TOXICOLOGY
Influence
AND
APPLIED
42,377-386
PHARMACOLOGY
of Cadmium on Zinc and Kidney: Role
Deparimeni
qf Public Health, Received
(1977)
Distribution in the of Metallothionein
NAOKI
SUCAWARA
Sapporo
Medical
February
College,
7. 1977; uccepred
May
S-l,
Mouse
W-l 7, Sapporo,
Liver
Japan
20, 1977
Influence of Cadmium on Zinc Distribution in the Mouse Liver and Kidney: Role of Metallothionein. SUGAWAKA. N. (1977). Touicol. Appl. Pharmacol. 42, 377~-386. Malt mice of the ICR strain were given 2.5 mg of Cd/kg by a SC injection of CdCl, and were sacrificed b) bleeding at 0.5.4, and 10 hr and 1.6. and 20 days. Cadmium was detected in the liver and kidney at 0.5 hr after the cadmium injection and the metal concentration increased progressively. In the kidney. zinc concentration at 20 days increased significantly in comparison with that of the control group. In the liver. the zinc concentration was not different from that in control mice up to 10 hr. but after 1 day. the concentration increased significantly. The proportion of cadmium in the liver and kidney supernatant increased with time up to 6 days after cadmium injection. The fact that cadmium content in the supernatant fraction increased with time to be more than YWU of the cadmium in the liver and kidney may suggest that the capacity of this fraction for cadmium accumulation increased wsith time. In the control group, about 78 and 68% ofthe total zinc content in the liver and kidney were present in the supernatant. After the injection. these values increased with time up to 6 days. At 20 daya after the injection. the decrease of the proportion of rinc was synchronous with the decrease of cadmium. The increased zinc in the liver supernatant may be due mainlv to the appearance of metallothionein which wab induced IIC ,WLW hy cadmium. At 1 hr after-the injection. about 63 and 55% of the cadmium in the aupernatsnt of the kidney and liver. respectively. were detected at the region corrcspondmg to the metallothionein fraction as separated on Sephadex G-75. However. at 4 hr. only about 4.6 and Y.3”0 of the soluble Lint was detected in this fraction of the kidney and hvcr. respectively. At later stage?. the uptake of cadmium into the metallothionein fraction incrcaaed io more than YO”~I. Ho\vever. this uptake of zinc was about 47 and 3 I ‘b at 6 davs m the liver and Lidne). H> further purification of the metallothioncin using a DEAE cellulose column. Tao distinct proteins (Peak II and Peak Ill). metallothioneins. were obtained. These protein\ contained Cd and Zn and were homogeneous as judged by the appearance of a smgle stainahlc hand on polyacrylamide gel disc electrophoresis. However. the ratio of CJ.!Zn was dependent on the length of time after the cadmium injection. At all times. the protein in Peak 11 occupied Imuch more cadmium and LIIIC‘ than that in Pcah 111.
It is well known that when animals are given cadmium by either the oral or parenteral route, cadmium is accumulated preferentially in the liver and the kidney, and that the metal is present as the bound form of a specific protein (thionein). The parallel relation between the content of cadmium and the content of metallothionein in the liver and kidney was found by Piotrowski et al. ( 1974). It is interesting that zinc has been shown to antagonize testicular weight loss induced by cadmium (Parizek. 19.57)and that the administration of zinc prior to a cadmium challenge produces a pronounced increaseof Cd LD50 in mice (Leber and Miya. 1976). However. the mechanism of interaction f ~‘p\‘Ipil: c /v-7I>\.