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Post-mortem and in vitro dimerization of metallothionein in cadmium-accumulated rat liver and kidney
7-l
Toxicology Letters, 16 (1983) 11-84 Elsevier
Biomedical
Press
POST-MORTEM AND IN VITRO DIMERIZATION OF METALLOTHIONEIN IN CADMIUM-ACCUMULATED KIDNEY
RAT LIVER AND
(Metallothionein dimer; high performance liquid chromatograph; absorption spectrophotometer; toxicity of cadmium) KAZUO
T. SUZUKI,
RIKO
OHNUKI
and KUMIKO
atomic
YAGUCHI
National Institute for Environmental Studies, Yatabe, Tsukuba, Ibaraki 305 (Japan) (Received
August
16th,
(Revision
received
September
(Accepted
October
6th,
1982) 17th, 1982)
1982)
SUMMARY Metallothionein
(MT) in the livers and kidneys
(Cd) was selectively the dimer
formation
cell damage
oxidized
to dimers
is facilitated
of rats that died after repeated
of MT with time after death.
in heavily
Cd-accumulated
injections
In vitro experiments
tissues by oxygen
of cadmium suggest
and temperature
that when
occurs.
INTRODUCTION
Increases in Cd/Zn ratio in MT [ 1,2], inter- and intramolecular oxidations of MT [2, 31, and in the concentrations of Cd-binding proteins related to, but with lower Mr values than, MT [2] together with an increase in distribution of Cd to the highMr protein fraction [2] are characteristic changes observed in the liver and kidneys with accumulation of Cd. The increase in Cd/Zn ratio in MT is assumed to be related to capacity of these organs for the biosynthesis of MT, and a higher Cd/Zn ratio is assumed to indicate that the capacity is closer to the limit [2]. Although increases of inter- and intramolecular oxidation products of MT are probably related to an increase in peroxidation products in the tissues with the accumulation of Cd, the exact oxidative mechanisms are unknown. The present paper examines the changes in the distribution profiles of Cd in the supernatant fractions of livers and kidneys of rats after death by repeated injections of Cd and Abbreviations:
ICP-AES,
inductively
coupled
plasma-atomic
metallothionein.
0378-4274/83/0000-OCKKl/$03.00
0 Elsevier
Biomedical
Press
emission
spectrometer;
MT,
78
relates these changes to the intermolecular oxidation of MT with the formation of dimers. Some in vitro experiments also are reported, which trace the changes after death. The results are discussed in relation to cell damage by Cd. MATERIALS
AND METHODS
Female Wistar rats, 6 to 7 weeks old, were purchased from Clea Japan (Tokyo) and were fed on a standard laboratory chow (MF diet, Oriental Yeast Co., Tokyo) and distilled water ad lib. A CdC12 solution in physiological saline was subcutaneously injected at a dose of 3.0 mg Cd/kg body weight, 4 times a week for 3 and 6 weeks into 10 and 30 rats, respectively. 3 out of the 30 rats died during the sixth week of treatment and 27 rats were killed by exsanguination under light ether anaesthesia 3 days after the last injection. The 10 rats were killed by electric shock (220 V) between the front legs 3 days after the last injection. Preparation of tissue homogenates and supernatants Portions of livers and kidneys were homogenized in 0.1 M Tris-HCl buffer solution (pH 7.4, 0.25 M glucose) using a polytron homogenizer either in an atmosphere of nitrogen gas or air. The homogenates were centrifuged at 170 000 x g for 1 h at 2°C. Determination
of metal concentrations
A 1.0 ml portion of each homogenate was digested with 1.0 ml of mixed acid (HNO3:HClOd = 5:l v/v) and concentrations of metals were determined by an inductively coupled plasma-atomic emission spectrometer (ICP-AES, Jarrell Ash Model 975 Plasma Atomcomp). Detection of distribution profiles of Cd in the supernatant fractions A 0.1 ml portion of each supernatant was applied to an SW column (TSK GEL SW3000,7.5 x 600 mm with a precolumn of 7.5 x 75 mm, Toyo Soda Co., Tokyo) which was attached to a high-performance liquid chromatograph (HLC803A, Toyo Soda Co.) and the column was eluted with 50 mM Tris-HCl buffer solution (pH 8.0 at 25°C dissolved gas was removed at 80°C under reduced pressure) containing 0.1% sodium azide at a flow rate of 1.O ml/min. Molecular absorbances at 254 and 280 nm, and atomic absorbance of Cd were determined with a dual wavelength UV detector (Altex 152) and a flame atomic absorption spectrophotometer (Hitachi AA170-50A) by connecting the outlet of the column to the UV detector and then to the nebulizer tube of the spectrophotometer (HPLC-AAS method [4]).
