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Toxicity of intravenously injected uranium in guinea pigs
TOXICOLOGY
AND
Toxicity
APPLIED
of
PHARMACOLOGY
7,
Intravenously
of
Animal
(1963)
Injected DANIEL
Department
32-36
Disease
R.
in Guinea
Pigs’
FARNELL
Research,
Received
Uranium
Auburn
December
University,
Auburn,
Alabama
17, 1963
The observation that uranium has an injurious effect on the kidneys is said to have been made as early as 1854 (MacNider, 1924). Since that time, uranium nephritis has been studied rather extensively in various animals, such as the rabbit, dog, rat, and mouse (MacNider, 1924; Voegtlin and Hodge, 1949; Tannenbaum, 1951; Stone et al., 1961). Dickson (1909) gave a detailed description in guinea pigs of both chronic and acute nephritis caused by subcutaneously injected uranium nitrate. The World War II Manhattan Project gave great impetus to toxicologic investigations of uranium, which was being used in large quantities in the development of the atomic bomb and in related activities (Voegtlin and Hodge, 1949 ; Tannenbaum, 195 1) . As a part of that project, a preliminary study was made of the effect of intravenously injected uranium nitrate hexahydrate in guinea pigs (Voegtlin and Hodge, 1949). The experiment reported in this paper arose during the process of establishing a convenient and reliable method for producing a nonfatal nephritis in guinea pigs. MATERIALS
AND
METHODS
Two separate experiments were performed. The first experiment was designed to determine renal lesions in guinea pigs given a single injection of uranium acetate, UO2 (CzH302)2*2H20. The second experiment consisted of a dose response test in which guinea pigs given varying single doses of uranium were observed over a period of 33 days to detect signs of toxicity. Experiment 1. Eight male guinea pigs ranging in size from 503 to 730 g were used in this experiment. The hair from the anterior surface of a forearm was removed, and 9570 alcohol was applied as an antiseptic. Uranium acetate in 0.8570 sodium chloride was injected into the cephalic vein by means of a l-ml tuberculin syringe and a 24-gauge hypodermic needle. Each guinea pig was weighed and the dosage of uranium was adjusted according to weight. The concentration of uranium acetate for each dose level was adjusted in an attempt to keep the total volume of fluid injected as uniform as practical, about 0.5 ml per animal. Two control animals were injected with saline only. The other animals received either 2.67, 5.35, or 10.69 mg of uranium acetate per kilogram of body weight. The amount of material administered to each guinea pig is shown in Table 1. 1 Publication no. 934, Publications Committee, School of Veterinary Medicine, sity, Auburn, Alabama. This study was conducted as a part of Hatch Project Experiment Station, Auburn University. 32
Auburn Univer146, Agricultural
TOXICITY
OF URANIUM
IN
GUINEA
PIGS
33
After injection, the animals were maintained on a stock guinea pig ration and tap water, which were available at all times. Lettuce was given daily as a supplement. On the fifth day after administration of uranium, all the animals were anesthetized in an ether chamber, bled from the heart, then killed. Each animal was necropsied, and the kidneys were placed in 10% formalin for histologic examination. The plasma was separated from the blood and frozen until used for the determination of plasma creatinine (Hawk et al., 1954). Paraffin sections (7 p) were prepared from the kidneys and stained with Mayer’s hematoxylin and eosin. Experiment 2. Ten guinea pigs ranging in weight from 680 to 962 g were used for this experiment. Uranium acetate was administered to each guinea pig as described for experiment 1. Following injection, the guinea pigs were maintained on the same diet as in the first experiment. The animals were frequently observed during the following 33 days, at the end of which time the experiment was concluded. RESULTS Results
of Experiment
1
Five days after injection of uranium, the kidneys of treated guinea pigs appeared somewhat enlarged and paler in color than those of the untreated animals. The cut surface of the kidneys from the animals that received uranium showed between the medulla and cortex a distinct demarcation that was not evident in the normal kidneys. Microscopically, there was considerable damage to kidney tissue from all dose levels of uranium. There were numerous tubular casts, both in the cortex and medulla. The majority of the casts were hyaline and bright pink-staining (almost as darkly staining as red blood cells), Other casts were finely granular and stained less intensely. In an occasional tubule there were free cells in the lumen or cellular casts containing leucocytes, erythrocytes, and/or epithelial cells. There were increased numbers of fibroblasts among tubules in the cortex. Also, there was a somewhat generalized increase in lymphoid cells, usually more noticeable in the vicinity of glomeruli, but occasionally in aggregations in the cortex not close to glomeruli. The tubules of the cortex of kidneys from treated animals appeared dilated as compared to kidneys from control animals. This lesion could have contributed to the abnormal demarcation between the medulla and cortex, as was seen grossly. Many of the tubules showed degenerative changes, such as pycnosis and fragmentation of cell nuclei, vacuolation and lysis of cytoplasm, and dislodgement of cells from the basement membrane. The presence of relatively abundant mitotic figures indicated active cellular regeneration. These mitotic changes were present both in tubular epithelial cells and in interstitial cells. Careful comparison with normal kidneys revealed an increase in fibrillar, pale, pinkstaining material among glomerular tuft capillaries. This material presumably was collagen, as described by Bencosme et al. (1960) in rat kidneys damaged by uranium. Further evidence of glomerular damage was the occasional presence in the capsular space of pink-staining homogeneous material of a composition similar in appearance to the hyaline casts seen in many of the tubules. The results of the creatinine determinations are shown in Table 1. There was a progressive increase in plasma creatinine with increasing doses of uranium.
