- Email: [email protected]
Effect of cadmium, mercury, and bismuth on the copper content in rat tissues
ENVIRONMENTAL
RESEARCH
19, 121-126
(1979)
Effect of Cadmium, Mercury, and Bismuth Copper Content in Rat Tissues’ JADWIGA
A. SZYMA~SKA
Department of Toxicological Rioanalysis, Medical
on the
AND ANDRZEJ J. ~ELAZOWSKI
Chemistry, Institute Academy, Narutowicza
of Environmental Research 120a 90-145 Lbdi Poland
and
Received July 18, 1978 In this study the effect of multiple subcutaneous administration of cadmium, mercury, and bismuth on the copper content in rat organs was investigated. In the case of all three metals a statistically significant increase in the whole-body copper retention was observed. In a majority of organs, only slight changes in the copper concentrations took place; on the other hand, there was a considerable elevation of the content of this metal in the kidneys, twofold after bismuth treatment and more than sixfold after treatment with cadmium or mercury. The increase in the copper concentration in the kidneys was paralleled by an augmentation in the level of kidney metallothionein-like proteins and by an increase in the copper content of the protein fraction of molecular weight 10,000, isolated from kidneys of rats exposed to cadmium, bismuth, or mercury. The metallothionein-like proteins play a key role in the binding of these three metals in the rat kidneys.
INTRODUCTION
Schroeder and Nanson ( 1974) and Julshamn et al. (1977) observed that exposure of rats to cadmium results in an increase in the level of copper in the kidneys. It results from the data of Suzuki et al. (1977) that this effect is due to kidney accumulation of cadmium in a form of a complex with the copper-containing metallothionein. Previously, we made similar observations in the case of bismuth (Piotrowski and Szymanska, 1976; Szymanska, 1977) and of mercury (Zelazowski and Piotrowski, 1978). The aim of the present paper was: (1) collection of comparable data for three metals: cadmium, bismuth and mercury; (2) determination of whether the copper cumulation in the kidneys because of exposure to these metals results in significant changes in the copper levels in other organs; and (3) elucidation of whether this phenomenon is conditioned by the same mechanism in the case of all three metals. MATERIAL AND METHODS
Groups of five to six male rats of the Wistar strain, weighing about 200 g, were repeatedly exposed to subcutaneous injections of “VdC12, BiQ, and *03HgC12, every other day, for a total of 2 weeks; doses of 2.0, 3.0, and 1.0 mg Me/kg, respectively, were applied. A control group consisted of animals receiving no metal. The copper content, mineralized with sulfuric and perchloric acid by a spectrophotometric method employing zinc dibenzyldithiocarbamate (Szymanska and Zychowicz, 1977), was estimated in tissues, blood, liver, kidneys, lungs, heart, spleen, brain, intestine, and skin and in bones and muscles jointly. ’ Supported by Grant 536/VI from the Division of Medical Sciences, Polish Academy of Sciences. 121 0013-9351/79/030121-06$02.00/0 Copyright All rights
@ 1979 by Academic~Pkess, Inc. of reproduction in any form resewed
122
SZYMANSKA
AND
ZELAZOWSKI
Proteins binding cadmium, bismuth, and mercury were isolated from kidneys of the control and exposed animals by fractionation of postmitochondrial supematants with acetone. The fractions rich in low molecular weight proteins (molecular weight of about 10,000) precipitated at 50-80% saturation with acetone (Zelazowski et al, 1978). The precipitates were dissolved and separated on Sephadex G-75 columns. In the column eluated protein was quantitated by absorbance measurements at 250 and 280 nm in a VSU-2 P spectrophotometer, and copper was estimated in combined fractions. Bismuth was determined in mineralized tissues or column eluates by a spectrophotometric method (Szymanska and Zychowicz, 1977). Mercury was estimated directly in the samples by a radiochemical method, measuring y-activity in a scintillation counter with NaJ/Tl crystal. Cadmium was also quantified by a radiochemical method, using a plastic SAD-12 crystal. The content of metallothionein-like proteins was estimated in liver and kidneys by a radiochemical method according to Zelazowski and Piotrowski (1977). RESULTS
