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Effect of cadmium on hepatic metallothionein level in early development of the rat
ENVIRONMENTAL
Effect
RESEARCH
24, 201-206 (1981)
of Cadmium on Hepatic Early Development
Metallothionein of the Rat
P. ASOKAN AND S.K. Industrial
Toxicology
Research
Centre, Lucknow-
Level
in
TANDON
Post Box No.
80. Mahatrnu
Gandhi
Mug.
1 India
Received May 27, 1980 The induction of hepatic metallothionein (MT) was investigated 24 hr after an intraperitoneal injection of 0.75 mg/kg Cd as CdCI,.H,O in rats of 7, 14, 21. and 90 days. Metallothionein in liver cytosolic fractions collected on Sephadex G-75 was characterized in terms of sulthydryl, total protein, Cd, and Zn contents. Most of the cytosolic Cd was associated with MT and the concentration of Cd was equal in the different age groups. The higher contents of sulthydryl, protein, and Zn both in control as well as in Cd-injected rats of 7 and 14 days than in those of 21 and 90 days indicate the presence of more native Znthionein in immature pups. However, the increase in sulthydryl and protein contents showed more prominent induction of MT in Cd-exposed animals of 21 and 90 days than in those of 7 and 14 days. The concentration of Cd was highest in liver followed by the other tissues. While hepatic accumulation of Cd was similar in all age groups, the renal accumulation increased significantly with age. The intestine and spleen of immature pups concentrated more Cd than those of mature animals. The accumulation of the metal did not differ significantly in heart and brain of the animals among the four groups.
INTRODUCTION
Cadmium induces metallothionein (MT) or similar proteins in liver and kidneys of different species (Piscator, 1964; Shaikh and Lucis, 1970; Squibb and Cousins, 1974; Nordberg et al., 1974; Kotsonis and Klaassen, 1978). A low-molecularweight Cd-binding protein in blood, both in erythrocytes and in plasma of Cdexposed mice (Nordberg, 1978), and Cd-, Cu-, and Zn-binding proteins in blood and urine of Cd-poisoned rats (Suzuki, 1978) have also been detected. This protein is probably MT which acts as a transport protein for Cd. The MT plays an important role in storage, transport, and detoxification of toxic metals (Nordberg ef al., 1971a; Webb, 1972; Suzuki and Yoshikawa, 1974; Webb and Magos, 1976; Probst et al., 1977; Yau and Mennear, 1977; Nordberg, 1978) and in the metabolism of Zn (Chen et al., 1974; Richards and Cousins, 1975; Sugawara, 1978). The exposure to heavy metals during the early postnatal period causes irreversible behavioral and biochemical changes (Chisolm, 1974; Rosen and Sorell. 1978). The administration of Cd in rats following birth decreases the growth rate and increases spontaneous locomotor activity (Rastogi er al., 1977). Cadmium chloride damages the central nervous system of the rat and rabbit only on exposure during early life (Gabbiani et al., 1967). About one-third of the lifetime burden of Cd accumulates in liver and kidneys by 3 years in humans (Henke er al., 1970). It was, therefore, of interest to investigate the induction of MT by Cd and its effect on the uptake and distribution of this metal during early life. 201 0013-9351/81/010201-06$02.00/O Copyright All rights
0 1981 by Academic Press. Inc of reproduction in any form reserved
202
ASOKAN
MATERIALS
Animals
AND
TANDON
AND
METHODS
and Treatment
