ENVIRONMENTAL
RESEARCH
Influence
14, 92 - 98 (1977)
of Age on Whole-Body Retention Distribution of llSmCd in the Rat D. KELLO
Institute
for
AND KRISTA
KOSTIAL
Medical Rrsrurch and Occ~rcpational Health. Yugo.\lav Sciences and Arts. 41000 Zagreb. Ytrgoslavia Received
August
and
Academy
oj
I I, 1976
The influence of age on whole-body retention and organ distribution of intraperitoneally applied ‘1SmCdC12 was studied in I-, 1-. 6-. and 52-week-old rats. The mean percent values of ‘rsmCd retention in the whole body decreased with increasing age. The distribution of lrs’“Cd in the body on the 14th day after application shows that in all age groups most of the cadmium is accumulated in the liver (45%56%) and kidneys (4-6s). In younger rats the percentage of cadmium in the kidney and blood was always higher than in older animals in contrast to the liver where it was lower. The percentage of the dose in the liver, kidneys, and blood represents however, a considerably lower fraction of the total whole-body retention in younger animals than in older rats. It is therefore concluded that age bears a significant influence on cadmium metabolism. This might be important for estimating cadmium body burden and critical organ exposure in the youngest age group.
INTRODUCTION
A consequence of a growing application of cadmium and its compounds has been an increased concentration of this toxic metal in the environment and accordingly, a danger to human health in general (WHO, 1972). Comprehensive studies indicate that the cadmium content in the body of an individual with an average exposure has reached almost one-fourth of the amount which is known to induce an impairment of the kidney functions (Friberg rt al., 1974). In our earlier studies we established that the gastrointestinal cadmium absorption in the neonatal period is about 80 times higher than in adult life (Kello and Kostial, 1977). However, with the experimental technique used, we could not determine how much of this effect was due to differences in intestinal absorption or to other changes in cadmium metabolism and distribution which depend on age. The results of our earlier experiments with lead show that young rats not only absorb significantly more lead from the gastrointestinal tract (Kostial et al., 1971), but also that they excrete less lead from the body (Kostial et nl., 1973), that they distribute it in the body differently (Momcilovic and Kostial, 1974), that they bind it in the bone more tightly (Kello et ul., 1975). and that a smaller fraction of lead can be removed with chelating agents than in adults (Jug0 et al., 1975). Consequently, we assumed that generally, age might have an influence on the metabolism of heavy metals, and that this aspect should be further investigated. The purpose of our studies therefore was to obtain more data on the retention and distribution of cadmium in relation to age in order to obtain additional parameters for estimating body burden and critical organ exposure to this toxic element in the youngest age group. 92 Copyright 0 1977 by Academic Press. Inc. All rights of reproducfmn m any form reserved.
ISSN 0013.9351
AGE
AND
MATERIAL
Cd
93
RETENTION
AND METHODS
We studied the fate of the intraperitoneally applied ‘lSmCd in albino rats aged 1, 3, 6, and 52 weeks. All animals received standard rat food and water, with the exception of l-week-old rats who were fed mother’s milk throughout the experiment. A single intraperitoneal dose of radioactive solution contained between 10 and 20 &i lls’nCd Cl, (specific activity by 0.55 1.O mCi/mgCd; purchased from “The Radiochemical Centre,” Amersham). In the period between the 1st and 14th day after radioisotope application at fixed time intervals the whole body radioactivity was measured in a two-crystal scintillation detector for y-radiation. On the 14th day of the experiment all animals were exsanguinated in ether narcosis and their kidneys and liver were removed. Fresh weight of liver, kidneys, and blood were determined and the activity was measured in an automatic sample changer. The weight of the weighed blood was corrected to the theoretical total whole blood content in animals of matching weight and age according to Belcher and Harris ( 1957). RESULTS
The results of body and organ weights presented in Table 1 show a higher gain in body than in organ weights with increasing age. The results of the mean percent values of **SmCd retention presented in Fig. 1 show a decrease in whole-body retention values with increasing age. The process of initial equilibration and elimination seems to be age dependent being practically absent in l-week-old animals and lasting about 3 days in the oldest group of rats
123456789
FIG. I. Whole-body retention as mean t SD. Age and number (10). n .