The copper content in micrograms/g of the individual tissues of control rats and of those exposed to bismuth, mercury, and cadmium is given in Table 1. The most significant changes in the copper concentration took place in the kidneys and were correlated with an increase in the level of metallothionein-like proteins (Fig. 1). A twofold increase in the copper content of the kidneys was noted after exposure to bismuth chloride (3.0 mg Bi/kg) while more than sixfold increases were observed following exposure to mercuric chloride (1 .O mg Hg/kg) and cadmium chloride (2.0 mg Cd/kg). Only slight elevations in the copper content occurred in some other
TABLE
1
DISTRIBUTION OFCOPPER FOLLOWING OF SEVEN DOSES OF BISMUTH,~ADMIUM,OR
Cu concentration, Organ Kidneys Liver Blood Lungs Heart Spleen Intestine Skin Brain Muscles + bones
Total (a9
Control 10.0 4.27 1.56 3.46 7.89 3.43 2.13 2.03 3.58
+ 2 f 2 * 2 t + +
2.80 1.35 0.29 2.44 2.21 1.54 1.13 0.82 1.10
Bismuth (7 x 3 mgikg) 20.7* 4.99 1.90* 2.95 6.19 1.5l3* 1.93 2.83 3.47
-t 4.70 4 0.88 % 0.21 +- 0.76 4 1.06 z!z0.37 t 0.47 f 0.44 4 1.45
ADMINISTRATION MERCURY~
&g
tissues (w/W)
Mercury (7 x 1 m&g) 60.8* 4.34 1.80 2.96 6.08 1.77* 2.08 2.61 3.24
f ? k 2 + + 2 f 2
7.30 0.65 0.37 1.25 0.67 0.16 0.57 0.42 0.38
Cadmium (7 x 2 mgikg) 65.7* 5.56 2.28* 2.46 6.56 2.02 1.75 3.06 2.94
f 0.90 2 0.69 f 0.43 -r- 0.50 f 0.90 2 0.28 2 1.28 2 0.78 2 0.28
1.07 * 0.39
1.34 4 0.27
1.20 -c 0.24
1.09 2 0.47
284 k 67
369* 2 29
378* + 48
421* f 62
a Means from five to six rats. * Statistically significant.
EFFECT
OF
Cd,
Hg, AND
Bi ON
FIG. 1. Dependence of the levels of metallothionein-like kidneys of control and exposed rats on the concentration
Cu CONTENT
proteins (O--O) and copper (O--O) of the administered metal.
123
in
organs, e.g., in the liver and skin. In the remaining organs the copper content decreased although the diminution was significant only for the spleen. The whole-body retention of copper showed increases in exposed animals, statistically significant for bismuth and mercury at P = 0.05 and for cadmium at P = 0.01. Results of molecular sieving of the kidney protein fraction precipitated at 50-80% saturation with acetone on Sephadex G-75 are shown in Fig. 2. For the control animals, the copper concentration in the protein fraction of molecular weight of about 10,000 was equal to 0.035 fig/cm3 eluate/g tissue. In the animals exposed to bismuth a twofold elevation of this concentration was observed, up to about 0.07 pglcm3/g, while in the animals receiving cadmium and mercury the copper level in the eluates increased to 0.10 and 0.25 pg/cm3/g, respectively. The highest rise in the level of kidney metallothionein-like proteins was found in the rats exposed to mercury (Table 2). DISCUSSION
The effect of cadmium on the organ distribution of copper has been already the subject of previous reports. Schroeder and Nanson (1974) demonstrated a more than twofold increase in the copper content of the kidneys following exposure of rats to cadmium, 50 ppm per OS, for 2-4 years. Julshamn et al. (1977) found only a tendency for elevation of the copper content in the kidneys and liver and a decrease in the copper concentration in the spleen. The present report confirms the latter findings. On the other hand, to our knowledge there are no data on the effect of mercury and bismuth on the copper metabolism. Our results demonstrate that the most
EFFECT
OF Cd, Hg, AND
Bi ON
TABLE THE
LEVEL
OF METALLOTHIONEIN-LIKE LIVER
AND
Kind of exposure Control Bi (7 x 3.0 mg/kg) Hg (7 x 1.0 mg/kg) Cd (7 x 2.0 mg/kg)
PROTEINS KIDNEY
125
Cu CONTENT
2 (EXPRESSED
OF CONTROL
AND
Kidney
AS METALLOTHIONEIN,
EXPOSED
IN
mg/g) IN
RATS”
Liver
0.15 2 0.02 0.50 ” 0.01*
0.10 Ifr 0.03 0.21 & 0.10**
1.242 0.13*
0.18 k 0.06*
0.81 t 0.03*
2.57t 0.25*
* Significant; P < 0.01. ** Not significant; P < 0.05.
