in Tables 1 and 2. The enzyme assays were performed at 3 days after administration of the nitrogen mustards and drug treatment and each value represents the average obtained on tissues from at least 4 animals. The data in Table 1 indicate that certain sulfur-containing compounds have the ability to counteract the changes in enzyme activity caused by 1.5 mg/kg of HN2 which is the LDjo of this compound for rats under the conditions of our experiments. In the untreated control animals this dose of HN2 caused an increase to 188% of normal in the adenosine triphosphatase activity of the spleen, and it reduced the cholinesterase activity of the intestine to 41% of normal, Mercaptoethylamine and reduced glutathione
600
400
Sodium diethyldithiocarhamate
Dimethylammonium dimethyldithiocarbamate
the mean.
1000
Cysteine
from
900
Glutathione
deviation
200
Mercaptoethylamine
a Average
-
Control
1.5
1.5
1.5
1.5
1.5
1.5
Dose of HN2 (w/kg) -
AGENTS ON ENZYME
Dose of dwz (w/k)
OF CHEMICAL
HN2
Treatment
EFFECTS
1
32.0 -r- 3.4
27.5 k 0.8
29.6 -+ 1.5
36.9 -c 3.4
36.6 k 1.6
41.9 ‘- 3.4
22.1 -c 2.0”
HN2-TREATED
145
124
134
167
166
12
4
116e4
112 IL 16
105 f
42 f
57 _c 12
48 ?I 13
117+6”
188
Activity (units)
Intestine
RATS
$7 of Control
triphosphatase
OF TISSUES FROM adenosine
Activity (units)
Spleen
ACTIWTIES
TABLS
99
96
90
36
49
41
-
yo of Control
cholinesterase
5 2 2 z1
E E
8 z z
$ Et
n9
740
BERNARD
E. HIETBRINK
AND
ANN
B. RAYMUND
caused a moderate reduction in the amount of nitrogen mustard-induced change in the adenosine triphosphatase activity of the spleen. Mercaptoethylamine also afforded someprotection to the intestine. Very substantial protection was provided to the spleen by cysteine, sodium diethyldithiocarbamate, and dimethylammonium dimethyldithiocarbamate, and these agents almost completely prevented the HN2-induced decrease in the cholinesteraseactivity of the intestine. Similar experiments on the influence of sulfur-containing compounds on the injury to the spleenand intestine causedby HNl demonstrated that most of the compounds almost completely prevented the changes in enzyme activity produced by the LDsO (0.75 mg/kg) of this nitrogen mustard. Additional experiments were, therefore, conducted to measure the ability of someof these compounds to reduce changesin enzyme activity caused by lethal dosesof HNl. The results of these experiments are summarized in Table 2, in which average values for groups of at least 4 animals are presented.These data indicate that 0.75 mg/kg of HNI causedan increase in the adenosinetriphosphatase activity of the spleensto 188% of normal and a decreasein the cholinesteraseactivity of the intestine to 31% of normal. All the compoundsthat were tested provided substantial protection against the changescausedby HNl in enzyme activities in the spleenand intestine. Glutathione was the least effective and sodium diethyldithiocarbamate was the most effective of the agents tested. The latter compound substantially reduced the changes in enzyme activity of the spleen and intestines after dosesof HNl as high as 5 mg/kg, which is more than 6 times the LDjo. Effect
of Combinations of Chemical Agents on the Enzyme Activities of
Tissuesof Nitrogen Mustard-Treated Rats After the ability of various sulfur-containing compounds to reduce the nitrogen mustard-induced changes in enzyme activities of the spleen and intestine was demonstrated, studies were undertaken to ascertain the protective influence of several combinations of these agents. In preliminary experiments it was found that most of the combinations tested almost completely eliminated the changes in enzyme activity in the spleen and intestine caused by 3.0 mg/kg of HNl or HN2. Thus, in order to obtain information on the relative efficacy of the various combinations, the protective compounds were administered before injection of 5 mg/kg of the nitrogen mustards. Groups each containing 4 animalswere sacrificed 3 days later for adenosinetriphosphatase assayson the spleensand cholinesterase
