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Toxicity of cadmium for the central nervous system
FXPFXIMENTAL
Toxicity
NEUROLOGY
of
Cadmium G.
Institut
18, 1.54-160
de Midicine
Received
(1967)
for
the
D.
BAIC,
GABBIANI,
Central AND
et de Chirurgie Expirimentales, Montreal, Canada February
2; Revision
Received
C.
Nervous
System
DBZIEL~ Universitt
March
de Montrecil,
6, 1967
In the rat and rabbit, cadmium chloride damages the central nervous system only when administered during the first days of life. The lesions are characterized by hemorrhagic suffusions together with destruction of fibers and cells. The granular layer of the cerebellar cortex is particularly affected.
Introduction In man, heavy metals are known to produce pathological changes of the nervous system (8). Since these are difficult to reproduce in experimental animals, particularly after the administration of inorganic salts (Z), knowledge of their pathogenesis and morphology is as yet incomplete. In adult animals of several species, such as the guinea pig, golden hamster, mouse and rat, the parenteral administration of cadmium salts produces lesions of the trigeminal and spinal sensory ganglia; these are mainly characterized by hemorrhagic suffusions, fiber and cell degeneration (3, 4). Here we report on the toxicity of cadmium for the central nervous system of newborn animals. We used the rat and rabbit, the first being sensitive and the second insensitive (according to our previous observations) to the action of cadmium on sensory ganglia. Materials
and
Methods
Two experiments were performed using male and female Sprague-Dawley rats and albino rabbits, both species suplied by the Robidoux Farm (Montreal). The number of animals per group, their age, body weight and day of treatment are indicated in the tables. Two rats and one rabbit per group served as untreated controls. Cadmium chloride (CdC12, Fisher Scientific Co.) was administered subcutaneously at the dose of 1 mg/lOO g body weight in 0.05 ml of water to the rats and 2 mg/lOO g body weight in 0.5 ml of water to the rabbits. Four days after CdCls injection the survivors were killed with chloroform. At autopsy, hemorrhagic changes were graded in terms of an arbi1 This work was supported by the Ministere de la Santb de la Province de QuCbec (subvention d’Ctablissement) and the John A. Hartford Foundation, Inc. We thank Dr. N. Mukai for his advice and supply of 1~x01 fast blue. 154
CADMIUM
TOXICITY
155
trary scale of 0 to 3, in which 0 = no hemorrhagic lesions, 1 = just visible hemorrhages, 2 = moderate, and 3 = most severe hemorrhages. The mean intensity of the lesions, together with the percentage of animals affected are listed in the tables. The brain, cerebellum and trigeminal ganglion of control and treated animals were fixed in neutral formalin or in Susa solution for staining with hematoxylin and phloxine, the PAS technique, and 1~x01 fast blue (7) counterstained either with cresyl violet or PAS. In our experience, keeping sensory ganglia in Susa solution for 24 hours provides a good preservation of the cells, whereas formalin does not. Results
As indicated in Table 1, the administration of comparable amounts of CdCla to newborn rats from the first to the twentieth days of life consistently induced lesions in the central nervous system (Fig. 1). Because of the thinness of the calvarium, the hemorrhagic suffusions were evident at external inspection, and after removing this bone, the cerebral and cerebellar surfaces appeared spotted with petechial hemorrhages which were occasionally widespread. Unexpectedly, the trigeminal and spinal ganglia were not affected prior to day 20; but on day 30, the only lesions of the nervous system produced by cadmium were those of the sensory ganglia, thus indicating that from then on, the animals had begun to react to cadmium as adults do. In the testis and ovary, we noted lesions (not mentioned in Table 1) similar to those already described (5, 6). In the newborn rabbit, the changes in the nervous system resembled those of the newborn rat. However, some differences were present in their distribution (Table 2). The cerebellar changes were more pronounced and extensive in the rabbit than in the rat (Fig. 2) and could be seen after treatment on day 30. The cerebrum showed hemorrhages only in the first 5 days of life. Changes in the trigeminal ganglion (similar to those seen