pronounced changes in the copper content induced by the three metals tested took place in the kidneys. An induced increase in the copper level, similar to that caused by cadmium, was also found after exposure to all three metals (by about 50% for cadmium and by about 30% for mercury and bismuth). A statistically significant diminution in the copper content was noted only in the spleen following exposure to bismuth and mercury. From among the published reports, only that of Suzuki et al. (1977) stated an increase in the copper content of the metallothionein-like protein fraction in the kidney of rats exposed to cadmium chloride. These authors found a correlation between duration of the exposure to the cadmium level in the fraction of low molecular weight proteins and the copper content of this fraction. Here we demonstrated an elevation in the copper content of the metallothionein-like protein fraction after exposure of rats to bismuth, mercury, and cadmium. The fraction characterized by an increased copper level also contained the highest concentrations of mercury, cadmium, and bismuth. Maxima of absorbances at 250 and 280 nm also occurred in this fraction. The ratio of A25,JA28,, amounted to 5 for the cadmium-induced metallothionein and was equal to 1.5- 1.6 for metallothioneinlike proteins isolated from kidneys of the control rats and of the animals exposed to bismuth and mercury. It results from the present data that the metallothionein-like proteins induced by cadmium, mercury, and bismuth in the kidneys, responsible for the binding of those metals in the kidneys, belong to copper proteins. Consequently, induction of these proteins as well as binding of the three exogenous metals in the kidneys requires a supply of equivalent amounts of copper. We suspected that the copper accumulation in the kidneys may take place at the expense of copper contained in other organs. This hypothesis was not confirmed since the increase in the copper content of the kidneys was accompanied by an elevation of the whole-body retention of copper, and no organ but the spleen showed a decrease in the concentration of this metal. One could assume that administration of cadmium, mercury, or bismuth changes the conditions of copper homeostasis so that an increase of the copper uptake in the intestine takes place. As proposed by Evans and LeBlanc (1976), low molecular weight proteins can also constitute an agent regulating the intestinal copper absorption. One should mention that some authors reported a decrease in the copper uptake following an oral cadmium administration (Evans e? al., 1970). Our results refer, on the other hand, to a subcutaneous metal administration. Apparently, a direct
126
SZYM.~NSK.~
AND
~ELAZ~WSKI
administration of the metal inductors per OS can induce biosynthesis of low molecular weight copper-binding proteins in the intestinal walls, and the produced proteins may constitute a factor limiting the copper uptake by its binding in the intestinal mucosa. It seems therefore that the final effect of the metals studied on copper retention depends on the method of their administration. ACKNOWLEDGMENTS The authors are indebted to Professor Jerzy K. Piotrowski cussion of the results.