Animal no.
530 604 730 507 549 643 505 553
Body wt. (grams)
0.51 0.55
1.069
1.069
1
TO MALE
TABLE
Volume of fluid administered (ml)
ADMINISTERED
0.53 0.60 0.73 0.51 0.55 0.64
ACETATE
0 0 0.267 0.267 0.535 0.535
70 Concentration of uranium acetate in 0.85% NaCl
URANIUM
GUINEA
0 0 2.67 2.67 5.35 5.35 10.69 10.69
Dose of uranium acetate administered (w/kg)
PIGS, EXPERIMENT 1
0 0 1.5 1.5 3.0 3.0 6.0 6.0
Dose of uranium (metal) administered (w/W
2 ?I F:
0.60 2.02 1.95 1.80 2.55 3 .oo 4.12
s M :(w/100 ml) 0.60
Plasma creatinine
TOXICITY
Results
of Experiment
OF
URANIUM
IN
GUINEA
35
PIGS
2
The data from Experiment 2 are shown in Table 2. Ten guinea pigs were observed for 33 days following injection with saline or various doses of uranium acetate. There were only two deaths during this period. The first occurred 8 days after injection at the highest dose of uranium, 6.0 mg/kg. The other death occurred at 14 days after injection at a dose of 3.0 mg/kg. Both of the animals that died had kidney lesions. Necropsies were not performed on the animals that survived. TABLE SURVIVAL
Animal no. 1 2 3 4 5 6 7 8 9 10
OF GUINEA
Sex
PIGS FOLLOWING
2
URANIUM
Weight (grams)
ADMINISTRATION, Dose of uranium (metal) administered (m&g)
EXPERINENT
2 Days survived following injection >33
780
0 0 1.5 1.5 1.5
F
883
3.0
>33
F F M
831
3.0
736 931
6.0
>33
F
827
6.0 6.0
>33
M F M F M
686 719 680 962
>33 >33 >33 >33 14 8
There were no deaths in the control animals or in those receiving a uranium dosage of I .5 mg/kg. DISCUSSION Uranium acetate produced definite kidney damage in all guinea pigs at doses of either 2.67, 5.35, or 10.69 mg/kg. These levels of uranium acetate correspond to levels of uranium metal of 1.5, 3.0, and 6.0 mg/kg, respectively. No fatalities occurred during 33 days from a dose of uranium acetate of 2.67 mg/kg. Also, less than one-half the animals given the two higher doses died during the same period. Dickson (1909), who used subcutaneously injected uranium nitrate, reported that a single dose of 5 mg/kg killed 5 out of 14 guinea pigs. This compares closely with the effect of intravenously injected uranium acetate. However, the dose of 2.67 mg/kg, a nonlethal level of uranium acetate, is much higher than the dose of 0.60 mg/kg, which has been reported to be the lethal dose of uranium nitrate hexahydrate injected intravenously in guinea pigs (Voegtlin and Hodge, 1949). Apparently, uranium nitrate hexahydrate is more toxic intravenously than an equal quantity of uranium acetate. The total content of uranium metal is slightly higher in the acetate compound; therefore, it seems possible that the nitrate ion is responsible for the greater toxicity of uranium nitrate hexahydrate. SUMMARY Kidney lesions and plasma creatinine were determined in a group of guinea pigs injected venously 5 days previously with doses of uranium acetate corresponding to either 1.5, 3.0, or of uranium metal per kilogram of body weight. Extensive lesions of nephritis, including
intra-
6.0mg both
36
DANIEL
R. FARNELL
glomerular and tubular damage, were seen at all three levels of uranium. Plasma creatinine increased noticeably as the uranium dosage increased. In another group of animals given uranium at the same dose levels as the first group, a dose of uranium acetate of 2.67 mg/kg (1.5 mg of uranium metal per kilogram) caused no deaths during a 33-day observation period. More than one-half of the animals survived the higher doses. An effective, nonlethal intravenous dose of uranium acetate for producing nephritis in guinea pigs is 2.67 mg/kg. ACKNOWLEDGMENT The
author
is grateful
for
technical
assistance
by Mrs.