19
1
21.0
I 20.2
254 nm
Retention
Time
(
min
1
Fig. 1. Distribution profiles of Cd in the kidney and liver supernatants of rats that died after repeated injections of Cd. Female rats were injected S.C. with Cd at a dose of 3.0 mg Cd/kg body weight, 4 times a week for 6 weeks and died during sixth week injection. Livers and kidneys of 3 dead rats were homogenized separately in IO ~01s. of buffer solution. Profiles A-C and D-F show the distribution profiles of Cd in the supernatants from the kidneys and livers on an SW column. Profiles A and D, B and E, and C and F are from each of the 3 rats. Concentrations of Cd in the livers and kidneys are as follows &g/g); A (134), B (124), C (145), D (286), E (246) and F (227). Profiles in the left upper panel show absorptions monitored at 254 and 280 nm for the kidney profile A. Peaks at 11.3, 20.2 (II) and 21.0 min (I) correspond to the void volume of the column, MT-II and MT-I, respectively. Peaks between 11.3 and 20.2 min (18.2 and 19.3 min) indicate MT dimers.
RESULTS AND DISCUSSION
Distribution profiles of Cd in the sup~rnatant fractions of livers and kidneys of rats that died during repeated injections of Cd are shown in Fig. 1. The distribution profiles of kidneys A, B and C are similar to those of livers D, E and F of the corresponding rats, respectively. The time interval between death and removal of the liver and kidneys, although unknown for any one of these rats, was tentatively estimated to be in the order: C(F) > B(E) > A(D) from the visible post-mortem changes. The distribution profile of Cd in the livers of rats which remained alive during the same injection treatment is shown in Fig. 2.
254 nm
II 20.2
Retention Fig. 2. Distribution
profile
Female rats were injected
Time
(
identified
1
of Cd in the liver supernatant
of rats killed after repeated
S.C. with Cd at a dose of 3.0 mg Cd/kg
and killed 3 days after the last injection. way as indicated
min
Livers of 3 rats were combined
in the legend to Fig. 1. The concentration
as follows;
11.3 (void volume
body weight,
of the column),
injections
of Cd.
4 times a week for 6 weeks
and homogenized
in the same
of Cd was 313 pg/g wet liver. Cd peaks are
18.6 (MT dimers),
20.2 (II, MT-II),
21.0 (I, MT-
I).