in adult rats) (4) were seen after treatment from the first to thirtieth days. Lesions of the testis, ovary and liver (not listed in Table 2) were also present. Since the cerebral and cerebellar changes are histologically similar in both species, we will describe them conjointly. As expected, in normal animals the fibers of the cerebrum and cerebellum were unmyelinized on the first day of life (as judged by 1~x01 fast blue stain) and subsequently myelinization gradually developed. In the cerebrum, the most striking lesions consisted of spotty hemorrhages affecting mainly the white matter and the deeper part of the gray matter. In the hemorrhagic areas of the gray matter several pyramidal cells had a very pale nucleus containing only the nucleolus, the nuclear membrane being thick and hyperchromatic. Nissl substance had disappeared. In addition, few pyramidal cells showed pycnotic nuclei and hyperchromatic cytoplasm. In the white matter, we observed hemorrhagic regions and also
156
GABBIANI,
BAIC,
AND
DiZIEL
various zones where the fibers had been destroyed and the neuroglia cells had pycnotic nuclei and cytolysis. In the cerebellum the hemorrhages were massive after treatment during the first days of life but more circumscribed in older animals. In the hemorrhagic areas the Purkinje cells had disintegrated. In the surrounding zones several of these cells had a large pale nucleus containing
FIG. 1. A. Brain of control rat 4 days of age. of the same age treated with CdCI,. FIG. 2. Central nervous system of a I?-day-old lesion is sharply limited to the cerebellum.
B. Hemorrhagic rabbit
treated
lesions with
in
a rat
CdCI,.
The
only the nucleolus to which some darkly stained chromatin was attached; the cytoplasm was homogenous. In the granular layer we noted large zones of cell destruction containing only pycnotic nuclei and some detritus (Fig. 3) : moreover many fibers had disappeared. Rabbits injected with Cd& on days 20 and 30 had spots of ceil necrosis in the granular layer with littIe or no hemorrhages. Discussion
The results confirm our previous observation (3) that cadmium is toxic for the nervous system. However, we have no explanation for the localization of cadmium-induced lesions in the cerebrum and cerebellum of newborn rats and in sensory ganglia of older rats. Cadmium damages the cerebrum, cerebellum and sensory ganglia of the rabbit only at an early age. These findings agree with the well-established clinical and pathological knowledge that encephalopathies caused by heavy metals are seen only in infants and very young children (8). The degree of damage to the central nervous system diminished with the increase of myelinization. Possibly myelinization plays a role in the development of the lesions. It is noteworthy that the cells of the granular layer of the cerebellum, which are the only ones strongly affected by cadmium in the new-
CADMIUM
TOXICITY
157
born, have an embryologic development similar to that of the cells of the sensory ganglia ( I). During work performed independently at this Institute, it was noted that in newborn rats lead acetate can induce hemorrhagic lesions of the cerebrum and cerebellum which resemble those seen after CdC& administration (Selye,
Fro. 3. A. Histologic aspect of cerebellar cortex of an ll-day-old rat. A layer of cells can still be noted at the periphery of the circumvolution and at that time no myelinated fibers are present. B. Lesions induced by CdCl, in a rat of the same age. Note the hemorrhagic spots (arrows) and the extensive necrosis of the granular layer (Susa, 1~x01 fast blue and cresyl violet; X 90).
Ferland and Shatter, personal communication). Moreover, in control experiments not reported here, we observed that in newborn rats, the chlorides of indium, therbium, thallium and mercury can produce changes of this type (Gabbiani, unpublished observation). However, these compounds must be given in a much higher dose than that of CdCls, the damage produced by the latter being much more constant and extensive. From our previous work, we know that the toxic action of cadmium on nervous sensory ganglia is specific (3) ; we cannot reach the same conclusion concerning its toxicity for the central nervous system of newborn animals, because essentially similar lesions can be produced by other metals. However, cadmium appears to be the most potent poison affecting the above-mentioned organs.