of intraperitoneally of animals: I week
10 n
IZ t3 DAYS
1L
applied llSfHCdCI, in rats of different age expressed (22). 0: 3 weeks (10). 0: 6 weeks (9). 0; 52 weeks
KELLO
AND
KOSTIAL
6: 2:
‘:
0
m
c,
AGE
AND
Cd
KE’L ESTION
95
after which period the retention curves become almost linear. In this second elimination period young rats seem to excrete even more cadmium than adults. The analysis of the distribution of iiF”Cd in the rat’s body on the 14th day after intraperitoneal application shows that regardless of age, by far the most cadmium is accumulated in the liver (Table 2). In this period the liver, although it makes only about 4% of the total body weight (Table 1) accumulates between 46 and 56% of the parenterally administered dose. The liver of young rats accumulates less cadmium than that of older animals. The kidney makes the second organ as far as the degree of “5”‘Cd retention during this period is concerned. Cadmium retention on the 14th day after intraperitoneal application amounts to 4-6%~ of the administered dose (Table 2). In young rats the kidney, in contrast to the liver, retains always more cadmium than in adults. The same holds for the blood, where the percentage in very young is approximately three times higher than in adult animals. Although young rats retain in the whole body a considerably higher percentage of the intraperitoneally applied “5”LCd, the percentage in the liver. kidney, and blood represents a considerably lower fraction of the total whole body retention in younger animals than in adults (Fig. 2). We could therefore conclude that the increased total cadmium burden in the young is due primarily to increased accumulation of this toxic metal in other parts of the body, but not at the site of injection, as proved in preliminary experiments. DISCUSSION
After intraperitoneal application the finding that young rats retain considerably more ‘IZ”‘Cd in the body is in agreement with data on the metabolism of some other heavy metals, i.e., mercury (Jugo, 1976), lead (Kostial rt trl., 1973), cerium (Inaba and Lengemann, 1972). and plutonium (Sikov and Mahlum. 1973). Although some authors ascribe such increased metal retention in the young to the immaturity of excretory organs (Stather. 1970). we assume that this might be primarily due to a different binding of cadmium in immature and adult rats immediately after a parenteral application. Our results show two phases of cadmium elimination in adult rats: a fast initial and a slow late one (Fig. I), which is in agreement with the results reported by other authors (Moore rt (I/., 1973). We might assume that during the initial fast phase cadmium in blood is bound to ligands of low molecular weight in contrast to the later phase when cadmium is supposed to be bound to larger molecules as found by Griffin and Matson (1972) in adult rats. At the early stage, an enhanced cadmium elimination could therefore be expected, owing to the important role played by the size of molecules to which cadmium is bound in circulation (Vestal and Heller, 196X: Foulkes, 1974). In immature animals. however, we observed only one slow phase of cadmium elimination. This might indicate that cadmium in the immature animals at this initial period might be bound to different ligands. most probably at the sites of intense synthesis of proteins, which is characteristic of this age. This hypothesis is supported by the fact that young rats showed a considerably lower cadmium retention in the liver. kidneys, and blood, and a higher retention in the rest of the body. The reason for this may be accounted for by a more intense growth of other
96
KELLO
AND
KOSTIAL
100 %
90
80
70
60
50
LO
30
20
10
m
LIVER
m
BLOOD
m
KIDNEYS
I-1
OTHER
FIG. 9. Retention of “‘“Cd in rats on 14th day after intraperitoneal percent of whole-body retention of cadmium.
TISSUES
application:
results
expressed
as
parts of the young rats’ body compared to critical organs like the liver and kidney. Sikov and Mahlum (1972) point out similar factors when explaining the differences in the metabolism of plutonium in young and old rats. This explanation is even more likely to be applicable to cadmium since cadmium metabolism proved to be closely associated with the synthesis of some proteins (Squibb and Cousins, 1974; Sabbioni and Marafante. 1975). The lower cadmium accumulation in the liver of young rats could be explained by several other factors too. It could be partly due to the faster rate of cadmium elimination from this organ at this age. On the other hand, it could be also caused by a decreased cadmium binding capacity of the liver in the young. This might be a result of a reduced synthesis of specific proteins, metallothionein to which cadmium is largely bound in the liver (Squibb and Cousins, 1974; Cherian, 1974). Thus, Pohlandt (1974) found that the capacity for the conversion of methionin into cysteine, the amino acid which makes about 30% of the liver metallothionein, is not sufficiently developed in young organism (Weser rt al., 1973). The assumption that young rats might not have enough cysteine for the synthesis of metal-
AGE
AND
Cd
97
RETENTION
lothionein is supported by Bernhart’s results (1970). He found that in young rats the need for the sulphur-containing amino acids is extremely strong (possibly because of hair growth). In the kidneys, on the other hand, in the same observation interval we found much more ilSmCd than in adult rats. This might be because of its specific redistribution in the young. The ability of cadmium to mobilize from the liver and other tissues and to accumulate in the kidneys is well known (Friberg et al., 1975). Such mobilization from other tissue as well as from the whole body might be much higher in the young than in the adult because of increased remodeling rate and could gradually bring about an earlier accumulation in the kidneys. This higher turnover of cadmium concentration in the blood. Gunn and Gould (1927), who found a significantly lower cadmium accumulation in the kidney of the young, obtained their results in an extremely short period after ‘iSnCd application (24 hours), when probably not enough time for its redistribution and accumulation in the kidney was allowed. From our results it is possible to conclude that age bears a significant influence on cadmium metabolism. This should be kept in mind when setting up standards for the maximum permissible exposure to cadmium of certain population groups the more so as the effect of its increased retention in young bodies is ultimately reflected in increased accumulation in critical organs, the kidneys. ACKNOWLEDGMENT This Agency
work
was partially
supported
by a Research
Grant
from
the U.S.