The differences that are apparent between the profiles in Figs. 1 and 2 may be attributed to (a) increased distribution of Cd to an area of shorter retention than MT and (b) changes in relative peak height of MT-I and -II. Cd peaks with shorter retention times than MT peaks can be identified as mixtures of three isomers; a dimer of MT-I (I-I), a dimer of MT-I and -11 (I-II) and a dimer of MT-II (II-II) [3]. The three dimers are eluted from the column in the order of II-II, I-II and I-I. Differences in retention times of the dimer peaks in Fig. 1 can be explained by the differences in composition. Thus a dimer peak of the shorter retention time (18.2 min) is rich in the dimer II-II, and a dimer peak of the longer retention time (19.3
81
min) is rich in the dimer I-I. The three dimers can be separated as individua1 peaks under different experimental conditions [3]. Changes in the relative peak height of MT-I and -II in Figs. 1 and 2 seem to have some relation to the changes in retention times of the dimer peaks. Although the peak height of MT-I was higher than that of MT-II in the liver supernatants prepared from animals immediately after death (Fig, 2), the former peak height
20.2
_iver tissue
Retentjon
Time
(
min
1
Fig. 3. Changes in the elution profiles of MT on an SW column with changes of storage conditions for homogenate and tissue of heavily Cd-accumulated liver. A heavily Cd-accumulated liver was obtained by injecting Cd into a rat at a dose of 3.0 mg Cd/kg body weight, 4 times a week for 6 weeks. The liver (12.2 g, concentration of Cd, 320 gg/g) was cut into portions (about 1 g each). Separate portions were homogenized in 3 ~01s. of buffer solution either under nitrogen gas (A and C) or under air (8 and D) and then stored either at -20°C (A and B) or at 20°C (C and D) for 24 h. The homogenates were rehomogenized after adding 7 vols. of the buffer and then centrifuged. Three portions of the liver were stored either at - 20°C (E) or at 20°C (F and G), and either under nitrogen gas (F) or under air (E and G) for 24 h, and then homogenized in 10 ~01s. buffer solution under nitrogen gas. The distribution profiles of Cd were determined on an SW column. Cd peaks were identified as follows; 11.3 (void volume of the column), 18.0 and 18.6 (MT dimers), 20.2 (II, MT-II), 21.0 min (I, MT-I).
82
Retention
Time
(
min
1
Fig. 4. Change with time post-mortem in the elution profiles of Cd in heavily Cd-accumulated rat liver. Female rats were injected S.C. with Cd at a dose of 3.0 mg Cd/kg body weight, 4 times a week for 3 weeks and killed by electric shock. death.
The rats were stored at 22’C at 0 (A), 1 (B), 2 (C), 4 (D), and 7 h (E) after
The livers were excised
atmosphere
of nitrogen
at these times
gas. Concentrations
and homogenized
in 9 ~01s. of buffer
of Cd in the supernatants
were as follows
solution
&g/ml);
in an
A (26.7),
B (23.8), C (29.2). D (26.2) and E (25.4). The distribution profiles of Cd were determined on an SW column. Cd peaks were identified as follows; 11.3 (void volume of the column), 17.9 and 18.6 (MT dimers),
20.2 (II, MT-II),
21.0 min (I, MT-I).
83
decreased more with time after death (Fig. 1). On the other hand, the dimer peaks in Fig. 1 were eluted faster as the time post mortem increased. These results suggest that MT-I is more easily dimerized than MT-II and a peak rich in the dimer I-I is more easily formed than the dimers II-II and I-II. The column used in the present study was washed with mercaptoethanol to remove metals bound non-selectively to the column. As a result, Cd bound loosely to the high-M, proteins in the supernatant fraction was adsorbed on the column and thus was not observed in this fraction of the distribution profiles [4]. Effects of oxygen and temperature on the dimerization of MT were studied using the livers of rats that survived the injection procedures described in the legends to Figs. 1 and 2. The rats were killed by exsanguination under light ether anaesthesia. Portions of the liver were stored in an atmosphere of either nitrogen gas (F) or air (E and G) and either at -20°C (E) or 20°C (F and G) for 24 h. After this time, the distribution profiles of Cd in the three groups were compared (Fig. 3). When liver tissues were cooled in a chilled buffer immediately after excision and stored at - 20°C without air, the distribution profile of Cd in the supernatant fraction of the liver tissues was indistinguishable from that obtained from fresh livers. From the distribution profiles E, F and G in Fig. 3, it can be concluded that the dimer peak is rich and the relative peak height of MT-I to -11 is low in the presence of air at 20°C. New Cd peaks with longer retention times than MT peaks appeared in the profiles obtained from the liver tissues stored at 20°C (F and G in Fig. 3). These new Cd peaks which although uncharacterized, probably were either intramolecular oxidation products or degradation products of MT, were stable to heat treatment (8O”C, 10 min) and the stability constants were high (the Cd was not adsorbed to the column). Although temperature and oxygen seem to alter the distribution profile of Cd in the liver tissue toward that observed after the post mortem changes (Fig. l), the changes in distribution profile G (Fig. 3) are not sufficient to explain the changes observed in Fig. 1. From the distribution profiles A to D (Fig. 3), however, it may be concluded that the dimer formation occurs more readily in the homogenate than in the whole tissue. In the former the dimer formation appeared to be catalysed by the particulate components since after removal of these by centrifugation it was not stimulated appreciably in the supernatant fraction (this profile is not shown). Changes in the distribution profiles of Cd in the liver supernatants of heavily Cdaccumulated rats were examined in animals that were kept atroom temperature for different periods of time after being killed by electric shock. As shown in Fig. 4, the dimer peak increased with time post mortem and the relative peak heights of MT-I and -11 also changed as expected from the retention times of the dimer peaks. Although the dimers of MT are slowly formed as an artefact during storage and isolation procedure, an increase of the dimer peak in heavily Cd-accumulated tissues cannot be explained only by the artefact. Probably some histological damages caused by Cd facilitate the dimer formation and the increase may be one of the signs of the Cd toxicity.