8.8 14.0
29.5 33.0
5 10
20 30
-.
-. ,_.
a Each of five groups h Degrees of damage
wt. (9) 5.6
Age (day) 1
60 0
100 100
Incid. (%) 100
PRODUCED
TABLE BY CdCI,
-
_
. .,.
- ._ -
1
2.0 0
3.0 2.4
Deg. (scale) 2.8
rats untreated.
100 0
100 100
Incid. (k, 100
lesionsh
NEWBORN
Cerebellum
Hemorrhagic
IN THE
consisted of ten rats treated with cadmium and two are expressed by arbitrary scale of 0 to 3.
0.8 0
3.0 2.0
3.0
Deg. (scale)
Cerebrum
LESIONS
The
RATS
latter,
controls,
0.8 1.6
0 0
0
Deg. (scale)
showed
Ganglion
0 0
0
_
20 30
80 60
60
(%,
Mort.
,.- -.
changes.
- .^
no pathologic
70 100
Incid. (70)
s. 2 P
5
ti 2 5 ? g ,g
0
10
20 30
8
10
a In addition to the cadmium-treated changes. 0 See Table 1.
9
55
158.7 306.3
5
10
65.2
46.7
1
5
wt. (9)
Age (day)
No.”
animals,
2.0 0.8 0 0 0
Deg. (scale)
TABLE
2
served
control
as untreated
60
100 100
loo
100
Incid. (%)
and did not show
0.8
60
1.0
1.0
3.0 1.0 1.0
100
Ganglion Deg. (scale)
100
100 100
Incid. (%I
lesionsb
RABBIT
2.2 1.6 2.0
2.0
Deg. (scale)
Cerebellum
Hemorrhagic
IN THE NEWBORN
each age group
0 0
0
SO
100
(k)
Incid.
BY CdCI,
from
Cerebrum
PRODUCED
one rabbit
LESIONS
any
pathologic
40
60
40 60 20
(%‘o)
Mort.
g
;;
l-l 8
8
0 % $
GABBIANI,
160
BAIC,
AND
DdZIEL
Since the beginning of this century, heavy metals have gradually lost much of their importance as therapeutic or toxic agents. However, the participation of several of them in the constitution of enzymes and in various essential physiological processes has heightened the interest of investigators in their metabolism. Although we cannot yet arrive at a definite conclusion regarding the mechanism of cadmium toxicity, the experimental model of encephalopathy which we have described, may furnish a useful test for the study of the action of heavy metals on the nervous system. References G. 1954. “Anatomia A., and H. NOETZEL. chronischer oraler Vergiftung
1.
CHIARUGI,
2.
ENDERS,
3.
GABBIANI,
dell’Uomo,” Vol. 4. Societa Editrice 1955. Spezifische Veranderungen mit Sublimat. Arch. E+ptZ. Pathol.
Libraria. Milano. im Kleinhirn bei Pharmakol. 225:
346-351.
G.
1966.
Action
of cadmium
chloride
on sensory
ganglia.
Experientia
22:
261-264.
4. 5. 6. 7. 8.
GABBIANI, G., A. GREGORY, and D. BAIC. Cadmium-induced selective lesions of sensory ganglia. J. Neuropathol. Exptl. Neural. (in press) KAR, A. B., R. P. DAS, and J. N. KARXUN. 1959. Ovarian changes in prepuberal rats after treatment with cadmium chloride. Acta Biol. Med. Germ. 3: 372-399. PARIZEK, J., and 2. ZAHOR. 1956. Effect of cadmium salts on testicular tissue. Nature 177: 1036. PURSE, E. A. 1961. “Histochemistry.” Churchill, London. ROBBINS, S. R. 1966. “Textbook of Pathology.” W. B. Saunders, Philadelphia, Pennsylvania.