Treatment
1.25 1.25 3.5
5.0 4.0
200
900 1000
600
400
5 Average deviation from the mean.
Control HNl Mercaptoethylamine Glutathione Cysteine Sodium diethyldithiocarbamate Dimethylammonium dimethyldithiocarbamate
Dose of HNl bdkd 0.75
Dose of drug (w/W -
32.4
29.5
135 147
2 1.5
137
132
129
188
% of ControI -
2.7
2
22.1 IL 2.0a 41.9 * 1.0 28.5 f 2.5 29.2 ? 2.6 29.8 2~ 4.6
Activity (units)
Spleen adenosine triphosphatase
73
-c
I4
77 f 18
62
66
Intestine cholinesterase Activity % of (units) ControI 117 t 6” 38 ‘- 6 31 77 k 6 66 65 2 4 56 85 2 15 73
TABLE 2 EFFECTS OF CHEMICAL AGENTS ON ENZYME ACTIVITIES OF TISSUES PROM HNI-TREATED RATS
z % $ i?l
2T 2 ;: z % 2 z i2 i2
1000
Cysteine plus mercaptoethylamine
a Average
deviation
Glutathione plus dimethylammonium dithiocarbamate
from
the mean.
dimethyl400
900
900 600
Glutathione sodium
plus diethyldithiocarbamate
400
dimethyl-
loo0
Cysteine plus dimethylammonium dithiocarbamate
600
loo0
plus diethyldithiocarbamate
Cysteine sodium
900
loo0
Cysteine plus glutathione
5.0
5.0
5 .o
5.0
s .o
5.0
1.5
200
-
(mg/W
HN2
Dose
AWNTS
-
Dose of drug (mg/kd
OF CHEMICAL
HNZ
OF COMBINATIONS
Control
Treatment
EFFECTS
TABLE
of
3
29.2
31.4
35.3
34.9
36.6
36.2
41.9
22.1
2.4
2.9
-r- 1.7
zk 1.1
& 2.0
-t
zk 3.0
f
+ 3.4
z!z 2.0a
Activity (unit.s)
adenosine
ACTIVITIES
Spleen
ON ENZYME
FROM
132
142
160
158
166
164
188
-
% of Control
triphosphatase
OF TISSUES
90 f
75 -I
57212
16
18
65 k 9
77
64
49
56
35
33
39 k 2
41k4
41
-
% of Control
cholinesterase
RATS
117&6= 48-c13
Activity (units)
Intestine
HNZ-TREATED
2
$
s m
5
z z z ?t
$
E M
3 T?*
a Average
deviation
from
the mean.
dimethyl-
plus diethyldithiocarbamate
dimethyldimethyldithio-
Glutathione plus dimethylammonium dithiocarbamate
Glutathione sodium
Cysteine plus ammonium carbamate
400
600 900
900
400
1000
600
1000
Cysteine sodium
plus diethyldithiocarhamate
-
Dose of drug (w/kg)
OF CHEMICAL
-
COMBINATIONS
Control
OF
HNl
Treatment
EFFECTS
TAB’LE
4
5.0
5.0
5.0
5.0
0.75
Dose of HNI bdkd -
adenosine
ACTIVITJES
1.0
24.8 k 1.5
24.8 2 2.0
27.3 ?z 0.7
25.0 2 0.5
41.9 *
22.1 k 2.oa
Activity (units)
Spleen
AGENTS ON ENZYME
112
112
123
113
188
-
76 of Control
triphosphatase
OF TISSUES FROM
94 f 12
108 c 4
114 * 14
92 -c- 13
38 f 6
117 k 65
Activity (units)
Intestine
HNl-TREATED
80
92
97
79
31
-
7%of Control
cholinesterase
RATS
744
BERNARD
E. HIETBRINK
AND
ANN
B. RAYMUND
assays on the intestines. The results of these measurements are shown in Tables. 3 and 4. The data in Table 3 show that all the combinations of sulfur-containing compounds tested were effective in reducing the degree of change in the enzyme activity of the spleen caused by 5 mg/kg of HN2. The combinations of glutathione with sodium diethyldithiocarbamate and glutathione with dimethylammonium dimethyldithiocarbamate were the most effective in preventing the HN2-induced changes in the tissues studied. The data in Table 4 show the influence of combinations of compounds on the changes in enzyme activity caused by HNl. The combination of cysteine or glutathione with sodium diethyldithiocarbamate or with dimethylammonium dimethyldithiocarbamate provided marked protection against HNl-induced changes in adenosine triphosphatase activity of the spleen and cholinesterase activity of the intestine. Eflect of Various Sulfur-Containing Nitrogen Mustards
Compounds on the Acute Toxicity
of