84
ACKNOWLEDGEMENT
We thank Dr. K. Kubota for continuous encouragement. REFERENCES 1 M. Sato and Y. Nagai, Mode of existence of cadmium in rat liver and kidney after prolonged subcutaneous administration, Toxicol. Appl. Pharmacol., 54 (1980) 90-99. 2 K.T. Suzuki, M. Yamamura, Y.K. Yamada and F. Shimizu, Distribution of cadmium in heavily cadmium-accumulated rat liver cytosols: Metallothionein and related cadmium-binding proteins, Toxicol. Lett., 8 (1981) 105-114. 3 K.T. Suzuki and M. Yamamura, Isolation and characterization of metallothionein dimers, Biochem. Pharmacol., 29 (1980) 689-692. 4 K.T. Suzuki, Direct connection of high speed liquid chromatograph (equipped with gel permeation column) to atomic absorption spectrophotometer for metalloprotein analysis: Metallothionein, Anal. Biochem., 102 (1980) 31-34.
Toxicology Letters, 16 (1983) 11-84 Elsevier
Biomedical
Press
POST-MORTEM AND IN VITRO DIMERIZATION OF METALLOTHIONEIN IN CADMIUM-ACCUMULATED KIDNEY
RAT LIVER AND
(Metallothionein dimer; high performance liquid chromatograph; absorption spectrophotometer; toxicity of cadmium) KAZUO
T. SUZUKI,
RIKO
OHNUKI
and KUMIKO
atomic
YAGUCHI
National Institute for Environmental Studies, Yatabe, Tsukuba, Ibaraki 305 (Japan) (Received
August
16th,
(Revision
received
September
(Accepted
October
6th,
1982) 17th, 1982)
1982)
SUMMARY Metallothionein
(MT) in the livers and kidneys
(Cd) was selectively the dimer
formation
cell damage
oxidized
to dimers
is facilitated
of rats that died after repeated
of MT with time after death.
in heavily
Cd-accumulated
injections
In vitro experiments
tissues by oxygen
of cadmium suggest
and temperature
that when
occurs.
INTRODUCTION
Increases in Cd/Zn ratio in MT [ 1,2], inter- and intramolecular oxidations of MT [2, 31, and in the concentrations of Cd-binding proteins related to, but with lower Mr values than, MT [2] together with an increase in distribution of Cd to the highMr protein fraction [2] are characteristic changes observed in the liver and kidneys with accumulation of Cd. The increase in Cd/Zn ratio in MT is assumed to be related to capacity of these organs for the biosynthesis of MT, and a higher Cd/Zn ratio is assumed to indicate that the capacity is closer to the limit [2]. Although increases of inter- and intramolecular oxidation products of MT are probably related to an increase in peroxidation products in the tissues with the accumulation of Cd, the exact oxidative mechanisms are unknown. The present paper examines the changes in the distribution profiles of Cd in the supernatant fractions of livers and kidneys of rats after death by repeated injections of Cd and Abbreviations:
ICP-AES,
inductively
coupled
plasma-atomic
metallothionein.