for his interest in this study and dis-
REFERENCES Evans, G. W., Majors, P. F., and Comatzer, W. E. (1970). Mechanisms for cadmium and zinc antagonism of copper metabolism. Biochem. Biophys. Res. Commun. 40, 1142-l 146. Evans, G. W., and LeBlanc, F. N. (1976). Copper-binding protein in rat intestine: Amino acid composition and function. Nutr. Rep. Int. 14, 281-288. Julshamn, K., Utne, F., and Braekkan, 0. R. (1977). Interactions of cadmium, zinc and iron in different organs and tissues of the rat. Acta Pharmacol. Toxicol. 41, 515-524. Piotrowski, J. K., and Szymanska, J. A. (1976). Influence of certain metals on the level of metallothionein-like proteins. J. Toxicol. Environ. Health 1, 991- 1002. Schroeder, H. A., and Nanson, A. P. (1974). Interactions of trace metals in rat tissues: Cadmium and nickel with zinc, chromium, copper, manganese. J. Nutr. 104, 167- 178. Suzuki, T. K., Kubota, K., and Takenaka, S. (1977). Copper in cadmium-exposed rat kidney metallothionein. Chem. Pharm. Bull. 25, 2794-2796. Szymanska, J. A. (1977). Ph.D. dissertation. Medical Academy. L6dt. Szymattska, J. A., and Zychowicz, M. (1977). Simultaneous spectrophotometric determination of bismuth and copper in biological material by using zinc dibenzyldithiocarbamate. Chem. Anal. 22, 985-988 (in Polish). Zelazowski, A. J., and Piotrowski, J. K. (1977). A modified procedure for determination of metallothionein-like proteins in animal tissues. Acta Biochim. Pal. 24, 97-103. Zelazowski, A. J., and Piotrowski, J. K. (1978). Further investigations on the inducible mercurybinding proteins in rat kidney. In “XVI Congr. Polish Biochem. Sot., Lbdt. Abstracts” (in Polish). Zelazowski, A. J., Piotrowski, J. K., Mogilnicka, E. M., Szymanska, J. A., and Kaszper, B. W. (1978). The preparation of metallothionein from equine renal cortex as a standard protein for analytical purposes. Bromatol. Chem. Toksykol. 11, 51-57 (in Polish).
RESEARCH
19, 121-126
(1979)
Effect of Cadmium, Mercury, and Bismuth Copper Content in Rat Tissues’ JADWIGA
A. SZYMA~SKA
Department of Toxicological Rioanalysis, Medical
on the
AND ANDRZEJ J. ~ELAZOWSKI
Chemistry, Institute Academy, Narutowicza
of Environmental Research 120a 90-145 Lbdi Poland
and
Received July 18, 1978 In this study the effect of multiple subcutaneous administration of cadmium, mercury, and bismuth on the copper content in rat organs was investigated. In the case of all three metals a statistically significant increase in the whole-body copper retention was observed. In a majority of organs, only slight changes in the copper concentrations took place; on the other hand, there was a considerable elevation of the content of this metal in the kidneys, twofold after bismuth treatment and more than sixfold after treatment with cadmium or mercury. The increase in the copper concentration in the kidneys was paralleled by an augmentation in the level of kidney metallothionein-like proteins and by an increase in the copper content of the protein fraction of molecular weight 10,000, isolated from kidneys of rats exposed to cadmium, bismuth, or mercury. The metallothionein-like proteins play a key role in the binding of these three metals in the rat kidneys.