Toxicity
NEUROLOGY
of
Cadmium G.
Institut
18, 1.54-160
de Midicine
Received
(1967)
for
the
D.
BAIC,
GABBIANI,
Central AND
et de Chirurgie Expirimentales, Montreal, Canada February
2; Revision
Received
C.
Nervous
System
DBZIEL~ Universitt
March
de Montrecil,
6, 1967
In the rat and rabbit, cadmium chloride damages the central nervous system only when administered during the first days of life. The lesions are characterized by hemorrhagic suffusions together with destruction of fibers and cells. The granular layer of the cerebellar cortex is particularly affected.
Introduction In man, heavy metals are known to produce pathological changes of the nervous system (8). Since these are difficult to reproduce in experimental animals, particularly after the administration of inorganic salts (Z), knowledge of their pathogenesis and morphology is as yet incomplete. In adult animals of several species, such as the guinea pig, golden hamster, mouse and rat, the parenteral administration of cadmium salts produces lesions of the trigeminal and spinal sensory ganglia; these are mainly characterized by hemorrhagic suffusions, fiber and cell degeneration (3, 4). Here we report on the toxicity of cadmium for the central nervous system of newborn animals. We used the rat and rabbit, the first being sensitive and the second insensitive (according to our previous observations) to the action of cadmium on sensory ganglia. Materials
and
Methods
Two experiments were performed using male and female Sprague-Dawley rats and albino rabbits, both species suplied by the Robidoux Farm (Montreal). The number of animals per group, their age, body weight and day of treatment are indicated in the tables. Two rats and one rabbit per group served as untreated controls. Cadmium chloride (CdC12, Fisher Scientific Co.) was administered subcutaneously at the dose of 1 mg/lOO g body weight in 0.05 ml of water to the rats and 2 mg/lOO g body weight in 0.5 ml of water to the rabbits. Four days after CdCls injection the survivors were killed with chloroform. At autopsy, hemorrhagic changes were graded in terms of an arbi1 This work was supported by the Ministere de la Santb de la Province de QuCbec (subvention d’Ctablissement) and the John A. Hartford Foundation, Inc. We thank Dr. N. Mukai for his advice and supply of 1~x01 fast blue. 154
CADMIUM
TOXICITY
155
trary scale of 0 to 3, in which 0 = no hemorrhagic lesions, 1 = just visible hemorrhages, 2 = moderate, and 3 = most severe hemorrhages. The mean intensity of the lesions, together with the percentage of animals affected are listed in the tables. The brain, cerebellum and trigeminal ganglion of control and treated animals were fixed in neutral formalin or in Susa solution for staining with hematoxylin and phloxine, the PAS technique, and 1~x01 fast blue (7) counterstained either with cresyl violet or PAS. In our experience, keeping sensory ganglia in Susa solution for 24 hours provides a good preservation of the cells, whereas formalin does not. Results
As indicated in Table 1, the administration of comparable amounts of CdCla to newborn rats from the first to the twentieth days of life consistently induced lesions in the central nervous system (Fig. 1). Because of the thinness of the calvarium, the hemorrhagic suffusions were evident at external inspection, and after removing this bone, the cerebral and cerebellar surfaces appeared spotted with petechial hemorrhages which were occasionally widespread. Unexpectedly, the trigeminal and spinal ganglia were not affected prior to day 20; but on day 30, the only lesions of the nervous system produced by cadmium were those of the sensory ganglia, thus indicating that from then on, the animals had begun to react to cadmium as adults do. In the testis and ovary, we noted lesions (not mentioned in Table 1) similar to those already described (5, 6). In the newborn rabbit, the changes in the nervous system resembled those of the newborn rat. However, some differences were present in their distribution (Table 2). The cerebellar changes were more pronounced and extensive in the rabbit than in the rat (Fig. 2) and could be seen after treatment on day 30. The cerebrum showed hemorrhages only in the first 5 days of life. Changes in the trigeminal ganglion (similar to those seen in adult rats) (4) were seen after treatment from the first