Dams together with their pups on the day of birth were obtained from the Industrial Toxicology Research Centre’s breeding colony; a uniform litter size of eight was established. They were fed on commercial pellet diet (Hind Liver Ltd., India) and water ad libitum. The animals at 7, 14, 21, and 90 days of age were given an intraperitoneal injection of 0.75 mg/kg Cd as CdCl,.H,O to induce MT. The controls received an equal volume of normal saline. Isolation and Characterization of Metallothionein The animals were sacrificed 24 hr after Cd administration and the liver pooled in each group and homogenized in 6 vol of chilled 0.25 M sucrose in 0.01 M Tris-HCl buffer (pH 8.3) using a Potter-Elvehjem Teflon homogenizer. The homogenate was centrifuged at 105,OOOgfor 1 hr to obtain an organelle-free cytosol fraction. Nine milliliters of cytosol was applied on a column of Sephadex G-75 (2.5 x 85 cm; Pharmacia Fine Chemicals, Uppsala, Sweden) which was precalibrated with blue dextran and cytochrome c. Proteins were eluted with Tris-HCl buffer at a flow rate of 25 ml&. Fractions of 10 ml were collected and absorptions recorded at 254 and 280 nm. The MT was characterized in terms of sullhydryl group, protein, Cd, and Zn contents. The sulfhydryl content of MT fractions was estimated with Ellman’s reagent (Boyne and Ellman, 1972). The protein in these fractions was precipitated with equal volumes of Tsuchiya’s reagent and assayed by the method of Piscator and Pettersson (1977) using protein from bovine serum albumin as standard. Zinc was estimated with a Perkin-Elmer 303 atomic absorption spectrometer (213.9 nm) and Cd was estimated calorimetrically (Saltzman, 1953). RESULTS
AND
DISCUSSION
Cadmium-induced rat liver MT was partially purified by gel filtration and characterized in terms of sulfhydryl group, protein, Cd, and Zn contents (Table 1). The MT fractions absorbed strongly at a wavelength of 250 nm, which is dependent on the Cd-mercaptide bond. However, none of the fraction absorbed at 280 nm probably due to the lack of aromatic amino acids in MT-like proteins (Nordberg, 1978). The higher sulthydryl, protein, and Zn contents both in control and in Cd-exposed rats of 7 and 14 days compared to those of 21 and 90 days indicates the presence of higher levels of Zn-thionein in neonates, as more than 70% of the sulfhydryl groups originate from MT-like proteins (Nagahashi et al., 1974). This confirms the occurrence of a high level of native MT in immature rats (Bell, 1979; Wong and Klaassen, 1979). The uptake of Cd, however, was similar at all four different ages. No significant change in sulfhydryl, protein, and Zn contents between Cd-exposed pups of 7 or 14 days and controls suggests no induction of hepatic MT by Cd and that Cd might have accumulated by binding to some of the free sites of native MT or similar proteins. The increases in sullhydryl and protein contents in Cd-treated rats of 21 and 90 days over the controls, however, were significantly higher than those of 7 and 14 days, indicating synthesis of more hepatic thionein in mature animals. The administration of Cd increases the amount
4.23 4.29 0.46 0.18
k 5 5 k
0.63” 0.75 0.06 0.01
Control
4.43 5.64 1.62 0.98
t + 2 k
0.26 0.39 0.21* 0.06*
4.18 3.98 2.37 2.11
_c 0.21 + 0.96 + 0.34 2 0.12
Control
FROM
TABLE
1
5.23 5.38 3.90 4.85
_c 0.45 k 0.33 t 0.44** + 0.19*
Experimental
Proteinb
ND? ND ND ND
Control 14.16 14.23 13.18 13.99
_t 1.09 2 0.87 ” 1.24 5 0.64
Experimental
Cadmium’
AND ZINC CONTENTS OF HEPATIC METALLOTHIONEIN CADMIUM-TREATED DEVELOPING RATS
PROTEIN, CADMIUM,
Experimental
Sulfhydryl”
TOTAL
” pmoles per fraction. h mg per fraction. c I*g per fraction. ” Values are mean + SE of five samples. ” ND, not detectable. *P < 0.001. **P < 0.01 vs control; Student’s r test.