Patricia
C. Logan
and Mrs.
Louise
Perez.
REFERENCES BENCOSME, S. A., STONE, R. S., LATTA, H., and MADDEN, S. C. (1960). Acute tubular and glomerular lesions in rat kidneys after uranium injury. A.M.A. Arch. Pathol. 69, 470-476. DICKSON, E. C. (1909). A report on the experimental production of chronic nephritis in animals by the use of uranium nitrate. Arch. Internal Med. 3, 375-410. HAWK, P. B., OSER, B. L., and SUMZMERSON, W. H. (1954). Practical Physiological Chemistry, 13th ed. McGraw-Hill, New York. MACNIDER, W. DEB. (1924). A review of acute experimental nephritis. Physiol. Rev. 4, 595-638. STONE, R. S., BENCOSME, S. A., LATTA, H., and MADDEN, S. C. (1961). Renal tubular fine structure studied during reaction to acute uranium injury. A.M.A. Arch. Pathol. 71, 160-174. TANNENBAUM, A. (Ed.) (1951). Toxicology of Uranium. McGraw-Hill, New York. VOEGTLIN, C., and HODGE, H. D. (Eds.) (1949). Pharmacology and Toxicology of Uranium Compounds. McGraw-Hill, New York.
AND
Toxicity
APPLIED
of
PHARMACOLOGY
7,
Intravenously
of
Animal
(1963)
Injected DANIEL
Department
32-36
Disease
R.
in Guinea
Pigs’
FARNELL
Research,
Received
Uranium
Auburn
December
University,
Auburn,
Alabama
17, 1963
The observation that uranium has an injurious effect on the kidneys is said to have been made as early as 1854 (MacNider, 1924). Since that time, uranium nephritis has been studied rather extensively in various animals, such as the rabbit, dog, rat, and mouse (MacNider, 1924; Voegtlin and Hodge, 1949; Tannenbaum, 1951; Stone et al., 1961). Dickson (1909) gave a detailed description in guinea pigs of both chronic and acute nephritis caused by subcutaneously injected uranium nitrate. The World War II Manhattan Project gave great impetus to toxicologic investigations of uranium, which was being used in large quantities in the development of the atomic bomb and in related activities (Voegtlin and Hodge, 1949 ; Tannenbaum, 195 1) . As a part of that project, a preliminary study was made of the effect of intravenously injected uranium nitrate hexahydrate in guinea pigs (Voegtlin and Hodge, 1949). The experiment reported in this paper arose during the process of establishing a convenient and reliable method for producing a nonfatal nephritis in guinea pigs. MATERIALS
AND
METHODS
Two separate experiments were performed. The first experiment was designed to determine renal lesions in guinea pigs given a single injection of uranium acetate, UO2 (CzH302)2*2H20. The second experiment consisted of a dose response test in which guinea pigs given varying single doses of uranium were observed over a period of 33 days to detect signs of toxicity. Experiment 1. Eight male guinea pigs ranging in size from 503 to 730 g were used in this experiment. The hair from the anterior surface of a forearm was removed, and 9570 alcohol was applied as an antiseptic. Uranium acetate in 0.8570 sodium chloride was injected into the cephalic vein by means of a l-ml tuberculin syringe and a 24-gauge hypodermic needle. Each guinea pig was weighed and the dosage of uranium was adjusted according to weight. The concentration of uranium acetate for each dose level was adjusted in an attempt to keep the total volume of fluid injected as uniform as practical, about 0.5 ml per animal. Two control animals were injected with saline only. The other animals received either 2.67, 5.35, or 10.69 mg of uranium acetate per kilogram of body weight. The amount of material administered to each guinea pig is shown in Table 1. 1 Publication no. 934, Publications Committee, School of Veterinary Medicine, sity, Auburn, Alabama. This study was conducted as a part of Hatch Project Experiment Station, Auburn University. 32