Acute toxicity studies were performed to ascertain whether a relationship exists between the effectiveness of the sulfur-containing chemicals in reducing changes in enzyme activity (Tables 1-4) and in preventing mortality caused by nitrogen mustards. For these measurements groups each containing 10 rats were treated with the same doses of the sulfur-containing protective agents that were used for the enzyme assays described above and were then given normally lethal doses of nitrogen mustards. The results of these measurements are summarized in Tables 5 and 6. The data in Table 5 indicate that cysteine was the most effective agent administered singly in preventing mortality from otherwise lethal doses of HN2. Only one of the animals given mercaptoethylamine (MEA) or glutathione prior to 4 mg/kg of HN2 survived whereas most of the rats injected with sodium diethyldithiocarbamate or dimethylammonium dimethyldithiocarabamate were able to tolerate this dose of nitrogen mustard. The combination of cysteine with MEA permitted survival of 60% of the animals given 7.5 mg/kg of HN2, but the animals given cysteine in combination with glutathione, sodium diethyldithiocarbamate or dimethylammonium dimethyldithiocarbamate were not able to tolerate 5 mg/kg of this nitrogen mustard as well as those which had received cysteine alone. Combination of glutathione with sodium diethyldithiocarbamate provided protection similar to that obtained with sodium diethyldithiocarabamate given singly. The data in Table 3 indicate that the combination of gluta-
ANTAGONISM
OF NITROGEN
745
MUSTARDS
thione and dimethylammonium dimethyldithiocarbamate is the most effective combination of those tested in preventing the changes in enzyme activity caused by 5 mg/kg of HN2. This combination prevented mortality of 90% of the animals given 7.5 mg/kg of HN2 and 40% of those given TABLE EFFECT
OF CHEMICAL GNEN
5
AGENTS
ON THE SUWIVAL
Doss
LETHAL
OF
OF RATS
HN2
Dose of drug tmdkd
Dose of HN2 (m&d
Mortality
% Mortality
Control
-
2.5
lO/lO
100
Mercaptoethylamine
200
4.0
9110
90
Glutathione
900
4.0
g/10
90
Cysteine
1000
5.0
o/10
Cysteine
1000
7.5
7/10
70
600
4.0
2/10
20
400
4.0
l/10
10
7.5
4/10
40
Drug
Sodium
treatment
diethyldithiocarhamate
Dimethylammonium dimethyldithiocarbamate
0
Cysteine plus mercaptoethylamine
1000
Cysteine plus glutathione
1000
900
5.0
2/lO
20
Cysteine plus sodium diethyldithiocarbamate
1000 600
5.0
2/10
20
Cysteine plus dimethylammonium dithiocarbamate
1000 5.0
4/10
40
4.0
2/10
20
7.5
l/10
10
6/10
60
Glutathione sodium
200
dimethyl400
plus diethyldithiocarbamate
Glutathione plus dimethylammonium dithiocarbamate
dimethyl-
Ghrtakhione plus dimethylammonium dithiocarbamate
dimethyl-
900 600 900 400 900
10.0
400
10 mg/kg of this nitrogen mustard. Thus, the combination of glutathione with dimethylammonium dimethyldithiocarbamate increased the LDrio of HN2 at least sixfold. The data presented in Table 6 show the effect of sulfur-containing compounds alone and in combination on the mortality of rats given lethal
746
BERNARD
E. HIETBRINK
AND
ANN
B. RAYMUND
doses of HNl. Mercaptoethylamine and glutathione were the least effective of the compounds tested in preventing HNl-induced mortality. Cysteine and the salts of dithiocarbamic acid were essentially equal in protective activity. All the combinations that were tested produced additive protective TABLE EFFECT
OF CHEMICAL GIVEN
6
AGENTS LETHAL
ON THE SURVIVAL DOSES
Dose of drug Drug
treatment
b-&k)
Control
OF RATS
OF HNl
%
Dose of HNl b-&kg)
Mortality
Mortality
1.5
10/10
100
Mercaptoethylamine
200
2.5
8/10
80
Glutathione
900
3.0
9/10
90
1000
5.0
2/10
20
600
5.0
2/10
20
400
5.0
4/10
40
7.5
2/10
20
7.5
l/l0
10
7.5
o/10
0
7.5
3/10
30
Cysteine Sodium
diethyldithiocarbamate
Dimethylammonium dithiocarbamate
dimethyl-
Cysteine plus sodium diethyldithiocarbamate
1000
Cysteine plus dimethylammonium dithiocarbamate
1000
Glutathione sodium
dimethyl400
plus diethyldithiocarbamate
Glutathione plus dimethylammonium dithiocarbamate
600
1000 600 900
dimethyl400
effects as measured by enzyme assays (Table 4), and the mortality studies illustrate that almost all the animals treated with these combinations were able to tolerate 7.5 mg/kg of HNI, which is 10 times the LDbO. DISCUSSION