0378-4274/83/0000-OCKKl/$03.00
0 Elsevier
Biomedical
Press
emission
spectrometer;
MT,
78
relates these changes to the intermolecular oxidation of MT with the formation of dimers. Some in vitro experiments also are reported, which trace the changes after death. The results are discussed in relation to cell damage by Cd. MATERIALS
AND METHODS
Female Wistar rats, 6 to 7 weeks old, were purchased from Clea Japan (Tokyo) and were fed on a standard laboratory chow (MF diet, Oriental Yeast Co., Tokyo) and distilled water ad lib. A CdC12 solution in physiological saline was subcutaneously injected at a dose of 3.0 mg Cd/kg body weight, 4 times a week for 3 and 6 weeks into 10 and 30 rats, respectively. 3 out of the 30 rats died during the sixth week of treatment and 27 rats were killed by exsanguination under light ether anaesthesia 3 days after the last injection. The 10 rats were killed by electric shock (220 V) between the front legs 3 days after the last injection. Preparation of tissue homogenates and supernatants Portions of livers and kidneys were homogenized in 0.1 M Tris-HCl buffer solution (pH 7.4, 0.25 M glucose) using a polytron homogenizer either in an atmosphere of nitrogen gas or air. The homogenates were centrifuged at 170 000 x g for 1 h at 2°C. Determination
of metal concentrations
A 1.0 ml portion of each homogenate was digested with 1.0 ml of mixed acid (HNO3:HClOd = 5:l v/v) and concentrations of metals were determined by an inductively coupled plasma-atomic emission spectrometer (ICP-AES, Jarrell Ash Model 975 Plasma Atomcomp). Detection of distribution profiles of Cd in the supernatant fractions A 0.1 ml portion of each supernatant was applied to an SW column (TSK GEL SW3000,7.5 x 600 mm with a precolumn of 7.5 x 75 mm, Toyo Soda Co., Tokyo) which was attached to a high-performance liquid chromatograph (HLC803A, Toyo Soda Co.) and the column was eluted with 50 mM Tris-HCl buffer solution (pH 8.0 at 25°C dissolved gas was removed at 80°C under reduced pressure) containing 0.1% sodium azide at a flow rate of 1.O ml/min. Molecular absorbances at 254 and 280 nm, and atomic absorbance of Cd were determined with a dual wavelength UV detector (Altex 152) and a flame atomic absorption spectrophotometer (Hitachi AA170-50A) by connecting the outlet of the column to the UV detector and then to the nebulizer tube of the spectrophotometer (HPLC-AAS method [4]).
19
1
21.0
I 20.2
254 nm
Retention
Time
(
min
1
Fig. 1. Distribution profiles of Cd in the kidney and liver supernatants of rats that died after repeated injections of Cd. Female rats were injected S.C. with Cd at a dose of 3.0 mg Cd/kg body weight, 4 times a week for 6 weeks and died during sixth week injection. Livers and kidneys of 3 dead rats were homogenized separately in IO ~01s. of buffer solution. Profiles A-C and D-F show the distribution profiles of Cd in the supernatants from the kidneys and livers on an SW column. Profiles A and D, B and E, and C and F are from each of the 3 rats. Concentrations of Cd in the livers and kidneys are as follows &g/g); A (134), B (124), C (145), D (286), E (246) and F (227). Profiles in the left upper panel show absorptions monitored at 254 and 280 nm for the kidney profile A. Peaks at 11.3, 20.2 (II) and 21.0 min (I) correspond to the void volume of the column, MT-II and MT-I, respectively. Peaks between 11.3 and 20.2 min (18.2 and 19.3 min) indicate MT dimers.