Environmental
Protection
REFERENCES Belcher. 8. H.. and Harris. E. B. ( 1957). Studies on plasma volume. red cell volume and total blood volume in young growing rats. J. Phvsiol. 139, 64-78. Bemhart. F. W. (1970). Comparison of the essential amino acid, nitrogen and calorie requirements of the weanling rat and breastfed infant. J. Nutr. 100, 461-466. Cherian. M. G. (1974). Isolation and purification of cadmium binding proteins from rat liver. Biochen~. Biophus. Res. 61, 920-926. Foulkes. E. S. (1974). Excretion and retention of cadmium. zinc. and mercury by rabbit kidney, A~cT. J. Physl. 227, l356- 1360. Friberg. L.. Piscator. M.. Nordberg. F. G., and Kjellstrom. T. (1974). “Cadmium in the Environment.” 2nd ed.. CRC Press. Cleveland. Friberg. L., Kjellstrom. T., Nordberg. G., and Piscator, M. (1975). “Cadmium in the Environment.” 3rd ed.. Environmental Protection Technology Series, EPA-65012-75-049. Griffin, R. M.. and Matson, W. R. (1972). The assessment of individual variability to trace metal insult: Low-molecular weight metal complexing agents as indicators of trace metal insult. Amer. I&. Hp. 33, 373-377. Gunn. A. S., and Gould. T. C. (1957). Selective accumulation of Cd”’ by cortex of rat kidney. Pro
RESEARCH
Influence
14, 92 - 98 (1977)
of Age on Whole-Body Retention Distribution of llSmCd in the Rat D. KELLO
Institute
for
AND KRISTA
KOSTIAL
Medical Rrsrurch and Occ~rcpational Health. Yugo.\lav Sciences and Arts. 41000 Zagreb. Ytrgoslavia Received
August
and
Academy
oj
I I, 1976
The influence of age on whole-body retention and organ distribution of intraperitoneally applied ‘1SmCdC12 was studied in I-, 1-. 6-. and 52-week-old rats. The mean percent values of ‘rsmCd retention in the whole body decreased with increasing age. The distribution of lrs’“Cd in the body on the 14th day after application shows that in all age groups most of the cadmium is accumulated in the liver (45%56%) and kidneys (4-6s). In younger rats the percentage of cadmium in the kidney and blood was always higher than in older animals in contrast to the liver where it was lower. The percentage of the dose in the liver, kidneys, and blood represents however, a considerably lower fraction of the total whole-body retention in younger animals than in older rats. It is therefore concluded that age bears a significant influence on cadmium metabolism. This might be important for estimating cadmium body burden and critical organ exposure in the youngest age group.
INTRODUCTION
A consequence of a growing application of cadmium and its compounds has been an increased concentration of this toxic metal in the environment and accordingly, a danger to human health in general (WHO, 1972). Comprehensive studies indicate that the cadmium content in the body of an individual with an average exposure has reached almost one-fourth of the amount which is known to induce an impairment of the kidney functions (Friberg rt al., 1974). In our earlier studies we established that the gastrointestinal cadmium absorption in the neonatal period is about 80 times higher than in adult life (Kello and Kostial, 1977). However, with the experimental technique used, we could not determine how much of this effect was due to differences in intestinal absorption or to other changes in cadmium metabolism and distribution which depend on age. The results of our earlier experiments with lead show that young rats not only absorb significantly more lead from the gastrointestinal tract (Kostial et al., 1971), but also that they excrete less lead from the body (Kostial et nl., 1973), that they distribute it in the body differently (Momcilovic and Kostial, 1974), that they bind it in the bone more tightly (Kello et ul., 1975). and that a smaller fraction of lead can be removed with chelating agents than in adults (Jug0 et al., 1975). Consequently, we assumed that generally, age might have an influence on the metabolism of heavy metals, and that this aspect should be further investigated. The purpose of our studies therefore was to obtain more data on the retention and distribution of cadmium in relation to age in order to obtain additional parameters for estimating body burden and critical organ exposure to this toxic element in the youngest age group. 92 Copyright 0 1977 by Academic Press. Inc. All rights of reproducfmn m any form reserved.