INTRODUCTION
Schroeder and Nanson ( 1974) and Julshamn et al. (1977) observed that exposure of rats to cadmium results in an increase in the level of copper in the kidneys. It results from the data of Suzuki et al. (1977) that this effect is due to kidney accumulation of cadmium in a form of a complex with the copper-containing metallothionein. Previously, we made similar observations in the case of bismuth (Piotrowski and Szymanska, 1976; Szymanska, 1977) and of mercury (Zelazowski and Piotrowski, 1978). The aim of the present paper was: (1) collection of comparable data for three metals: cadmium, bismuth and mercury; (2) determination of whether the copper cumulation in the kidneys because of exposure to these metals results in significant changes in the copper levels in other organs; and (3) elucidation of whether this phenomenon is conditioned by the same mechanism in the case of all three metals. MATERIAL AND METHODS
Groups of five to six male rats of the Wistar strain, weighing about 200 g, were repeatedly exposed to subcutaneous injections of “VdC12, BiQ, and *03HgC12, every other day, for a total of 2 weeks; doses of 2.0, 3.0, and 1.0 mg Me/kg, respectively, were applied. A control group consisted of animals receiving no metal. The copper content, mineralized with sulfuric and perchloric acid by a spectrophotometric method employing zinc dibenzyldithiocarbamate (Szymanska and Zychowicz, 1977), was estimated in tissues, blood, liver, kidneys, lungs, heart, spleen, brain, intestine, and skin and in bones and muscles jointly. ’ Supported by Grant 536/VI from the Division of Medical Sciences, Polish Academy of Sciences. 121 0013-9351/79/030121-06$02.00/0 Copyright All rights
@ 1979 by Academic~Pkess, Inc. of reproduction in any form resewed
122
SZYMANSKA
AND
ZELAZOWSKI
Proteins binding cadmium, bismuth, and mercury were isolated from kidneys of the control and exposed animals by fractionation of postmitochondrial supematants with acetone. The fractions rich in low molecular weight proteins (molecular weight of about 10,000) precipitated at 50-80% saturation with acetone (Zelazowski et al, 1978). The precipitates were dissolved and separated on Sephadex G-75 columns. In the column eluated protein was quantitated by absorbance measurements at 250 and 280 nm in a VSU-2 P spectrophotometer, and copper was estimated in combined fractions. Bismuth was determined in mineralized tissues or column eluates by a spectrophotometric method (Szymanska and Zychowicz, 1977). Mercury was estimated directly in the samples by a radiochemical method, measuring y-activity in a scintillation counter with NaJ/Tl crystal. Cadmium was also quantified by a radiochemical method, using a plastic SAD-12 crystal. The content of metallothionein-like proteins was estimated in liver and kidneys by a radiochemical method according to Zelazowski and Piotrowski (1977). RESULTS
The copper content in micrograms/g of the individual tissues of control rats and of those exposed to bismuth, mercury, and cadmium is given in Table 1. The most significant changes in the copper concentration took place in the kidneys and were correlated with an increase in the level of metallothionein-like proteins (Fig. 1). A twofold increase in the copper content of the kidneys was noted after exposure to bismuth chloride (3.0 mg Bi/kg) while more than sixfold increases were observed following exposure to mercuric chloride (1 .O mg Hg/kg) and cadmium chloride (2.0 mg Cd/kg). Only slight elevations in the copper content occurred in some other
TABLE
1
DISTRIBUTION OFCOPPER FOLLOWING OF SEVEN DOSES OF BISMUTH,~ADMIUM,OR
Cu concentration, Organ Kidneys Liver Blood Lungs Heart Spleen Intestine Skin Brain Muscles + bones
Total (a9
Control 10.0 4.27 1.56 3.46 7.89 3.43 2.13 2.03 3.58
+ 2 f 2 * 2 t + +
2.80 1.35 0.29 2.44 2.21 1.54 1.13 0.82 1.10
Bismuth (7 x 3 mgikg) 20.7* 4.99 1.90* 2.95 6.19 1.5l3* 1.93 2.83 3.47
-t 4.70 4 0.88 % 0.21 +- 0.76 4 1.06 z!z0.37 t 0.47 f 0.44 4 1.45
ADMINISTRATION MERCURY~
&g
tissues (w/W)
Mercury (7 x 1 m&g) 60.8* 4.34 1.80 2.96 6.08 1.77* 2.08 2.61 3.24
f ? k 2 + + 2 f 2
7.30 0.65 0.37 1.25 0.67 0.16 0.57 0.42 0.38
Cadmium (7 x 2 mgikg) 65.7* 5.56 2.28* 2.46 6.56 2.02 1.75 3.06 2.94
f 0.90 2 0.69 f 0.43 -r- 0.50 f 0.90 2 0.28 2 1.28 2 0.78 2 0.28
1.07 * 0.39
1.34 4 0.27
1.20 -c 0.24
1.09 2 0.47
284 k 67
369* 2 29
378* + 48
421* f 62
a Means from five to six rats. * Statistically significant.