7 14 21 90
Age (days)
SULFHYDRYL,
99.11 112.82 21.96 18.31
Zinc’
2 25.33 + 12.33 _t 4.62 + 6.35
Control
FRACTIONS
103.90 121.68 32.93 26.86
k t 2 i
13.70 2.91 4.76 4.14
Experimental
204
ASOKAN
AND TANDON
of Cd-binding protein in rat and rabbit liver which plays an important role in protection against Cd toxicity (Piscator, 1964; Shaikh and Lucis, 1970). Since the concentration of hepatic Zn-thionein decreases with age (Ohtake et al., 1978; Bell, 1979), the Cd-exposed rats postweaning produce more hepatic thionein to prevent Cd toxicity, which explains the increase in sulfhydryl and protein contents in the animals of 21 and 90 days. The concentration of Cd 24 hr after injection was in the order: liver > intestine > kidney = spleen > heart > brain in the rats of7 and 14 days and liver > kidney > intestine > spleen > heart > brain in those of 21 and 90 days, which is similar to an earlier observation (Kotsonis and Klaassen, 1977) (Table 2). Soon after absorption the metal appears in liver from where it is distributed to other tissues. The time of its redistribution and accumulation in other tissues after injection was obviously very short in the present study (Kello and Kostial, 1977). Though the total hepatic Cd ought to increase with the age owing to growth of the tissue, the metal concentration in the liver was similar at all four different ages (Gunn and Gould, 1957). This suggests a critical concentration or a quick equilibrium for the hepatic Cd which appears independent of age. The renal accumulation of Cd, however, was very low in immature rats probably due to the smaller number of nephrons, but it increased significantly with age (Gunn and Gould, 1957). The kidney is the critical tissue for Cd and the developed organ concentrates and retains far more Cd owing to the induction of MT. The uptake of Cd by intestine and spleen was higher in the pups of 7 and 14 days. The inability of immature animals to eliminate Cd from the intestine or a less prominent exfoliation of intestinal epithelial cells in these animals might be responsible for the higher concentration of Cd in their intestines. Further, the spleen of infants plays a role in erythropoiesis and Cd tends to concentrate quickly in erythrocytes bound either to hemoglobin or to a MT-like protein (Carlson and TABLE UPTAKE
OF CADMIUM
IN DIFFERENT
2
TISSUES OF CADMIUM-TREATED
DEVELOPING
RATS
Age (days) at injection Tissue Liver Kidney Intestine Spleen Heart Brain
7
14
21
90
10.31 + 0.91” (7) 0.66 rt 0.04 (7 2.03 t 0.05 (9) O.% 2 0.08
10.47 2 0.53 (8) 0.82 2 0.04** (7) 2.35 r 0.15 (9) 0.75 f 0.03**
9.35 + 0.18 (7) 1.18 k 0.07* (8) 1.10 + 0.10* (7 0.62 2 0.06 (7) 0.32 2 0.04 (5) 0.06 2 0.009 (4)
10.54 2 0.71 (6) 2.61 f 0.20* (6) 1.24 k 0.04 (4) 0.62 + 0.04 (5) 0.56 f 0.07** (4) 0.05 -c 0.007 (4)
(8)
0.30 2 0.04 (7) 0.05 f 0.002
03)
(8)
0.21 k 0.07 (6) 0.06 f 0.007
(8)
n Values are mean (pg Cd/g fresh tissue) + SE of the number of samples given in parentheses. *P c 0.001, **P < 0.05 when compared to the value in the previous column as evaluated by Student’s I test.