Auburn Univer146, Agricultural
TOXICITY
OF URANIUM
IN
GUINEA
PIGS
33
After injection, the animals were maintained on a stock guinea pig ration and tap water, which were available at all times. Lettuce was given daily as a supplement. On the fifth day after administration of uranium, all the animals were anesthetized in an ether chamber, bled from the heart, then killed. Each animal was necropsied, and the kidneys were placed in 10% formalin for histologic examination. The plasma was separated from the blood and frozen until used for the determination of plasma creatinine (Hawk et al., 1954). Paraffin sections (7 p) were prepared from the kidneys and stained with Mayer’s hematoxylin and eosin. Experiment 2. Ten guinea pigs ranging in weight from 680 to 962 g were used for this experiment. Uranium acetate was administered to each guinea pig as described for experiment 1. Following injection, the guinea pigs were maintained on the same diet as in the first experiment. The animals were frequently observed during the following 33 days, at the end of which time the experiment was concluded. RESULTS Results
of Experiment
1
Five days after injection of uranium, the kidneys of treated guinea pigs appeared somewhat enlarged and paler in color than those of the untreated animals. The cut surface of the kidneys from the animals that received uranium showed between the medulla and cortex a distinct demarcation that was not evident in the normal kidneys. Microscopically, there was considerable damage to kidney tissue from all dose levels of uranium. There were numerous tubular casts, both in the cortex and medulla. The majority of the casts were hyaline and bright pink-staining (almost as darkly staining as red blood cells), Other casts were finely granular and stained less intensely. In an occasional tubule there were free cells in the lumen or cellular casts containing leucocytes, erythrocytes, and/or epithelial cells. There were increased numbers of fibroblasts among tubules in the cortex. Also, there was a somewhat generalized increase in lymphoid cells, usually more noticeable in the vicinity of glomeruli, but occasionally in aggregations in the cortex not close to glomeruli. The tubules of the cortex of kidneys from treated animals appeared dilated as compared to kidneys from control animals. This lesion could have contributed to the abnormal demarcation between the medulla and cortex, as was seen grossly. Many of the tubules showed degenerative changes, such as pycnosis and fragmentation of cell nuclei, vacuolation and lysis of cytoplasm, and dislodgement of cells from the basement membrane. The presence of relatively abundant mitotic figures indicated active cellular regeneration. These mitotic changes were present both in tubular epithelial cells and in interstitial cells. Careful comparison with normal kidneys revealed an increase in fibrillar, pale, pinkstaining material among glomerular tuft capillaries. This material presumably was collagen, as described by Bencosme et al. (1960) in rat kidneys damaged by uranium. Further evidence of glomerular damage was the occasional presence in the capsular space of pink-staining homogeneous material of a composition similar in appearance to the hyaline casts seen in many of the tubules. The results of the creatinine determinations are shown in Table 1. There was a progressive increase in plasma creatinine with increasing doses of uranium.
Animal no.