ISSN 0013.9351
AGE
AND
MATERIAL
Cd
93
RETENTION
AND METHODS
We studied the fate of the intraperitoneally applied ‘lSmCd in albino rats aged 1, 3, 6, and 52 weeks. All animals received standard rat food and water, with the exception of l-week-old rats who were fed mother’s milk throughout the experiment. A single intraperitoneal dose of radioactive solution contained between 10 and 20 &i lls’nCd Cl, (specific activity by 0.55 1.O mCi/mgCd; purchased from “The Radiochemical Centre,” Amersham). In the period between the 1st and 14th day after radioisotope application at fixed time intervals the whole body radioactivity was measured in a two-crystal scintillation detector for y-radiation. On the 14th day of the experiment all animals were exsanguinated in ether narcosis and their kidneys and liver were removed. Fresh weight of liver, kidneys, and blood were determined and the activity was measured in an automatic sample changer. The weight of the weighed blood was corrected to the theoretical total whole blood content in animals of matching weight and age according to Belcher and Harris ( 1957). RESULTS
The results of body and organ weights presented in Table 1 show a higher gain in body than in organ weights with increasing age. The results of the mean percent values of **SmCd retention presented in Fig. 1 show a decrease in whole-body retention values with increasing age. The process of initial equilibration and elimination seems to be age dependent being practically absent in l-week-old animals and lasting about 3 days in the oldest group of rats
123456789
FIG. I. Whole-body retention as mean t SD. Age and number (10). n .
of intraperitoneally of animals: I week
10 n
IZ t3 DAYS
1L
applied llSfHCdCI, in rats of different age expressed (22). 0: 3 weeks (10). 0: 6 weeks (9). 0; 52 weeks
KELLO
AND
KOSTIAL
6: 2:
‘:
0
m
c,
AGE
AND
Cd
KE’L ESTION
95
after which period the retention curves become almost linear. In this second elimination period young rats seem to excrete even more cadmium than adults. The analysis of the distribution of iiF”Cd in the rat’s body on the 14th day after intraperitoneal application shows that regardless of age, by far the most cadmium is accumulated in the liver (Table 2). In this period the liver, although it makes only about 4% of the total body weight (Table 1) accumulates between 46 and 56% of the parenterally administered dose. The liver of young rats accumulates less cadmium than that of older animals. The kidney makes the second organ as far as the degree of “5”‘Cd retention during this period is concerned. Cadmium retention on the 14th day after intraperitoneal application amounts to 4-6%~ of the administered dose (Table 2). In young rats the kidney, in contrast to the liver, retains always more cadmium than in adults. The same holds for the blood, where the percentage in very young is approximately three times higher than in adult animals. Although young rats retain in the whole body a considerably higher percentage of the intraperitoneally applied “5”LCd, the percentage in the liver. kidney, and blood represents a considerably lower fraction of the total whole body retention in younger animals than in adults (Fig. 2). We could therefore conclude that the increased total cadmium burden in the young is due primarily to increased accumulation of this toxic metal in other parts of the body, but not at the site of injection, as proved in preliminary experiments. DISCUSSION
After intraperitoneal application the finding that young rats retain considerably more ‘IZ”‘Cd in the body is in agreement with data on the metabolism of some other heavy metals, i.e., mercury (Jugo, 1976), lead (Kostial rt trl., 1973), cerium (Inaba and Lengemann, 1972). and plutonium (Sikov and Mahlum. 1973). Although some authors ascribe such increased metal retention in the young to the immaturity of excretory organs (Stather. 1970). we assume that this might be primarily due to a different binding of cadmium in immature and adult rats immediately after a parenteral application. Our results show two phases of cadmium elimination in adult rats: a fast initial and a slow late one (Fig. I), which is in agreement with the results reported by other authors (Moore rt (I/., 1973). We might assume that during the initial fast phase cadmium in blood is bound to ligands of low molecular weight in contrast to the later phase when cadmium is supposed to be bound to larger molecules as found by Griffin and Matson (1972) in adult rats. At the early stage, an enhanced cadmium elimination could therefore be expected, owing to the important role played by the size of molecules to which cadmium is bound in circulation (Vestal and Heller, 196X: Foulkes, 1974). In immature animals. however, we observed only one slow phase of cadmium elimination. This might indicate that cadmium in the immature animals at this initial period might be bound to different ligands. most probably at the sites of intense synthesis of proteins, which is characteristic of this age. This hypothesis is supported by the fact that young rats showed a considerably lower cadmium retention in the liver. kidneys, and blood, and a higher retention in the rest of the body. The reason for this may be accounted for by a more intense growth of other
96
KELLO
AND
KOSTIAL
100 %
90
80
70
60
50
LO
30
20
10
m
LIVER
m
BLOOD
m
KIDNEYS
I-1
OTHER
FIG. 9. Retention of “‘“Cd in rats on 14th day after intraperitoneal percent of whole-body retention of cadmium.