A qualitative similarity between the nitrogen mustards and X-irradiation in their ability to inhibit citrate synthesis (DuBois et al., 1951, 1952) and to increase the adenosine triphosphatase activity of the hematopoietic tissues (DuBois et al., 1956) has been demonstrated previously in this laboratory. The results of studies described in this communication have shown that the nitrogen mustards, like X-irradiation (Burn et al., 1952 ; Doull and Cummings, 1953) cause a substantial decrease in the cholinesterase ac-
ANTAGONISM
OF NITROGEN
MUSTARDS
747
tivity of the small intestine of rats. The ability of chemical compounds to modify the radiation-induced changes in the adenosine triphosphatase activity of the spleens and cholinesterase activity of the small intestine of rats has been employed with satisfactory results for quantitating the protective activity of chemical agents against radiation damage to these tissues (Hietbrink et al., 1961). In view of the marked similarity in the biochemical effects of nitrogen mustards and X-irradiation, it was reasoned that the sameenzyme assaysshould constitute a suitable method of searching for agents capable of protecting against the toxicity of nitrogen mustards. The results of the present study demonstrated that several sulfur-containing compounds reduce the degree of nitrogen mustard-induced changes in the enzyme activities of the spleen and intestine. The protective action of cysteine, mercaptoethylamine (Goldenthal et al., 1959)) and glutathione (Therkelsen, 1958) on the toxicity of HN2 has been well established. In the present study the use of enzyme assays to test a number of other chemicals for protective activity resulted in the finding that derivatives of dithiocarbamic acid substantially reduce the injurious effects of the nitrogen mustards on the spleensand intestines of rats. A comparison of dimethylammonium dimethyldithiocarbamate and sodium diethyldithiocarbamate with cysteine, mercaptoethylamine, and glutathione indicated that these derivatives of dithiocarbamic acid exceed glutathione and mercaptoethylamine in protective activity to the spleen and intestine and they are similar to cysteine in protective activity against damage to these tissues. The results of acute toxicity measurementsdemonstrated that the enzyme assaysprovide a reliable indication of the protective activity of sulfur-containing compounds against nitrogen mustard lethality. The administration of certain combinations of sulfur-containing compounds before injection of HNl or HN2 provided more protection to the spleen and intestine than could be achieved by one compound alone. Enzyme assaysindicated that several of the combinations provided substantial reductions in the biological effects of high dosesof nitrogen mustards. The data indicated that in most instances combinations of cysteine or glutathione with derivatives of dithiocarbamic acid were considerably more effective in reducing the acute toxicity of HNl than the acute toxicity of HN2. In this connection most of the animals given these combinations were able to tolerate dosesof HN1 equivalent to about 10 times the normal LDjo while this treatment permitted survival of a majority of the rats given 3 to 5 times the LDjo of HN2. The exact mechanismsresponsible for the protective activity of sulfur-containing compounds are not com-
748
BERNARD
E. HIETBRINK
AND
ANN
B. RAYMUND
