Chem.-Biol. Interactions, 15 (1976) 327--336
327
© E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press, A m s t e r d a m - - Printed in The N e t h e r l a n d s
THE BIOSYNTHESIS OF M E T A L L O T H I O N E I N IN RAT L I V E R AND KIDNEY A F T E R ADMINISTRATION OF CADMIUM
ZAHIR A. SHAIKH and J. CRISPIN SMITH Department of Pharmacology and Toxicology. University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 (U.S.A.)
(Received May 21st, 1976) (Revision received August 26th, 1976) ( A c c e p t e d August 31st, 1976)
SUMMARY The biosynthesis of the cadmium-binding protein, metallothionein, was studied in rat liver and kidney after injection of cadmium chloride. A simplified procedure for the isolation of metallothionein from liver and kidney tissues was devised. It was found that the concentration of a subcutaneously injected dose of 30 pmoles of l°9CdCl~/kg in the liver reached the maximum within 36 h. Thereafter, a slow decrease in the concentration of the isotope was noted during the 3 week period. In the kidney, the isotope was taken up in two phases. During the first phase the uptake was faster and lasted for a b o u t 4 days. The second phase of 1°9Cd accumulation showed a slower increase in the concentration of the isotope. In both liver and kidney tissues 75--80% of the 1°9Cd was associated with metallothionein. Amino acid incorporation studies revealed that active biosynthesis of metallothionein t o o k place in the kidney as well as in the liver of cadmium-exposed rats. The turnover of 3SS-labeled metallothionein was also investigated and the half-lives of the hepatic and the renal metallothionein were found to be 2.8 and 5 days, respectively.
INTRODUCTION In laboratory animals orally or parenterally administered cadmium (Cd) accumulates principally in the liver and kidney [ 1--3]. A protein, containing 4 to 6% Cd by weight, was discovered by Vallee and coworkers in equine and human kidneys [4--6]. This protein, which also contained lesser quantities of zinc and copper, was named m e t a l l o t h i o n e i n by Kiigi and Vallee [5] because of its metal-binding capacity and abundance of cysteinyl residues. In studies of rabbits chronically exposed to Cd, Piscator [7] observed that the
328 concentration of a liver protein, similar in molecular weight to metallothionein, was increased. Shaikh and Lucis [8,9] first presented the evidence that exposure to Cd stimulates the synthesis of the Cd-binding protein in liver and kidney tissues of mice and rats. Since then, several investigators have described the p h e n o m e n o n in chickens [10], mice [11], rats [10,12-17], and rabbits [18]. In an earlier report, Shaikh and Lucis [2] described the metabolic fate of a tracer dose of 1°9Cd. The present investigation was designed to study the time-course of uptake of a subtoxic dose of 1°9Cd and its effect on the concentration of metallothionein in liver and kidney. Friberg et al. [3] advanced the interesting hypothesis that metallothionein is produced in the liver and slowly released to the blood; it is then filtered through the glomeruli and reabsorbed by the renal tubules. This theory implies that the protein is synthesized only in the liver. The validity of this hypothesis was tested by studying the incorporation of 3SS-labeled cysteine into hepatic and renal metallothioneins. According to Webb [14] metallothionein is resistant to attacks by proteases in vitro. To determine whether metallothionein is catabolized in vivo, the hepatic and renal proteins were labeled with 3SS-cysteine and disappearance of the label from both proteins was followed. An abstract describing the findings has been published elsewhere [ 19]. MATERIALS AND METHODS Male rats of Wistar strain averaging 150--200 g were obtained from Microbiological Associates, Bethesda, Md. The animals were housed in galvanized cages and fed Charles River rat chow and tap water ad libitum. Carrier-free 1°9CDC12 was purchased from New England Nuclear and L-[3SS]cystine (specific activity 200 gCi/pmole) was