TISSUES
application:
results
expressed
as
parts of the young rats’ body compared to critical organs like the liver and kidney. Sikov and Mahlum (1972) point out similar factors when explaining the differences in the metabolism of plutonium in young and old rats. This explanation is even more likely to be applicable to cadmium since cadmium metabolism proved to be closely associated with the synthesis of some proteins (Squibb and Cousins, 1974; Sabbioni and Marafante. 1975). The lower cadmium accumulation in the liver of young rats could be explained by several other factors too. It could be partly due to the faster rate of cadmium elimination from this organ at this age. On the other hand, it could be also caused by a decreased cadmium binding capacity of the liver in the young. This might be a result of a reduced synthesis of specific proteins, metallothionein to which cadmium is largely bound in the liver (Squibb and Cousins, 1974; Cherian, 1974). Thus, Pohlandt (1974) found that the capacity for the conversion of methionin into cysteine, the amino acid which makes about 30% of the liver metallothionein, is not sufficiently developed in young organism (Weser rt al., 1973). The assumption that young rats might not have enough cysteine for the synthesis of metal-
AGE
AND
Cd
97
RETENTION
lothionein is supported by Bernhart’s results (1970). He found that in young rats the need for the sulphur-containing amino acids is extremely strong (possibly because of hair growth). In the kidneys, on the other hand, in the same observation interval we found much more ilSmCd than in adult rats. This might be because of its specific redistribution in the young. The ability of cadmium to mobilize from the liver and other tissues and to accumulate in the kidneys is well known (Friberg et al., 1975). Such mobilization from other tissue as well as from the whole body might be much higher in the young than in the adult because of increased remodeling rate and could gradually bring about an earlier accumulation in the kidneys. This higher turnover of cadmium concentration in the blood. Gunn and Gould (1927), who found a significantly lower cadmium accumulation in the kidney of the young, obtained their results in an extremely short period after ‘iSnCd application (24 hours), when probably not enough time for its redistribution and accumulation in the kidney was allowed. From our results it is possible to conclude that age bears a significant influence on cadmium metabolism. This should be kept in mind when setting up standards for the maximum permissible exposure to cadmium of certain population groups the more so as the effect of its increased retention in young bodies is ultimately reflected in increased accumulation in critical organs, the kidneys. ACKNOWLEDGMENT This Agency
work
was partially
supported
by a Research
Grant
from
the U.S.
Environmental
Protection
REFERENCES Belcher. 8. H.. and Harris. E. B. ( 1957). Studies on plasma volume. red cell volume and total blood volume in young growing rats. J. Phvsiol. 139, 64-78. Bemhart. F. W. (1970). Comparison of the essential amino acid, nitrogen and calorie requirements of the weanling rat and breastfed infant. J. Nutr. 100, 461-466. Cherian. M. G. (1974). Isolation and purification of cadmium binding proteins from rat liver. Biochen~. Biophus. Res. 61, 920-926. Foulkes. E. S. (1974). Excretion and retention of cadmium. zinc. and mercury by rabbit kidney, A~cT. J. Physl. 227, l356- 1360. Friberg. L.. Piscator. M.. Nordberg. F. G., and Kjellstrom. T. (1974). “Cadmium in the Environment.” 2nd ed.. CRC Press. Cleveland. Friberg. L., Kjellstrom. T., Nordberg. G., and Piscator, M. (1975). “Cadmium in the Environment.” 3rd ed.. Environmental Protection Technology Series, EPA-65012-75-049. Griffin, R. M.. and Matson, W. R. (1972). The assessment of individual variability to trace metal insult: Low-molecular weight metal complexing agents as indicators of trace metal insult. Amer. I&. Hp. 33, 373-377. Gunn. A. S., and Gould. T. C. (1957). Selective accumulation of Cd”’ by cortex of rat kidney. Pro