EFFECT
OF
CADMIUM
ON
HEPATIC
METALLOTHIONEIN
205
Friberg, 1957; Nordberg et al., 1971b) which may be related to the comparatively higher uptake of this metal by spleen of neonates. The concentration of Cd in the heart and the brain was lowest of all the vital organs examined and was more or less similar at all stages of the animal’s development probably due to the poor affinity of these tissues toward Cd or the short period allowed for distribution of the metal after the injection. However, only the adult heart was able to concentrate a significant amount of the metal within 24 hr of its administration. It may be concluded that regardless of age, liver takes up more Cd than any other tissue which is incorporated into MT and the mechanism of this incorporation appears to be different in immature and mature animals. The level of native Zn-thionein shows the importance of the regulatory mechanism of Zn distribution in early development of the animals. Cadmium has a pronounced effect on the catabolism of MT related to Zn (Feldman et al., 1978a, b) and therefore further studies are needed to investigate the effect of Cd on the metabolism of Zn-thionein and the distribution of Zn during early development. ACKNOWLEDGMENTS The authors are grateful to the Director, Industrial Toxicology Research Centre, Lucknow, for his interest and advice. Thanks are due Mr. M. Ashquin and Mr. Ashok Kumar for technical assistance.
REFERENCES Bell, J. U. (1979). Native metallothionein levels in rat hepatic cytosol during perinatal development. Toxicol. Appl. Pharmacol. 50, 101- 107. Boyne, A. F., and Ellman, G. L. (1972). A methodology for analysis of tissue sulthydryl components. Anal.
Biochem.
46, 639-653.
Carlson. L. A., and Friberg, L. (1957). The distribution of cadmium in blood after repeated exposure. Stand. J. C/in. Lab. Invest. 9, 1-4. Chen, R. W., Eakin, D. J.. and Whanger, P. D. (1974). Biological function of metallothionein. II. Its role in zinc metabolism in the rat. Nutr. Rep. Int. 10, 195-200. Chisolm. J. J., Jr. (1974). Heavy metals exposures: Toxicity from metal-metal interactions and behavioral effects. Pediatrics 53, 841. Feldman, S. F., Failla, M. L., and Cousins, R. J. (1978a). Degradation of rat liver metallothionein in rlitro.
Biochim.
Biophys.
Acta
544, 638-646.
Feldman, S. F., Squibb, K. S., and Cousins, R. J. (1978b). Degradation of cadmium-thionein in rat liver and kidney. J. Toxicol. Environ. Health 4, 805-813. Gabbiani. G., Bait, D.. and Deziel, C. (1967). Toxicity ofcadmium in the central nervous system. E~rp. Neural. 18, 154- 160. Gunn, S. A., and Gould, T. C. (1957). Selective accumulation of ““Cd by cortex of rat kidney, Proc. Sot.
Exp.
Biol.
Med.
96, 820-823.
Henke, G., Sachs, H. W., and Bohn, G. (1970). Cadmium determination in the liver and kidneys of children and juveniles by means of neutron activation analysis. Arch. Toxico!. 26, 8- 16. Kello, D.. and Kostial, K. (1977). Influence of age on whole body retention and distribution of ‘lsmCd in rat. Enrriron. Res. 14, 92-98. Kotsonis. F. N.. and Klaassen, C. D. (1977). Toxicity and distribution of cadmium administered to rats at sublethal doses. Toxicol. Appl. Pharmacol. 41, 667-680. Kotsonis. F. N., and Klaassen, C. D. (1978). The relationship of metallothionein to the toxicity of cadmium after prolonged oral adminstration to rats. Toxicol. Appl. Pharmucol. 46, !9-54. Nagahashi. M., Wada, O., Ono, T., Yamaguchi, N., and Takeo, K. (1974). Dose response relationships between given doses of heavy metals and sulfhydryl group contents in the special protein fractions extracted from tissues of mice. Ind. Health 12, 31-36. Nordberg, G. F., Piscator, M., and Lind, B. (1971a). Distribution of cadmium among protein fractions of mouse liver. Acfa Pharmacol. Toxicol. 29, 456-470.
206
ASOKAN
AND TANDON
Nordberg, Cl. F., Piscator, M., and Nordberg, M. (1971b). On the distribution Acta
Pharmacol.
Toxicol.
of cadmium in blood.
30, 289-295.