obtained from Amersham Searle Corporation. Cadmium chloride solution was prepared from ultrapure-grade salt provided by Ventron Corporation. An injection solution of 15 pmoles Cd/ml (specific activity 0.25 pCi/pmole) was prepared from CdC12 and 1°9CdC12 stock solutions after dilution with isotonic saline. Injections were made subcutaneously in the interscapular region and the injected dose was 30 #moles Cd/kg. Radiolabeled cystine was diluted with isotonic saline to 100 pCi/ml and injected intraperitoneally in the a m o u n t of 20 pCi/rat. The animals were sacrificed by exsanguination. Liver and kidneys were removed, washed in chilled isotonic saline, weighed and either fractionated immediately or frozen and analyzed later. The procedure used in this study to isolate metallothionein was a modification of that described earlier by Shaikh and Lucis [20]. The modification consisted of heat-treating the homogenate to coagulate the high molecular weight proteins and to stop enzymatic reactions. It has been reported earlier [14] that metallothionein is heat stable. This was confirmed in our laboratory and advantage was taken of this property of the protein. A 20% (w/v) homogenate of the tissue was prepared in chilled 1 mM Tris--HC1 buffer, pH 8.6, using a glass homogenizer and a teflon pestle. The homogenate was heated at 85°C in a water
329 bath for 10 min, cooled, and centrifuged at 10 000 g for 10 min at 4°C. The supernatant was collected and the residue was suspended in an equal volume of Tris buffer and centrifuged again. The combined supernatants accounted for 75--80% of the tissue ~°9Cd, which is very similar to the a m o u n t of ~°9Cd recovered in the soluble fraction of the homogenate reported earlier [20]. In the present procedure the supernatant was obtained more rapidly and contained smaller amounts of contaminating proteins. To isolate metallothionein, the supernatant was fractionated by gel filtration chromatography. 10. ml of the liver supernatant and 5 ml of the kidney supernatant were applied respectively to 2.6 X 95 cm and 1.6 X 95 cm glass columns packed with Sephadex G-75 (Pharmacia). The samples were eluted from the columns with Tris buffer containing 0.02% sodium azide and the eluates were monitored at 254 nm and 278 nm simultaneously. Flow rate of the 2.6 )< 95 cm column was maintained at 28 ml/h and that of the 1.6 )< 95 cm column at 13 ml/h with the help of peristaltic pumps. 15 min fractions were collected. Fractions corresponding to elution volume/void volume ratio of 1.6 to 2.1 contained almost all of the '°9Cd applied to the columns. These were pooled and designated as crude metallothionein. Protein concentration was measured by the m e t h o d of Lowry et al. [21]. In unexposed animals the crude metallothionein fraction contains very little metallothionein [14,20]. Additional protein of this molecular weight produced after Cd exposure, however, is all metallothionein [13,14,16,20]. We established earlier that by ion-exchange chromatography, the crude protein could be separated into several components of metallothionein [20]. Heterogeneity of metallothionein has also been reported by others [10,13, 17,18,22]. Since the purpose of the present investigation was to measure total metallothionein produced after Cd exposure, further fractionation by ion-exchange chromatography was n o t carried out. The contaminating proteins in the crude preparation amounted to 0.9 mg/g liver and 0.6 mg/g kidney. These values were subtracted from those estimated for the crude metallothionein to arrive at metallothionein content. For assaying ~°9Cd, 1 ml aliquots of homogenate, supernatant and column fractions were counted in a well-type Packard gamma spectrometer at an efficiency of 57%. Before assay of 3ss in homogenates and supernatants, 0.2 ml aliquots of the samples were digested with 1 ml of Soluene (Packard) at 60°C. After digestion, the samples were neutralized with 50 pl of glacial acetic acid. Aquasol (NEN) was used as a scintillation cocktail for tissue samples as well as for the column fractions. The counting was performed in a Packard liquid scintillation spectrometer. Quench corrections were made using the external standard ratio method. RESULTS The initial part of the present study was directed toward elucidation of the tissue distribution of a subtoxic dose of Cd with time and the effect of increasing tissue concentration of Cd on the level of metallothionein. The