to thirtieth days. Lesions of the testis, ovary and liver (not listed in Table 2) were also present. Since the cerebral and cerebellar changes are histologically similar in both species, we will describe them conjointly. As expected, in normal animals the fibers of the cerebrum and cerebellum were unmyelinized on the first day of life (as judged by 1~x01 fast blue stain) and subsequently myelinization gradually developed. In the cerebrum, the most striking lesions consisted of spotty hemorrhages affecting mainly the white matter and the deeper part of the gray matter. In the hemorrhagic areas of the gray matter several pyramidal cells had a very pale nucleus containing only the nucleolus, the nuclear membrane being thick and hyperchromatic. Nissl substance had disappeared. In addition, few pyramidal cells showed pycnotic nuclei and hyperchromatic cytoplasm. In the white matter, we observed hemorrhagic regions and also
156
GABBIANI,
BAIC,
AND
DiZIEL
various zones where the fibers had been destroyed and the neuroglia cells had pycnotic nuclei and cytolysis. In the cerebellum the hemorrhages were massive after treatment during the first days of life but more circumscribed in older animals. In the hemorrhagic areas the Purkinje cells had disintegrated. In the surrounding zones several of these cells had a large pale nucleus containing
FIG. 1. A. Brain of control rat 4 days of age. of the same age treated with CdCI,. FIG. 2. Central nervous system of a I?-day-old lesion is sharply limited to the cerebellum.
B. Hemorrhagic rabbit
treated
lesions with
in
a rat
CdCI,.
The
only the nucleolus to which some darkly stained chromatin was attached; the cytoplasm was homogenous. In the granular layer we noted large zones of cell destruction containing only pycnotic nuclei and some detritus (Fig. 3) : moreover many fibers had disappeared. Rabbits injected with Cd& on days 20 and 30 had spots of ceil necrosis in the granular layer with littIe or no hemorrhages. Discussion
The results confirm our previous observation (3) that cadmium is toxic for the nervous system. However, we have no explanation for the localization of cadmium-induced lesions in the cerebrum and cerebellum of newborn rats and in sensory ganglia of older rats. Cadmium damages the cerebrum, cerebellum and sensory ganglia of the rabbit only at an early age. These findings agree with the well-established clinical and pathological knowledge that encephalopathies caused by heavy metals are seen only in infants and very young children (8). The degree of damage to the central nervous system diminished with the increase of myelinization. Possibly myelinization plays a role in the development of the lesions. It is noteworthy that the cells of the granular layer of the cerebellum, which are the only ones strongly affected by cadmium in the new-
CADMIUM
TOXICITY
157
born, have an embryologic development similar to that of the cells of the sensory ganglia ( I). During work performed independently at this Institute, it was noted that in newborn rats lead acetate can induce hemorrhagic lesions of the cerebrum and cerebellum which resemble those seen after CdC& administration (Selye,
Fro. 3. A. Histologic aspect of cerebellar cortex of an ll-day-old rat. A layer of cells can still be noted at the periphery of the circumvolution and at that time no myelinated fibers are present. B. Lesions induced by CdCl, in a rat of the same age. Note the hemorrhagic spots (arrows) and the extensive necrosis of the granular layer (Susa, 1~x01 fast blue and cresyl violet; X 90).
Ferland and Shatter, personal communication). Moreover, in control experiments not reported here, we observed that in newborn rats, the chlorides of indium, therbium, thallium and mercury can produce changes of this type (Gabbiani, unpublished observation). However, these compounds must be given in a much higher dose than that of CdCls, the damage produced by the latter being much more constant and extensive. From our previous work, we know that the toxic action of cadmium on nervous sensory ganglia is specific (3) ; we cannot reach the same conclusion concerning its toxicity for the central nervous system of newborn animals, because essentially similar lesions can be produced by other metals. However, cadmium appears to be the most potent poison affecting the above-mentioned organs.