RESULTS AND DISCUSSION
Distribution profiles of Cd in the sup~rnatant fractions of livers and kidneys of rats that died during repeated injections of Cd are shown in Fig. 1. The distribution profiles of kidneys A, B and C are similar to those of livers D, E and F of the corresponding rats, respectively. The time interval between death and removal of the liver and kidneys, although unknown for any one of these rats, was tentatively estimated to be in the order: C(F) > B(E) > A(D) from the visible post-mortem changes. The distribution profile of Cd in the livers of rats which remained alive during the same injection treatment is shown in Fig. 2.
254 nm
II 20.2
Retention Fig. 2. Distribution
profile
Female rats were injected
Time
(
identified
1
of Cd in the liver supernatant
of rats killed after repeated
S.C. with Cd at a dose of 3.0 mg Cd/kg
and killed 3 days after the last injection. way as indicated
min
Livers of 3 rats were combined
in the legend to Fig. 1. The concentration
as follows;
11.3 (void volume
body weight,
of the column),
injections
of Cd.
4 times a week for 6 weeks
and homogenized
in the same
of Cd was 313 pg/g wet liver. Cd peaks are
18.6 (MT dimers),
20.2 (II, MT-II),
21.0 (I, MT-
I).
The differences that are apparent between the profiles in Figs. 1 and 2 may be attributed to (a) increased distribution of Cd to an area of shorter retention than MT and (b) changes in relative peak height of MT-I and -II. Cd peaks with shorter retention times than MT peaks can be identified as mixtures of three isomers; a dimer of MT-I (I-I), a dimer of MT-I and -11 (I-II) and a dimer of MT-II (II-II) [3]. The three dimers are eluted from the column in the order of II-II, I-II and I-I. Differences in retention times of the dimer peaks in Fig. 1 can be explained by the differences in composition. Thus a dimer peak of the shorter retention time (18.2 min) is rich in the dimer II-II, and a dimer peak of the longer retention time (19.3
81
min) is rich in the dimer I-I. The three dimers can be separated as individua1 peaks under different experimental conditions [3]. Changes in the relative peak height of MT-I and -II in Figs. 1 and 2 seem to have some relation to the changes in retention times of the dimer peaks. Although the peak height of MT-I was higher than that of MT-II in the liver supernatants prepared from animals immediately after death (Fig, 2), the former peak height
20.2
_iver tissue
Retentjon
Time
(
min
1
Fig. 3. Changes in the elution profiles of MT on an SW column with changes of storage conditions for homogenate and tissue of heavily Cd-accumulated liver. A heavily Cd-accumulated liver was obtained by injecting Cd into a rat at a dose of 3.0 mg Cd/kg body weight, 4 times a week for 6 weeks. The liver (12.2 g, concentration of Cd, 320 gg/g) was cut into portions (about 1 g each). Separate portions were homogenized in 3 ~01s. of buffer solution either under nitrogen gas (A and C) or under air (8 and D) and then stored either at -20°C (A and B) or at 20°C (C and D) for 24 h. The homogenates were rehomogenized after adding 7 vols. of the buffer and then centrifuged. Three portions of the liver were stored either at - 20°C (E) or at 20°C (F and G), and either under nitrogen gas (F) or under air (E and G) for 24 h, and then homogenized in 10 ~01s. buffer solution under nitrogen gas. The distribution profiles of Cd were determined on an SW column. Cd peaks were identified as follows; 11.3 (void volume of the column), 18.0 and 18.6 (MT dimers), 20.2 (II, MT-II), 21.0 min (I, MT-I).
82
Retention
Time
(
min
1
Fig. 4. Change with time post-mortem in the elution profiles of Cd in heavily Cd-accumulated rat liver. Female rats were injected S.C. with Cd at a dose of 3.0 mg Cd/kg body weight, 4 times a week for 3 weeks and killed by electric shock. death.
The rats were stored at 22’C at 0 (A), 1 (B), 2 (C), 4 (D), and 7 h (E) after
The livers were excised
atmosphere
of nitrogen
at these times
gas. Concentrations
and homogenized
in 9 ~01s. of buffer
of Cd in the supernatants
were as follows
solution
&g/ml);
in an
A (26.7),
B (23.8), C (29.2). D (26.2) and E (25.4). The distribution profiles of Cd were determined on an SW column. Cd peaks were identified as follows; 11.3 (void volume of the column), 17.9 and 18.6 (MT dimers),
20.2 (II, MT-II),
21.0 min (I, MT-I).