\i.,~krn,i PVC,\ hli ill ‘\I,r,;i,,, 0.05. by a one-way ANOVA]. In the kidney, this concentration increased with time and was about 7% of the injected dose at 20 days after the injection. Stall et al. (1976) reported that when cadmium, 3.41 mg/kg, was injected SC to rats, the concentration in the liver at 12 hr increasedsignificantly relative to that at 1, 24. and 72 hr. Both organs in the control group (not treated with cadmium) contained too small an amount of cadmium to determine the concentration accurately. In the kidney. zinc concentration at 20 days increased significantly (p < 0.01 by Scheffe’s test) in comparison with that of the control group. In the liver, zinc concentration was not different from that in the control group up to 10 hr [F(3, 29) = 0.28, p > 0.051. but after 1 day this concentration increased significantly IF(3. 31) r28.4. p < 0.0 11and at 6 and 20 days was about two times that in the control group. Cadmium and zinc in the 30,OOOgsuperrzatanf. The proportion of cadmium and zinc in the 30,OOOgsupernatant of both organs is shown in Table 2. The proportion of cadmium in the liver and kidney supernatant increased with time up to 6 days after the cadmium injection. The proportion of zinc in the control group was about 78 and 68% in the liver and kidney. respectively. After the injection. these values increased with time. At 20 days after the injection, the decrease of the proportion of zinc was synchronous with the decreaseof cadmium. Typical separation profiles for the 3O.OOOgsupernatantsof both organs on Sephadex G-75 are shown in Fig. 1. The proportions of cadmium and zinc in the 30,OOOg supernatant proteins are shown in Table 3. As illustrated in Fig. 1. two or four peaks were obtained by the cadmium or zinc tracer, respectively. The proportions of zinc in Fraction I and Fraction II of the kidney supernatant were combined, becauseFraction I and Fraction II could not be separated distinctly by the column. The elution region of Fraction III was near that of cytochrome c. Following Sephadex separation, cadmium could not be detected clearly at the region corresponding to the metallothionein fraction (Fraction III) at 0.5 hr after the injection. At 4 hr after the injection. about 63 and 55% of the cadmium in the supernatant of the kidney and liver, respectively, were detected at the region corresponding to the metallothionein fraction and the remainder of the cadmium was present in the proteins near the void volume. At later stages. the distribution to metallothionein increasedwith time to about 99 and 98% in the kidney and liver at 20 days. While the proportion of zinc in Fraction III increased up to 20 days in the kidney and reached about 37% of zinc in the supernatant, in the liver this proportion reached a
C/G)
Lug)
Kidney Cd/g
Zn/g
i 2.2 (9)
15.0 & 2.0 (9)
21.7
Control
k 0.1
(3)
16.0 + 0.3 (3)
I.8
5.3 f 2.6 (6)" 22.1 + 1.9 (6)'
0.5 Hr -
_-.
13.0
t
1.3 (9)
3.0 + 0.6 (9)
injection -___
17.0
+
weighI.
The
numbers
13.1 2 2.1 (9)
* 3.7 (9) f 5.1 (9)"
1 Day
1.2 (9)
22.1 29.1
~-~
+ 7.5 (8) -c x.5 (8)
represent
11.4 & 3.2 (8) 17.0 _+ 3.0(S)
25.8 41.3
6 Days
in parentheses
~~
TREAWDWITHCAIIMIUM
5.0 f 0.8 (9)
+ 2.8 (9)h i 3.1 (9)
10 Hr
after __~-~
MICE
3.1 f 0.9 (9)
23.3 22.0
~~___
Time
ANDKIDNEYOF
15.9 -t 6.0 (9) 20.4 & 5.2 (9)
4 Hr
~~~~-
LIVEK
1
” Data arc presented as means + SII. The tissue weight of the organs is expressed as the \vet h F(3.3 1) 0.73.~ \ 0.05 (from the IO-hr to the 20.day group). 0.28. p .y 0.05 (from the control tn the IO-hr group). C F(3.29) d F(3.3 I ) = 2X.4. p < 0.0 I (the control group. and from the I -day to the ZO-da!: group). e p < 0.01 wpective to control. by Scheffe’s test.
(,uug)
(,ug)
Zn/g
--
Cd/g
Liver
Organ
.-.
CADMIUMANDZINCINTHE
TABLE
+ 5.3 (9) 1 4.9 (9)
the number
of mice
12.9 i 1.6 (8) 20.7 t 2.3 (8)'
22.9 33.4
20 Days
F -z ZG
z-
s-
5
INFLUENCE
OF
CADMIUM
TABLE CADMIUM
AND ZINC
IN THE SUPERNATANT Percentage
Organ Liver Kidney
of
381
ZINC
2
FRACTION
LIVER AND KIDNEY
OF
Cd or Zn in the soluble
Cd
Zn
Cd
Zn
Cd
Zn
Cd
Zn
Cd
Zn
78.2 67.7
78.5 70.0
86.5 86.0
82.2 72.5
89.8 86.6
79.6 70.8
96.3 93.7
88.7 77.8
96.9 94.8
91.9 78.3
95.4 91.8
88.0 74.1
+ pp. Y 100) represents
1 Day
fraction
0.5 Hr --~ Cd Zn
” Each value (aup./sup.