530 604 730 507 549 643 505 553
Body wt. (grams)
0.51 0.55
1.069
1.069
1
TO MALE
TABLE
Volume of fluid administered (ml)
ADMINISTERED
0.53 0.60 0.73 0.51 0.55 0.64
ACETATE
0 0 0.267 0.267 0.535 0.535
70 Concentration of uranium acetate in 0.85% NaCl
URANIUM
GUINEA
0 0 2.67 2.67 5.35 5.35 10.69 10.69
Dose of uranium acetate administered (w/kg)
PIGS, EXPERIMENT 1
0 0 1.5 1.5 3.0 3.0 6.0 6.0
Dose of uranium (metal) administered (w/W
2 ?I F:
0.60 2.02 1.95 1.80 2.55 3 .oo 4.12
s M :(w/100 ml) 0.60
Plasma creatinine
TOXICITY
Results
of Experiment
OF
URANIUM
IN
GUINEA
35
PIGS
2
The data from Experiment 2 are shown in Table 2. Ten guinea pigs were observed for 33 days following injection with saline or various doses of uranium acetate. There were only two deaths during this period. The first occurred 8 days after injection at the highest dose of uranium, 6.0 mg/kg. The other death occurred at 14 days after injection at a dose of 3.0 mg/kg. Both of the animals that died had kidney lesions. Necropsies were not performed on the animals that survived. TABLE SURVIVAL
Animal no. 1 2 3 4 5 6 7 8 9 10
OF GUINEA
Sex
PIGS FOLLOWING
2
URANIUM
Weight (grams)
ADMINISTRATION, Dose of uranium (metal) administered (m&g)
EXPERINENT
2 Days survived following injection >33
780
0 0 1.5 1.5 1.5
F
883
3.0
>33
F F M
831
3.0
736 931
6.0
>33
F
827
6.0 6.0
>33
M F M F M
686 719 680 962
>33 >33 >33 >33 14 8
There were no deaths in the control animals or in those receiving a uranium dosage of I .5 mg/kg. DISCUSSION Uranium acetate produced definite kidney damage in all guinea pigs at doses of either 2.67, 5.35, or 10.69 mg/kg. These levels of uranium acetate correspond to levels of uranium metal of 1.5, 3.0, and 6.0 mg/kg, respectively. No fatalities occurred during 33 days from a dose of uranium acetate of 2.67 mg/kg. Also, less than one-half the animals given the two higher doses died during the same period. Dickson (1909), who used subcutaneously injected uranium nitrate, reported that a single dose of 5 mg/kg killed 5 out of 14 guinea pigs. This compares closely with the effect of intravenously injected uranium acetate. However, the dose of 2.67 mg/kg, a nonlethal level of uranium acetate, is much higher than the dose of 0.60 mg/kg, which has been reported to be the lethal dose of uranium nitrate hexahydrate injected intravenously in guinea pigs (Voegtlin and Hodge, 1949). Apparently, uranium nitrate hexahydrate is more toxic intravenously than an equal quantity of uranium acetate. The total content of uranium metal is slightly higher in the acetate compound; therefore, it seems possible that the nitrate ion is responsible for the greater toxicity of uranium nitrate hexahydrate. SUMMARY Kidney lesions and plasma creatinine were determined in a group of guinea pigs injected venously 5 days previously with doses of uranium acetate corresponding to either 1.5, 3.0, or of uranium metal per kilogram of body weight. Extensive lesions of nephritis, including
intra-
6.0mg both
36
DANIEL
R. FARNELL
glomerular and tubular damage, were seen at all three levels of uranium. Plasma creatinine increased noticeably as the uranium dosage increased. In another group of animals given uranium at the same dose levels as the first group, a dose of uranium acetate of 2.67 mg/kg (1.5 mg of uranium metal per kilogram) caused no deaths during a 33-day observation period. More than one-half of the animals survived the higher doses. An effective, nonlethal intravenous dose of uranium acetate for producing nephritis in guinea pigs is 2.67 mg/kg. ACKNOWLEDGMENT The
author
is grateful
for
technical
assistance
by Mrs.
Patricia
C. Logan
and Mrs.
Louise
Perez.
REFERENCES BENCOSME, S. A., STONE, R. S., LATTA, H., and MADDEN, S. C. (1960). Acute tubular and glomerular lesions in rat kidneys after uranium injury. A.M.A. Arch. Pathol. 69, 470-476. DICKSON, E. C. (1909). A report on the experimental production of chronic nephritis in animals by the use of uranium nitrate. Arch. Internal Med. 3, 375-410. HAWK, P. B., OSER, B. L., and SUMZMERSON, W. H. (1954). Practical Physiological Chemistry, 13th ed. McGraw-Hill, New York. MACNIDER, W. DEB. (1924). A review of acute experimental nephritis. Physiol. Rev. 4, 595-638. STONE, R. S., BENCOSME, S. A., LATTA, H., and MADDEN, S. C. (1961). Renal tubular fine structure studied during reaction to acute uranium injury. A.M.A. Arch. Pathol. 71, 160-174. TANNENBAUM, A. (Ed.) (1951). Toxicology of Uranium. McGraw-Hill, New York. VOEGTLIN, C., and HODGE, H. D. (Eds.) (1949). Pharmacology and Toxicology of Uranium Compounds. McGraw-Hill, New York.