83
decreased more with time after death (Fig. 1). On the other hand, the dimer peaks in Fig. 1 were eluted faster as the time post mortem increased. These results suggest that MT-I is more easily dimerized than MT-II and a peak rich in the dimer I-I is more easily formed than the dimers II-II and I-II. The column used in the present study was washed with mercaptoethanol to remove metals bound non-selectively to the column. As a result, Cd bound loosely to the high-M, proteins in the supernatant fraction was adsorbed on the column and thus was not observed in this fraction of the distribution profiles [4]. Effects of oxygen and temperature on the dimerization of MT were studied using the livers of rats that survived the injection procedures described in the legends to Figs. 1 and 2. The rats were killed by exsanguination under light ether anaesthesia. Portions of the liver were stored in an atmosphere of either nitrogen gas (F) or air (E and G) and either at -20°C (E) or 20°C (F and G) for 24 h. After this time, the distribution profiles of Cd in the three groups were compared (Fig. 3). When liver tissues were cooled in a chilled buffer immediately after excision and stored at - 20°C without air, the distribution profile of Cd in the supernatant fraction of the liver tissues was indistinguishable from that obtained from fresh livers. From the distribution profiles E, F and G in Fig. 3, it can be concluded that the dimer peak is rich and the relative peak height of MT-I to -11 is low in the presence of air at 20°C. New Cd peaks with longer retention times than MT peaks appeared in the profiles obtained from the liver tissues stored at 20°C (F and G in Fig. 3). These new Cd peaks which although uncharacterized, probably were either intramolecular oxidation products or degradation products of MT, were stable to heat treatment (8O”C, 10 min) and the stability constants were high (the Cd was not adsorbed to the column). Although temperature and oxygen seem to alter the distribution profile of Cd in the liver tissue toward that observed after the post mortem changes (Fig. l), the changes in distribution profile G (Fig. 3) are not sufficient to explain the changes observed in Fig. 1. From the distribution profiles A to D (Fig. 3), however, it may be concluded that the dimer formation occurs more readily in the homogenate than in the whole tissue. In the former the dimer formation appeared to be catalysed by the particulate components since after removal of these by centrifugation it was not stimulated appreciably in the supernatant fraction (this profile is not shown). Changes in the distribution profiles of Cd in the liver supernatants of heavily Cdaccumulated rats were examined in animals that were kept atroom temperature for different periods of time after being killed by electric shock. As shown in Fig. 4, the dimer peak increased with time post mortem and the relative peak heights of MT-I and -11 also changed as expected from the retention times of the dimer peaks. Although the dimers of MT are slowly formed as an artefact during storage and isolation procedure, an increase of the dimer peak in heavily Cd-accumulated tissues cannot be explained only by the artefact. Probably some histological damages caused by Cd facilitate the dimer formation and the increase may be one of the signs of the Cd toxicity.
84
ACKNOWLEDGEMENT
We thank Dr. K. Kubota for continuous encouragement. REFERENCES 1 M. Sato and Y. Nagai, Mode of existence of cadmium in rat liver and kidney after prolonged subcutaneous administration, Toxicol. Appl. Pharmacol., 54 (1980) 90-99. 2 K.T. Suzuki, M. Yamamura, Y.K. Yamada and F. Shimizu, Distribution of cadmium in heavily cadmium-accumulated rat liver cytosols: Metallothionein and related cadmium-binding proteins, Toxicol. Lett., 8 (1981) 105-114. 3 K.T. Suzuki and M. Yamamura, Isolation and characterization of metallothionein dimers, Biochem. Pharmacol., 29 (1980) 689-692. 4 K.T. Suzuki, Direct connection of high speed liquid chromatograph (equipped with gel permeation column) to atomic absorption spectrophotometer for metalloprotein analysis: Metallothionein, Anal. Biochem., 102 (1980) 31-34.