10Hr
OF MICE
Influence
AND
APPLIED
42,377-386
PHARMACOLOGY
of Cadmium on Zinc and Kidney: Role
Deparimeni
qf Public Health, Received
(1977)
Distribution in the of Metallothionein
NAOKI
SUCAWARA
Sapporo
Medical
February
College,
7. 1977; uccepred
May
S-l,
Mouse
W-l 7, Sapporo,
Liver
Japan
20, 1977
Influence of Cadmium on Zinc Distribution in the Mouse Liver and Kidney: Role of Metallothionein. SUGAWAKA. N. (1977). Touicol. Appl. Pharmacol. 42, 377~-386. Malt mice of the ICR strain were given 2.5 mg of Cd/kg by a SC injection of CdCl, and were sacrificed b) bleeding at 0.5.4, and 10 hr and 1.6. and 20 days. Cadmium was detected in the liver and kidney at 0.5 hr after the cadmium injection and the metal concentration increased progressively. In the kidney. zinc concentration at 20 days increased significantly in comparison with that of the control group. In the liver. the zinc concentration was not different from that in control mice up to 10 hr. but after 1 day. the concentration increased significantly. The proportion of cadmium in the liver and kidney supernatant increased with time up to 6 days after cadmium injection. The fact that cadmium content in the supernatant fraction increased with time to be more than YWU of the cadmium in the liver and kidney may suggest that the capacity of this fraction for cadmium accumulation increased wsith time. In the control group, about 78 and 68% ofthe total zinc content in the liver and kidney were present in the supernatant. After the injection. these values increased with time up to 6 days. At 20 daya after the injection. the decrease of the proportion of rinc was synchronous with the decrease of cadmium. The increased zinc in the liver supernatant may be due mainlv to the appearance of metallothionein which wab induced IIC ,WLW hy cadmium. At 1 hr after-the injection. about 63 and 55% of the cadmium in the aupernatsnt of the kidney and liver. respectively. were detected at the region corrcspondmg to the metallothionein fraction as separated on Sephadex G-75. However. at 4 hr. only about 4.6 and Y.3”0 of the soluble Lint was detected in this fraction of the kidney and hvcr. respectively. At later stage?. the uptake of cadmium into the metallothionein fraction incrcaaed io more than YO”~I. Ho\vever. this uptake of zinc was about 47 and 3 I ‘b at 6 davs m the liver and Lidne). H> further purification of the metallothioncin using a DEAE cellulose column. Tao distinct proteins (Peak II and Peak Ill). metallothioneins. were obtained. These protein\ contained Cd and Zn and were homogeneous as judged by the appearance of a smgle stainahlc hand on polyacrylamide gel disc electrophoresis. However. the ratio of CJ.!Zn was dependent on the length of time after the cadmium injection. At all times. the protein in Peak 11 occupied Imuch more cadmium and LIIIC‘ than that in Pcah 111.
It is well known that when animals are given cadmium by either the oral or parenteral route, cadmium is accumulated preferentially in the liver and the kidney, and that the metal is present as the bound form of a specific protein (thionein). The parallel relation between the content of cadmium and the content of metallothionein in the liver and kidney was found by Piotrowski et al. ( 1974). It is interesting that zinc has been shown to antagonize testicular weight loss induced by cadmium (Parizek. 19.57)and that the administration of zinc prior to a cadmium challenge produces a pronounced increaseof Cd LD50 in mice (Leber and Miya. 1976). However. the mechanism of interaction f ~‘p\‘Ipil: c /v-7I>\.