8.8 14.0
29.5 33.0
5 10
20 30
-.
-. ,_.
a Each of five groups h Degrees of damage
wt. (9) 5.6
Age (day) 1
60 0
100 100
Incid. (%) 100
PRODUCED
TABLE BY CdCI,
-
_
. .,.
- ._ -
1
2.0 0
3.0 2.4
Deg. (scale) 2.8
rats untreated.
100 0
100 100
Incid. (k, 100
lesionsh
NEWBORN
Cerebellum
Hemorrhagic
IN THE
consisted of ten rats treated with cadmium and two are expressed by arbitrary scale of 0 to 3.
0.8 0
3.0 2.0
3.0
Deg. (scale)
Cerebrum
LESIONS
The
RATS
latter,
controls,
0.8 1.6
0 0
0
Deg. (scale)
showed
Ganglion
0 0
0
_
20 30
80 60
60
(%,
Mort.
,.- -.
changes.
- .^
no pathologic
70 100
Incid. (70)
s. 2 P
5
ti 2 5 ? g ,g
0
10
20 30
8
10
a In addition to the cadmium-treated changes. 0 See Table 1.
9
55
158.7 306.3
5
10
65.2
46.7
1
5
wt. (9)
Age (day)
No.”
animals,
2.0 0.8 0 0 0
Deg. (scale)
TABLE
2
served
control
as untreated
60
100 100
loo
100
Incid. (%)
and did not show
0.8
60
1.0
1.0
3.0 1.0 1.0
100
Ganglion Deg. (scale)
100
100 100
Incid. (%I
lesionsb
RABBIT
2.2 1.6 2.0
2.0
Deg. (scale)
Cerebellum
Hemorrhagic
IN THE NEWBORN
each age group
0 0
0
SO
100
(k)
Incid.
BY CdCI,
from
Cerebrum
PRODUCED
one rabbit
LESIONS
any
pathologic
40
60
40 60 20
(%‘o)
Mort.
g
;;
l-l 8
8
0 % $
GABBIANI,
160
BAIC,
AND
DdZIEL
Since the beginning of this century, heavy metals have gradually lost much of their importance as therapeutic or toxic agents. However, the participation of several of them in the constitution of enzymes and in various essential physiological processes has heightened the interest of investigators in their metabolism. Although we cannot yet arrive at a definite conclusion regarding the mechanism of cadmium toxicity, the experimental model of encephalopathy which we have described, may furnish a useful test for the study of the action of heavy metals on the nervous system. References G. 1954. “Anatomia A., and H. NOETZEL. chronischer oraler Vergiftung
1.
CHIARUGI,
2.
ENDERS,
3.
GABBIANI,
dell’Uomo,” Vol. 4. Societa Editrice 1955. Spezifische Veranderungen mit Sublimat. Arch. E+ptZ. Pathol.
Libraria. Milano. im Kleinhirn bei Pharmakol. 225:
346-351.
G.
1966.
Action
of cadmium
chloride
on sensory
ganglia.
Experientia
22:
261-264.
4. 5. 6. 7. 8.
GABBIANI, G., A. GREGORY, and D. BAIC. Cadmium-induced selective lesions of sensory ganglia. J. Neuropathol. Exptl. Neural. (in press) KAR, A. B., R. P. DAS, and J. N. KARXUN. 1959. Ovarian changes in prepuberal rats after treatment with cadmium chloride. Acta Biol. Med. Germ. 3: 372-399. PARIZEK, J., and 2. ZAHOR. 1956. Effect of cadmium salts on testicular tissue. Nature 177: 1036. PURSE, E. A. 1961. “Histochemistry.” Churchill, London. ROBBINS, S. R. 1966. “Textbook of Pathology.” W. B. Saunders, Philadelphia, Pennsylvania.