EFFECT
OF
Cd,
Hg, AND
Bi ON
FIG. 1. Dependence of the levels of metallothionein-like kidneys of control and exposed rats on the concentration
Cu CONTENT
proteins (O--O) and copper (O--O) of the administered metal.
123
in
organs, e.g., in the liver and skin. In the remaining organs the copper content decreased although the diminution was significant only for the spleen. The whole-body retention of copper showed increases in exposed animals, statistically significant for bismuth and mercury at P = 0.05 and for cadmium at P = 0.01. Results of molecular sieving of the kidney protein fraction precipitated at 50-80% saturation with acetone on Sephadex G-75 are shown in Fig. 2. For the control animals, the copper concentration in the protein fraction of molecular weight of about 10,000 was equal to 0.035 fig/cm3 eluate/g tissue. In the animals exposed to bismuth a twofold elevation of this concentration was observed, up to about 0.07 pglcm3/g, while in the animals receiving cadmium and mercury the copper level in the eluates increased to 0.10 and 0.25 pg/cm3/g, respectively. The highest rise in the level of kidney metallothionein-like proteins was found in the rats exposed to mercury (Table 2). DISCUSSION
The effect of cadmium on the organ distribution of copper has been already the subject of previous reports. Schroeder and Nanson (1974) demonstrated a more than twofold increase in the copper content of the kidneys following exposure of rats to cadmium, 50 ppm per OS, for 2-4 years. Julshamn et al. (1977) found only a tendency for elevation of the copper content in the kidneys and liver and a decrease in the copper concentration in the spleen. The present report confirms the latter findings. On the other hand, to our knowledge there are no data on the effect of mercury and bismuth on the copper metabolism. Our results demonstrate that the most
EFFECT
OF Cd, Hg, AND
Bi ON
TABLE THE
LEVEL
OF METALLOTHIONEIN-LIKE LIVER
AND
Kind of exposure Control Bi (7 x 3.0 mg/kg) Hg (7 x 1.0 mg/kg) Cd (7 x 2.0 mg/kg)
PROTEINS KIDNEY
125
Cu CONTENT
2 (EXPRESSED
OF CONTROL
AND
Kidney
AS METALLOTHIONEIN,
EXPOSED
IN
mg/g) IN
RATS”
Liver
0.15 2 0.02 0.50 ” 0.01*
0.10 Ifr 0.03 0.21 & 0.10**
1.242 0.13*
0.18 k 0.06*
0.81 t 0.03*
2.57t 0.25*
* Significant; P < 0.01. ** Not significant; P < 0.05.