Nordberg, M. (1978). Studies on metallothionein and cadmium. Environ. Res. 15, 381-404. Nordberg, M., Trojanowska, B., and Nordberg, G. F. (1974). Studies on metal-binding proteins of low molecular weight from renal tissue of rabbits exposed to cadmium or mercury. Environ. Physiol. Biochem. 4, 149- 158. Ohtake, H., Hasegawa, K., and Koga, M. (1978). Zinc binding protein in the livers of neonatal normal and partially hepatectomized rats. Biochem. J. 174, 995- 1005. Piscator, M. (1964). On cadmium in normal human kidney together with a report on the isolation of metallothionein from livers of cadmium exposed rabbits. Nord. Hyg. Tidskr. 45, 76-82. Piscator, M., and Pettersson, B. (1977). Chronic cadmium poisoning diagnosis and prevention. In “Clinical Chemistry and Chemical Toxicology of Metals” (S. S. Brown, Ed.), pp. 143-155. Elsevier/North-Holland, Amsterdam. Probst, G. S., Bousquet, W. F., and Miya, T. S. (1977). Correlation of hepatic metallothionein concentrations with acute cadmium toxicity in the mouse. Toxicol. Appl. Pharmacol. 39, 61-67. Rastogi, R. B., Merali, Z., and Singhal, R. L. (1977). Cadmium alters behaviour and the biosynthetic capacity for catecholamines and serotonin in neonatal rat brain. J. Neurochem. 28, 789-794. Richards, M. P., and Cousins, R. J. (1975). Mammalian zinc homeostasis: Requirement for RNA and metallothionein synthesis. Biochem. Biophys. Res. Commun. 64, 1215-1223. Rosen, J. F., and SorelI, M. (1978). The metabolism and subclinical effects of lead in children. In “The Biogeochemistry of Lead in the Environment” (Nriagu, Ed.), pp. 151-172. Elsevier/NorthHolland, Amsterdam. Saltzman, B. E. (1953). Calorimetric microdetermination with dithizone with improved separation of interfering metals. Anal. Chem. 25, 493-497. Shaikh, Z., and Lucis, 0. J. (1970). Induction of cadmium binding protein. Fed. Proc. 29, 298 (Abstract No. 301) Squibb, K. S., and Cousins, R. J. (1974). Control of cadmium binding protein synthesis in rat liver. Environ. Sugawara,
Physiol.
Biochem.
4, 24-30.
N. (1978). Zinc distribution and metallothionein in the liver and kidneys of mice treated with cadmium. J. Toxicol. Sci. 3, 153- 162. Suzuki, Y. (1978). A further purification of the low molecular weight cadmium, copper and zinc binding proteins in the blood and urine of cadmium poisoned rats. Ind. Health. 16, 91-94. Suzuki, Y., and Yoshikawa, H. (1974). Role of metallothionein in the liver in protection against cadmium toxicity. Znd. Health 12, 141- 151. Webb, M. (1972). Protection by zinc against cadmium toxicity. Biochem. Pharmacol. 21, 2767-2771. Webb, M., and Magos, L. (1976). Cadmium thionein and the protection against the nephrotoxicity of mercury. Chem. Biol. Interact. 14, 357-369. Wong, K. L., and Klaassen, C. D. (1979). Isolation and characterisation of liver metallothionein in newborn rats. Toxicol. Appl. Pharmacol. 48, A115. Yau, E. T., and Mennear, J. H. (1977). Pancreatic metallothionein: Protection against cadmium induced inhibition of insulin secretory activity. Toxicol. Appl. Pharmacol. 39, 515-520.
Effect
RESEARCH
24, 201-206 (1981)
of Cadmium on Hepatic Early Development
Metallothionein of the Rat
P. ASOKAN AND S.K. Industrial
Toxicology
Research
Centre, Lucknow-
Level
in
TANDON
Post Box No.
80. Mahatrnu
Gandhi
Mug.