330 uptake of l°9Cd by liver is shown in Fig. 1. Maximum concentration was reached within 36 h after injection. Thereafter, slow depletion of the isotope was noted during the 3-week period. The liver was further fractionated to separate metallothionein and it was found that 75--80% of the tissue 1°9Cd was associated with this protein after 6 h and remained bound for 22 days (Fig. 1). Estimation of metallothionein revealed that its concentration increased with the uptake of Cd by the tissue (Fig. 1). This increase in metallothionein content accounted for the binding and long retention time of Cd in the liver. Fig. 2 illustrates the uptake of 1°9Cd by kidney, its effect on metallothionein concentration, and its binding with the protein in this organ. Continuous accumulation of the isotope was found in the kidney for 22 days. However, a faster rate of uptake was observed during the first 4 days after injection than later on. Total accumulation in 4 days was 80% of that obtained at the end of 22 days. Increase in Cd concentration of the tissue caused the level of metallothionein to rise in a pattern similar to that of ~°gCd accumulation. Renal metallothionein was responsible for sequestering nearly twothirds of the renal Cd (Fig. 2). To investigate whether metallothionein was being synthesized de novo in response to Cd challenge, the animals were primed with Cd for 22 h and then given a dose of [3ss] cystine to label the proteins. The control animals received no Cd. Animals were sacrificed at various time intervals after the injection of [3SS]cystine and incorporation of the label into hepatic and renal metallothionein was determined. It was found t h a t the hepatic as well as the renal metallothionein t o o k up the 3ss label within 2 h after cystine injection. Other cellular proteins also showed a similar pattern. The biosynthesis of metallothionein seemed to be a continuous process and even 3 weeks after
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327
© E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press, A m s t e r d a m - - Printed in The N e t h e r l a n d s
THE BIOSYNTHESIS OF M E T A L L O T H I O N E I N IN RAT L I V E R AND KIDNEY A F T E R ADMINISTRATION OF CADMIUM
ZAHIR A. SHAIKH and J. CRISPIN SMITH Department of Pharmacology and Toxicology. University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 (U.S.A.)
(Received May 21st, 1976) (Revision received August 26th, 1976) ( A c c e p t e d August 31st, 1976)
SUMMARY The biosynthesis of the cadmium-binding protein, metallothionein, was studied in rat liver and kidney after injection of cadmium chloride. A simplified procedure for the isolation of metallothionein from liver and kidney tissues was devised. It was found that the concentration of a subcutaneously injected dose of 30 pmoles of l°9CdCl~/kg in the liver reached the maximum within 36 h. Thereafter, a slow decrease in the concentration of the isotope was noted during the 3 week period. In the kidney, the isotope was taken up in two phases. During the first phase the uptake was faster and lasted for a b o u t 4 days. The second phase of 1°9Cd accumulation showed a slower increase in the concentration of the isotope. In both liver and kidney tissues 75--80% of the 1°9Cd was associated with metallothionein. Amino acid incorporation studies revealed that active biosynthesis of metallothionein t o o k place in the kidney as well as in the liver of cadmium-exposed rats. The turnover of 3SS-labeled metallothionein was also investigated and the half-lives of the hepatic and the renal metallothionein were found to be 2.8 and 5 days, respectively.