pronounced changes in the copper content induced by the three metals tested took place in the kidneys. An induced increase in the copper level, similar to that caused by cadmium, was also found after exposure to all three metals (by about 50% for cadmium and by about 30% for mercury and bismuth). A statistically significant diminution in the copper content was noted only in the spleen following exposure to bismuth and mercury. From among the published reports, only that of Suzuki et al. (1977) stated an increase in the copper content of the metallothionein-like protein fraction in the kidney of rats exposed to cadmium chloride. These authors found a correlation between duration of the exposure to the cadmium level in the fraction of low molecular weight proteins and the copper content of this fraction. Here we demonstrated an elevation in the copper content of the metallothionein-like protein fraction after exposure of rats to bismuth, mercury, and cadmium. The fraction characterized by an increased copper level also contained the highest concentrations of mercury, cadmium, and bismuth. Maxima of absorbances at 250 and 280 nm also occurred in this fraction. The ratio of A25,JA28,, amounted to 5 for the cadmium-induced metallothionein and was equal to 1.5- 1.6 for metallothioneinlike proteins isolated from kidneys of the control rats and of the animals exposed to bismuth and mercury. It results from the present data that the metallothionein-like proteins induced by cadmium, mercury, and bismuth in the kidneys, responsible for the binding of those metals in the kidneys, belong to copper proteins. Consequently, induction of these proteins as well as binding of the three exogenous metals in the kidneys requires a supply of equivalent amounts of copper. We suspected that the copper accumulation in the kidneys may take place at the expense of copper contained in other organs. This hypothesis was not confirmed since the increase in the copper content of the kidneys was accompanied by an elevation of the whole-body retention of copper, and no organ but the spleen showed a decrease in the concentration of this metal. One could assume that administration of cadmium, mercury, or bismuth changes the conditions of copper homeostasis so that an increase of the copper uptake in the intestine takes place. As proposed by Evans and LeBlanc (1976), low molecular weight proteins can also constitute an agent regulating the intestinal copper absorption. One should mention that some authors reported a decrease in the copper uptake following an oral cadmium administration (Evans e? al., 1970). Our results refer, on the other hand, to a subcutaneous metal administration. Apparently, a direct
126
SZYM.~NSK.~
AND
~ELAZ~WSKI
administration of the metal inductors per OS can induce biosynthesis of low molecular weight copper-binding proteins in the intestinal walls, and the produced proteins may constitute a factor limiting the copper uptake by its binding in the intestinal mucosa. It seems therefore that the final effect of the metals studied on copper retention depends on the method of their administration. ACKNOWLEDGMENTS The authors are indebted to Professor Jerzy K. Piotrowski cussion of the results.
for his interest in this study and dis-
REFERENCES Evans, G. W., Majors, P. F., and Comatzer, W. E. (1970). Mechanisms for cadmium and zinc antagonism of copper metabolism. Biochem. Biophys. Res. Commun. 40, 1142-l 146. Evans, G. W., and LeBlanc, F. N. (1976). Copper-binding protein in rat intestine: Amino acid composition and function. Nutr. Rep. Int. 14, 281-288. Julshamn, K., Utne, F., and Braekkan, 0. R. (1977). Interactions of cadmium, zinc and iron in different organs and tissues of the rat. Acta Pharmacol. Toxicol. 41, 515-524. Piotrowski, J. K., and Szymanska, J. A. (1976). Influence of certain metals on the level of metallothionein-like proteins. J. Toxicol. Environ. Health 1, 991- 1002. Schroeder, H. A., and Nanson, A. P. (1974). Interactions of trace metals in rat tissues: Cadmium and nickel with zinc, chromium, copper, manganese. J. Nutr. 104, 167- 178. Suzuki, T. K., Kubota, K., and Takenaka, S. (1977). Copper in cadmium-exposed rat kidney metallothionein. Chem. Pharm. Bull. 25, 2794-2796. Szymanska, J. A. (1977). Ph.D. dissertation. Medical Academy. L6dt. Szymattska, J. A., and Zychowicz, M. (1977). Simultaneous spectrophotometric determination of bismuth and copper in biological material by using zinc dibenzyldithiocarbamate. Chem. Anal. 22, 985-988 (in Polish). Zelazowski, A. J., and Piotrowski, J. K. (1977). A modified procedure for determination of metallothionein-like proteins in animal tissues. Acta Biochim. Pal. 24, 97-103. Zelazowski, A. J., and Piotrowski, J. K. (1978). Further investigations on the inducible mercurybinding proteins in rat kidney. In “XVI Congr. Polish Biochem. Sot., Lbdt. Abstracts” (in Polish). Zelazowski, A. J., Piotrowski, J. K., Mogilnicka, E. M., Szymanska, J. A., and Kaszper, B. W. (1978). The preparation of metallothionein from equine renal cortex as a standard protein for analytical purposes. Bromatol. Chem. Toksykol. 11, 51-57 (in Polish).