1 India
Received May 27, 1980 The induction of hepatic metallothionein (MT) was investigated 24 hr after an intraperitoneal injection of 0.75 mg/kg Cd as CdCI,.H,O in rats of 7, 14, 21. and 90 days. Metallothionein in liver cytosolic fractions collected on Sephadex G-75 was characterized in terms of sulthydryl, total protein, Cd, and Zn contents. Most of the cytosolic Cd was associated with MT and the concentration of Cd was equal in the different age groups. The higher contents of sulthydryl, protein, and Zn both in control as well as in Cd-injected rats of 7 and 14 days than in those of 21 and 90 days indicate the presence of more native Znthionein in immature pups. However, the increase in sulthydryl and protein contents showed more prominent induction of MT in Cd-exposed animals of 21 and 90 days than in those of 7 and 14 days. The concentration of Cd was highest in liver followed by the other tissues. While hepatic accumulation of Cd was similar in all age groups, the renal accumulation increased significantly with age. The intestine and spleen of immature pups concentrated more Cd than those of mature animals. The accumulation of the metal did not differ significantly in heart and brain of the animals among the four groups.
INTRODUCTION
Cadmium induces metallothionein (MT) or similar proteins in liver and kidneys of different species (Piscator, 1964; Shaikh and Lucis, 1970; Squibb and Cousins, 1974; Nordberg et al., 1974; Kotsonis and Klaassen, 1978). A low-molecularweight Cd-binding protein in blood, both in erythrocytes and in plasma of Cdexposed mice (Nordberg, 1978), and Cd-, Cu-, and Zn-binding proteins in blood and urine of Cd-poisoned rats (Suzuki, 1978) have also been detected. This protein is probably MT which acts as a transport protein for Cd. The MT plays an important role in storage, transport, and detoxification of toxic metals (Nordberg ef al., 1971a; Webb, 1972; Suzuki and Yoshikawa, 1974; Webb and Magos, 1976; Probst et al., 1977; Yau and Mennear, 1977; Nordberg, 1978) and in the metabolism of Zn (Chen et al., 1974; Richards and Cousins, 1975; Sugawara, 1978). The exposure to heavy metals during the early postnatal period causes irreversible behavioral and biochemical changes (Chisolm, 1974; Rosen and Sorell. 1978). The administration of Cd in rats following birth decreases the growth rate and increases spontaneous locomotor activity (Rastogi er al., 1977). Cadmium chloride damages the central nervous system of the rat and rabbit only on exposure during early life (Gabbiani et al., 1967). About one-third of the lifetime burden of Cd accumulates in liver and kidneys by 3 years in humans (Henke er al., 1970). It was, therefore, of interest to investigate the induction of MT by Cd and its effect on the uptake and distribution of this metal during early life. 201 0013-9351/81/010201-06$02.00/O Copyright All rights
0 1981 by Academic Press. Inc of reproduction in any form reserved
202
ASOKAN
MATERIALS
Animals
AND
TANDON
AND
METHODS
and Treatment