pletely understood, but Goldenthal et al. (1959) have demonstrated that cysteine combines with HN2 in vivo and that the protection provided by this compound is at least partly due to this chemical inactivation. Thus the ability of these agents to protect against the toxicity of HNl to a greater degree than against HN2 may be due to a difference in the rate at which these toxic agents react with sulfur-containing compounds. SUMMARY Measurements of the effect of nitrogen mustards on the cholinesterase activity of the small intestine of rats indicated that HNI and HN2 cause a decrease in enzyme activity of the intestine that is maximal at 2-4 days after administration and is reversible after sublethal doses of the nitrogen mustards. Modification of nitrogen mustard-induced changes in the adenosine triphosphatase activity of the spleens and the cholinesterase activity of the small intestines of rats was employed as a means of evaluating protective activity provided by sulfur-containing compounds against injury by nitrogen mustards to these tissues. Cysteine, dimethylammonium dimethyldithiocarbamate, and sodium diethyldithiocarbamate, when given before the nitrogen mustards, were more effective than mercaptoethylamine or reduced glutathione in reducing the biological effects of HNl and HN2 on the spleen and intestine of rats. Acute toxicity studies showed that the enzyme assays were valid indicators of the degree of protection provided by these compounds. Combinations of cysteine with mercaptoethylamine or glutathione were more effective in preventing HNZ-induced mortality than when these sulfur-containing compounds were administered alone. Most of the animals given combinations of cysteine or glutathione with the derivatives of dithiocarbamic acid were able to survive after doses of HNl as great as 10 times the normal LD,,. The results of this study demonstrated the applicability of enzyme assays on tissues known to be damaged by a particular toxic agent as valuable tools in the search for chemical protective agents. REFERENCES J. H., KORDICK, P., and MOLE, R. H. (1952). The effect of x-irradiation on the response of the intestine to acetylcholine and on its content of “pseudo” cholinesterase. Brit. J. Pharmacol. 7, 58-66. DOULL, J., and CUMMINGS, 0. K. (1953). Studies on the effects of x-irradiation on the neurohumoral mechanisms of the gastro-intestinal tract, hematopoietic tissues and various other tissues of rats. USAF Radiation Lab. Quart. Progr. Kept. NO.
BURN,
8, 28-49. DUBOIS, K.
P., and MANGUN, G. H. (1947). Effect of hexaethyl tetraphosphate on choline esterase in vitro and in vivo. PYOC. So&. Exptl. Biol. Med. %, 137-139. DIJBOIS, K. P., and PETERSEN, D. F. (1954). Adenosine triphosphatase and 5nucleotidase activity of hematopoietic tissues of irradiated animals. Am. J. Pkysiol. 176, 282-286. DuRors, K. P., and POTTER, V. R. (1943). The assay of animal tissues for respiratory enzymes. III. Adenosinetriphosphatase. J. Biol. Chem. 160, 185-195.
ANTAGONISM
OF NITROGEN
MUSTARDS
749
DUBOIS, K. P., COCHRAN, K. W., and DOULL, J. (1951). Inhibition of citric acid synthesis in vivo by x-irradiation. Proc. Sot. Exptl. Biol. Med. 76, 422-427. DUBOIS, K. P., DEROIN, J., and COCHRAN, K. W. (1952). Influence of nitrogen mustards on citric acid synthesis in vivo. Proc. Sot. Exptl. Biol. Med. 81, 230-234. DUBOIS, K. P., PETERSEN, D. F., and ZINS, G. R. (1956). Phosphatase activity of hematopoietic tissues of nitrogen mustard-treated animals. Proc. SOL Exptl. Biol. Med. 91, 244-248. FISKE, C. H., and SUBBAROW, Y. (1926). The calorimetric determination of phosphorus. J. Biol. Chem. 66, 375-400. GOLDENTHAL, E. J., NADKARNI, M. V., and SMITH, P. K. (1959). A study of comparative protection against lethality of triethylenemelamine, nitrogen mustard and x-irradiation in mice. Radiation Res. 10, 571-583. HIETBRINK, B. E., RAYMUND, A. B., ZINS, G. R., and DUBOIS, K. P. (1961). Enzymatic measurement of the radioprotective activity of chemical agents. Toxicol. Appl. Pharmacol. 3, 267-277. PETERSEN, D. F., and DUBOIS, K. P. (1955). Modification of the radiation-induced increases in phosphatase activity of hematopoietic tissues by chemical agents. Am. J. Physiol. 181, 513-518. THERKELSEN, A. J. (1958). Studies of the mechanism of the protective action of sulfhydryl compounds and amines against nitrogen mustard (HN2) and roentgen irradiation in mice. Biochem. Pharmacol. 1. 258-266.