INTRODUCTION In laboratory animals orally or parenterally administered cadmium (Cd) accumulates principally in the liver and kidney [ 1--3]. A protein, containing 4 to 6% Cd by weight, was discovered by Vallee and coworkers in equine and human kidneys [4--6]. This protein, which also contained lesser quantities of zinc and copper, was named m e t a l l o t h i o n e i n by Kiigi and Vallee [5] because of its metal-binding capacity and abundance of cysteinyl residues. In studies of rabbits chronically exposed to Cd, Piscator [7] observed that the
328 concentration of a liver protein, similar in molecular weight to metallothionein, was increased. Shaikh and Lucis [8,9] first presented the evidence that exposure to Cd stimulates the synthesis of the Cd-binding protein in liver and kidney tissues of mice and rats. Since then, several investigators have described the p h e n o m e n o n in chickens [10], mice [11], rats [10,12-17], and rabbits [18]. In an earlier report, Shaikh and Lucis [2] described the metabolic fate of a tracer dose of 1°9Cd. The present investigation was designed to study the time-course of uptake of a subtoxic dose of 1°9Cd and its effect on the concentration of metallothionein in liver and kidney. Friberg et al. [3] advanced the interesting hypothesis that metallothionein is produced in the liver and slowly released to the blood; it is then filtered through the glomeruli and reabsorbed by the renal tubules. This theory implies that the protein is synthesized only in the liver. The validity of this hypothesis was tested by studying the incorporation of 3SS-labeled cysteine into hepatic and renal metallothioneins. According to Webb [14] metallothionein is resistant to attacks by proteases in vitro. To determine whether metallothionein is catabolized in vivo, the hepatic and renal proteins were labeled with 3SS-cysteine and disappearance of the label from both proteins was followed. An abstract describing the findings has been published elsewhere [ 19]. MATERIALS AND METHODS Male rats of Wistar strain averaging 150--200 g were obtained from Microbiological Associates, Bethesda, Md. The animals were housed in galvanized cages and fed Charles River rat chow and tap water ad libitum. Carrier-free 1°9CDC12 was purchased from New England Nuclear and L-[3SS]cystine (specific activity 200 gCi/pmole) was obtained from Amersham Searle Corporation. Cadmium chloride solution was prepared from ultrapure-grade salt provided by Ventron Corporation. An injection solution of 15 pmoles Cd/ml (specific activity 0.25 pCi/pmole) was prepared from CdC12 and 1°9CdC12 stock solutions after dilution with isotonic saline. Injections were made subcutaneously in the interscapular region and the injected dose was 30 #moles Cd/kg. Radiolabeled cystine was diluted with isotonic saline to 100 pCi/ml and injected intraperitoneally in the a m o u n t of 20 pCi/rat. The animals were sacrificed by exsanguination. Liver and kidneys were removed, washed in chilled isotonic saline, weighed and either fractionated immediately or frozen and analyzed later. The procedure used in this study to isolate metallothionein was a modification of that described earlier by Shaikh and Lucis [20]. The modification consisted of heat-treating the homogenate to coagulate the high molecular weight proteins and to stop enzymatic reactions. It has been reported earlier [14] that metallothionein is heat stable. This was confirmed in our laboratory and advantage was taken of this property of the protein. A 20% (w/v) homogenate of the tissue was prepared in chilled 1 mM Tris--HC1 buffer, pH 8.6, using a glass homogenizer and a teflon pestle. The homogenate was heated at 85°C in a water
329 bath for 10 min, cooled, and centrifuged at 10 000 g for 10 min at 4°C. The supernatant was collected and the residue was suspended in an equal volume of Tris buffer and centrifuged again. The combined supernatants accounted for 75--80% of the tissue ~°9Cd, which is very similar to the a m o u n t of ~°9Cd recovered in the soluble fraction of the homogenate reported earlier [20]. In the present procedure the supernatant was obtained more rapidly and contained smaller amounts of contaminating proteins. To isolate metallothionein, the supernatant was fractionated by gel filtration chromatography. 10. ml of the liver supernatant and 5 ml of the kidney supernatant were applied respectively to 2.6 X 95 cm and 1.6 X 95 cm glass columns packed with Sephadex G-75 (Pharmacia). The samples were eluted from the columns with Tris buffer containing 0.02% sodium azide and the eluates were monitored at 254 nm and 278 nm simultaneously. Flow rate of the 2.6 )< 95 cm column was maintained at 28 ml/h and that of the 1.6 )< 95 cm column at 13 ml/h with the help of peristaltic pumps. 