Dams together with their pups on the day of birth were obtained from the Industrial Toxicology Research Centre’s breeding colony; a uniform litter size of eight was established. They were fed on commercial pellet diet (Hind Liver Ltd., India) and water ad libitum. The animals at 7, 14, 21, and 90 days of age were given an intraperitoneal injection of 0.75 mg/kg Cd as CdCl,.H,O to induce MT. The controls received an equal volume of normal saline. Isolation and Characterization of Metallothionein The animals were sacrificed 24 hr after Cd administration and the liver pooled in each group and homogenized in 6 vol of chilled 0.25 M sucrose in 0.01 M Tris-HCl buffer (pH 8.3) using a Potter-Elvehjem Teflon homogenizer. The homogenate was centrifuged at 105,OOOgfor 1 hr to obtain an organelle-free cytosol fraction. Nine milliliters of cytosol was applied on a column of Sephadex G-75 (2.5 x 85 cm; Pharmacia Fine Chemicals, Uppsala, Sweden) which was precalibrated with blue dextran and cytochrome c. Proteins were eluted with Tris-HCl buffer at a flow rate of 25 ml&. Fractions of 10 ml were collected and absorptions recorded at 254 and 280 nm. The MT was characterized in terms of sullhydryl group, protein, Cd, and Zn contents. The sulfhydryl content of MT fractions was estimated with Ellman’s reagent (Boyne and Ellman, 1972). The protein in these fractions was precipitated with equal volumes of Tsuchiya’s reagent and assayed by the method of Piscator and Pettersson (1977) using protein from bovine serum albumin as standard. Zinc was estimated with a Perkin-Elmer 303 atomic absorption spectrometer (213.9 nm) and Cd was estimated calorimetrically (Saltzman, 1953). RESULTS
AND
DISCUSSION
Cadmium-induced rat liver MT was partially purified by gel filtration and characterized in terms of sulfhydryl group, protein, Cd, and Zn contents (Table 1). The MT fractions absorbed strongly at a wavelength of 250 nm, which is dependent on the Cd-mercaptide bond. However, none of the fraction absorbed at 280 nm probably due to the lack of aromatic amino acids in MT-like proteins (Nordberg, 1978). The higher sulthydryl, protein, and Zn contents both in control and in Cd-exposed rats of 7 and 14 days compared to those of 21 and 90 days indicates the presence of higher levels of Zn-thionein in neonates, as more than 70% of the sulfhydryl groups originate from MT-like proteins (Nagahashi et al., 1974). This confirms the occurrence of a high level of native MT in immature rats (Bell, 1979; Wong and Klaassen, 1979). The uptake of Cd, however, was similar at all four different ages. No significant change in sulfhydryl, protein, and Zn contents between Cd-exposed pups of 7 or 14 days and controls suggests no induction of hepatic MT by Cd and that Cd might have accumulated by binding to some of the free sites of native MT or similar proteins. The increases in sullhydryl and protein contents in Cd-treated rats of 21 and 90 days over the controls, however, were significantly higher than those of 7 and 14 days, indicating synthesis of more hepatic thionein in mature animals. The administration of Cd increases the amount
4.23 4.29 0.46 0.18
k 5 5 k
0.63” 0.75 0.06 0.01
Control
4.43 5.64 1.62 0.98
t + 2 k
0.26 0.39 0.21* 0.06*
4.18 3.98 2.37 2.11
_c 0.21 + 0.96 + 0.34 2 0.12
Control
FROM
TABLE
1
5.23 5.38 3.90 4.85
_c 0.45 k 0.33 t 0.44** + 0.19*
Experimental
Proteinb
ND? ND ND ND
Control 14.16 14.23 13.18 13.99
_t 1.09 2 0.87 ” 1.24 5 0.64
Experimental
Cadmium’
AND ZINC CONTENTS OF HEPATIC METALLOTHIONEIN CADMIUM-TREATED DEVELOPING RATS
PROTEIN, CADMIUM,
Experimental
Sulfhydryl”
TOTAL
” pmoles per fraction. h mg per fraction. c I*g per fraction. ” Values are mean + SE of five samples. ” ND, not detectable. *P < 0.001. **P < 0.01 vs control; Student’s r test.