\i.,~krn,i PVC,\ hli ill ‘\I,r,;i,,, 0.05. by a one-way ANOVA]. In the kidney, this concentration increased with time and was about 7% of the injected dose at 20 days after the injection. Stall et al. (1976) reported that when cadmium, 3.41 mg/kg, was injected SC to rats, the concentration in the liver at 12 hr increasedsignificantly relative to that at 1, 24. and 72 hr. Both organs in the control group (not treated with cadmium) contained too small an amount of cadmium to determine the concentration accurately. In the kidney. zinc concentration at 20 days increased significantly (p < 0.01 by Scheffe’s test) in comparison with that of the control group. In the liver, zinc concentration was not different from that in the control group up to 10 hr [F(3, 29) = 0.28, p > 0.051. but after 1 day this concentration increased significantly IF(3. 31) r28.4. p < 0.0 11and at 6 and 20 days was about two times that in the control group. Cadmium and zinc in the 30,OOOgsuperrzatanf. The proportion of cadmium and zinc in the 30,OOOgsupernatant of both organs is shown in Table 2. The proportion of cadmium in the liver and kidney supernatant increased with time up to 6 days after the cadmium injection. The proportion of zinc in the control group was about 78 and 68% in the liver and kidney. respectively. After the injection. these values increased with time. At 20 days after the injection, the decrease of the proportion of zinc was synchronous with the decreaseof cadmium. Typical separation profiles for the 3O.OOOgsupernatantsof both organs on Sephadex G-75 are shown in Fig. 1. The proportions of cadmium and zinc in the 30,OOOg supernatant proteins are shown in Table 3. As illustrated in Fig. 1. two or four peaks were obtained by the cadmium or zinc tracer, respectively. The proportions of zinc in Fraction I and Fraction II of the kidney supernatant were combined, becauseFraction I and Fraction II could not be separated distinctly by the column. The elution region of Fraction III was near that of cytochrome c. Following Sephadex separation, cadmium could not be detected clearly at the region corresponding to the metallothionein fraction (Fraction III) at 0.5 hr after the injection. At 4 hr after the injection. about 63 and 55% of the cadmium in the supernatant of the kidney and liver, respectively, were detected at the region corresponding to the metallothionein fraction and the remainder of the cadmium was present in the proteins near the void volume. At later stages. the distribution to metallothionein increasedwith time to about 99 and 98% in the kidney and liver at 20 days. While the proportion of zinc in Fraction III increased up to 20 days in the kidney and reached about 37% of zinc in the supernatant, in the liver this proportion reached a
C/G)
Lug)
Kidney Cd/g
Zn/g
i 2.2 (9)
15.0 & 2.0 (9)
21.7
Control
k 0.1
(3)
16.0 + 0.3 (3)
I.8
5.3 f 2.6 (6)" 22.1 + 1.9 (6)'
0.5 Hr -
_-.
13.0
t
1.3 (9)
3.0 + 0.6 (9)
injection -___
17.0
+
weighI.
The
numbers
13.1 2 2.1 (9)
* 3.7 (9) f 5.1 (9)"
1 Day
1.2 (9)
22.1 29.1
~-~
+ 7.5 (8) -c x.5 (8)
represent
11.4 & 3.2 (8) 17.0 _+ 3.0(S)
25.8 41.3
6 Days
in parentheses
~~
TREAWDWITHCAIIMIUM
5.0 f 0.8 (9)
+ 2.8 (9)h i 3.1 (9)
10 Hr
after __~-~
MICE
3.1 f 0.9 (9)
23.3 22.0
~~___
Time
ANDKIDNEYOF
15.9 -t 6.0 (9) 20.4 & 5.2 (9)
4 Hr
~~~~-
LIVEK
1
” Data arc presented as means + SII. The tissue weight of the organs is expressed as the \vet h F(3.3 1) 0.73.~ \ 0.05 (from the IO-hr to the 20.day group). 0.28. p .y 0.05 (from the control tn the IO-hr group). C F(3.29) d F(3.3 I ) = 2X.4. p < 0.0 I (the control group. and from the I -day to the ZO-da!: group). e p < 0.01 wpective to control. by Scheffe’s test.
(,uug)
(,ug)
Zn/g
--
Cd/g
Liver
Organ
.-.
CADMIUMANDZINCINTHE
TABLE
+ 5.3 (9) 1 4.9 (9)
the number
of mice
12.9 i 1.6 (8) 20.7 t 2.3 (8)'
22.9 33.4
20 Days
F -z ZG
z-
s-
5
INFLUENCE
OF
CADMIUM
TABLE CADMIUM
AND ZINC
IN THE SUPERNATANT Percentage
Organ Liver Kidney
of
381
ZINC
2
FRACTION
LIVER AND KIDNEY
OF
Cd or Zn in the soluble
Cd
Zn
Cd
Zn
Cd
Zn
Cd
Zn
Cd
Zn
78.2 67.7
78.5 70.0
86.5 86.0
82.2 72.5
89.8 86.6
79.6 70.8
96.3 93.7
88.7 77.8
96.9 94.8
91.9 78.3
95.4 91.8
88.0 74.1
+ pp. Y 100) represents
1 Day
fraction
0.5 Hr --~ Cd Zn
” Each value (aup./sup.
10Hr
OF MICE