15 min fractions were collected. Fractions corresponding to elution volume/void volume ratio of 1.6 to 2.1 contained almost all of the '°9Cd applied to the columns. These were pooled and designated as crude metallothionein. Protein concentration was measured by the m e t h o d of Lowry et al. [21]. In unexposed animals the crude metallothionein fraction contains very little metallothionein [14,20]. Additional protein of this molecular weight produced after Cd exposure, however, is all metallothionein [13,14,16,20]. We established earlier that by ion-exchange chromatography, the crude protein could be separated into several components of metallothionein [20]. Heterogeneity of metallothionein has also been reported by others [10,13, 17,18,22]. Since the purpose of the present investigation was to measure total metallothionein produced after Cd exposure, further fractionation by ion-exchange chromatography was n o t carried out. The contaminating proteins in the crude preparation amounted to 0.9 mg/g liver and 0.6 mg/g kidney. These values were subtracted from those estimated for the crude metallothionein to arrive at metallothionein content. For assaying ~°9Cd, 1 ml aliquots of homogenate, supernatant and column fractions were counted in a well-type Packard gamma spectrometer at an efficiency of 57%. Before assay of 3ss in homogenates and supernatants, 0.2 ml aliquots of the samples were digested with 1 ml of Soluene (Packard) at 60°C. After digestion, the samples were neutralized with 50 pl of glacial acetic acid. Aquasol (NEN) was used as a scintillation cocktail for tissue samples as well as for the column fractions. The counting was performed in a Packard liquid scintillation spectrometer. Quench corrections were made using the external standard ratio method. RESULTS The initial part of the present study was directed toward elucidation of the tissue distribution of a subtoxic dose of Cd with time and the effect of increasing tissue concentration of Cd on the level of metallothionein. The
330 uptake of l°9Cd by liver is shown in Fig. 1. Maximum concentration was reached within 36 h after injection. Thereafter, slow depletion of the isotope was noted during the 3-week period. The liver was further fractionated to separate metallothionein and it was found that 75--80% of the tissue 1°9Cd was associated with this protein after 6 h and remained bound for 22 days (Fig. 1). Estimation of metallothionein revealed that its concentration increased with the uptake of Cd by the tissue (Fig. 1). This increase in metallothionein content accounted for the binding and long retention time of Cd in the liver. Fig. 2 illustrates the uptake of 1°9Cd by kidney, its effect on metallothionein concentration, and its binding with the protein in this organ. Continuous accumulation of the isotope was found in the kidney for 22 days. However, a faster rate of uptake was observed during the first 4 days after injection than later on. Total accumulation in 4 days was 80% of that obtained at the end of 22 days. Increase in Cd concentration of the tissue caused the level of metallothionein to rise in a pattern similar to that of ~°gCd accumulation. Renal metallothionein was responsible for sequestering nearly twothirds of the renal Cd (Fig. 2). To investigate whether metallothionein was being synthesized de novo in response to Cd challenge, the animals were primed with Cd for 22 h and then given a dose of [3ss] cystine to label the proteins. The control animals received no Cd. Animals were sacrificed at various time intervals after the injection of [3SS]cystine and incorporation of the label into hepatic and renal metallothionein was determined. It was found t h a t the hepatic as well as the renal metallothionein t o o k up the 3ss label within 2 h after cystine injection. Other cellular proteins also showed a similar pattern. The biosynthesis of metallothionein seemed to be a continuous process and even 3 weeks after
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HOMOGENATE •
MET~LLOTHIONEIN
@ ~ lO 0
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?°
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01234
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7 8 I'5 DAYSAFTERCADMIUMINJECTION
Fig. 1. U p t a k e a n d d i s t r i b u t i o n o f tration.
I
l°9Cd in liver
a n d its e f f e c t o n m e t a l l o t h i o n e i n c o n c e n -
331
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