7 14 21 90
Age (days)
SULFHYDRYL,
99.11 112.82 21.96 18.31
Zinc’
2 25.33 + 12.33 _t 4.62 + 6.35
Control
FRACTIONS
103.90 121.68 32.93 26.86
k t 2 i
13.70 2.91 4.76 4.14
Experimental
204
ASOKAN
AND TANDON
of Cd-binding protein in rat and rabbit liver which plays an important role in protection against Cd toxicity (Piscator, 1964; Shaikh and Lucis, 1970). Since the concentration of hepatic Zn-thionein decreases with age (Ohtake et al., 1978; Bell, 1979), the Cd-exposed rats postweaning produce more hepatic thionein to prevent Cd toxicity, which explains the increase in sulfhydryl and protein contents in the animals of 21 and 90 days. The concentration of Cd 24 hr after injection was in the order: liver > intestine > kidney = spleen > heart > brain in the rats of7 and 14 days and liver > kidney > intestine > spleen > heart > brain in those of 21 and 90 days, which is similar to an earlier observation (Kotsonis and Klaassen, 1977) (Table 2). Soon after absorption the metal appears in liver from where it is distributed to other tissues. The time of its redistribution and accumulation in other tissues after injection was obviously very short in the present study (Kello and Kostial, 1977). Though the total hepatic Cd ought to increase with the age owing to growth of the tissue, the metal concentration in the liver was similar at all four different ages (Gunn and Gould, 1957). This suggests a critical concentration or a quick equilibrium for the hepatic Cd which appears independent of age. The renal accumulation of Cd, however, was very low in immature rats probably due to the smaller number of nephrons, but it increased significantly with age (Gunn and Gould, 1957). The kidney is the critical tissue for Cd and the developed organ concentrates and retains far more Cd owing to the induction of MT. The uptake of Cd by intestine and spleen was higher in the pups of 7 and 14 days. The inability of immature animals to eliminate Cd from the intestine or a less prominent exfoliation of intestinal epithelial cells in these animals might be responsible for the higher concentration of Cd in their intestines. Further, the spleen of infants plays a role in erythropoiesis and Cd tends to concentrate quickly in erythrocytes bound either to hemoglobin or to a MT-like protein (Carlson and TABLE UPTAKE
OF CADMIUM
IN DIFFERENT
2
TISSUES OF CADMIUM-TREATED
DEVELOPING
RATS
Age (days) at injection Tissue Liver Kidney Intestine Spleen Heart Brain
7
14
21
90
10.31 + 0.91” (7) 0.66 rt 0.04 (7 2.03 t 0.05 (9) O.% 2 0.08
10.47 2 0.53 (8) 0.82 2 0.04** (7) 2.35 r 0.15 (9) 0.75 f 0.03**
9.35 + 0.18 (7) 1.18 k 0.07* (8) 1.10 + 0.10* (7 0.62 2 0.06 (7) 0.32 2 0.04 (5) 0.06 2 0.009 (4)
10.54 2 0.71 (6) 2.61 f 0.20* (6) 1.24 k 0.04 (4) 0.62 + 0.04 (5) 0.56 f 0.07** (4) 0.05 -c 0.007 (4)
(8)
0.30 2 0.04 (7) 0.05 f 0.002
03)
(8)
0.21 k 0.07 (6) 0.06 f 0.007
(8)
n Values are mean (pg Cd/g fresh tissue) + SE of the number of samples given in parentheses. *P c 0.001, **P < 0.05 when compared to the value in the previous column as evaluated by Student’s I test.
EFFECT
OF
CADMIUM
ON
HEPATIC
METALLOTHIONEIN
205
Friberg, 1957; Nordberg et al., 1971b) which may be related to the comparatively higher uptake of this metal by spleen of neonates. The concentration of Cd in the heart and the brain was lowest of all the vital organs examined and was more or less similar at all stages of the animal’s development probably due to the poor affinity of these tissues toward Cd or the short period allowed for distribution of the metal after the injection. However, only the adult heart was able to concentrate a significant amount of the metal within 24 hr of its administration. It may be concluded that regardless of age, liver takes up more Cd than any other tissue which is incorporated into MT and the mechanism of this incorporation appears to be different in immature and mature animals. The level of native Zn-thionein shows the importance of the regulatory mechanism of Zn distribution in early development of the animals. Cadmium has a pronounced effect on the catabolism of MT related to Zn (Feldman et al., 1978a, b) and therefore further studies are needed to investigate the effect of Cd on the metabolism of Zn-thionein and the distribution of Zn during early development. ACKNOWLEDGMENTS The authors are grateful to the Director, Industrial Toxicology Research Centre, Lucknow, for his interest and advice. Thanks are due Mr. M. Ashquin and Mr. Ashok Kumar for technical assistance.
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