Toxicology, 60 (1990) 151--159 Elsevier Scientific Publishers Ireland Ltd.
Metallothionein production: similar responsiveness of avian liver and kidney to chronic cadmium administration A . M . S c h e u h a m m e r a,* a n d D . M . T e m p l e t o n b ~Environment Canada, Canadian Wildlife Service, 100 Gamelin Boulevard, Ottawa, Ontario KIA OH3 and ~University of Toronto, Department of Clinical Biochemistry, Banting Institute, 100 College Street, Toronto, Ontario M5G 1L5 (Canada) (Received May 22nd, 1989; accepted September 6th, 1989)
Summary The accumulation of hepatic and renal Cd, Zn, Cu, and metallothionein (MT) was investigated in ringed turtle doves (Streptopelia risoria) chronically exposed to 3 different concentrations of dietary Cd. When only tissue-Cd was considered as an inducer of MT, kidney was found to be 35070 as responsive as liver in producing MT. However, when all potentially relevant inducing metals (Cd + Zn + Cu) were taken into account, kidney was found to be 85°7o as responsive as liver. The greater production of MT/mol Cd in liver was accounted for mainly by a greater co-accumulation of Zn/mol Cd in liver than in kidney. We conclude that the apparent tissue specificity in expression of MT may be overestimated by failure to consider fluctuations in multiple inducers. Variability in tissue-MT concentrations after chronic dietary Cd administration is best accounted for by a consideration of tissueCd, -Zn, and -Cu, rather than tissue-Cd alone. Key words: Birds; Cadmium; Copper; Liver; Kidney; Metallothionein synthesis; Zinc
Introduction T h e s y n t h e s i s o f m e t a l l o t h i o n e i n ( M T ) c a n b e i n d u c e d b y several m e t a l s . T h e most effective of these are the essential elements Zn and Cu, and the non-essential m e t a l s C d a n d H g [1,2]. A l t h o u g h s e v e r a l o t h e r m e t a l i o n s c a n c a u s e m e a s u r a b l e i n c r e a s e s in tissue M T , t h e s e i n c r e a s e s a r e g e n e r a l l y l o w in c o m p a r i s o n to those produced by the more effective inducers. Various organs have been comp a r e d w i t h r e s p e c t to t h e i r M T " i n d u c i b i l i t y " o r " i n d u c t i o n p o t e n t i a l " , t h a t is, t h e a m o u n t o f M T t h e y p r o d u c e in r e s p o n s e to a d m i n i s t r a t i o n o f a g i v e n i n d u c ing m e t a l . O l a f s o n [3] f o u n d t h e r e s p o n s e o f liver to b e a p p r o x i m a t e l y t w i c e t h a t o f k i d n e y a f t e r s.c. a d m i n i s t r a t i o n o f e i t h e r C d 2÷ o r Z n 2÷ to m i c e . I n d u c i b i l i t y was *To whom correspondence should be sent. 0300-483x/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
151
determined in relation to the dose administered, however, and not the concentration of metal accumulated by the respective tissues. Onosaka and Cherian [4] studied the accumulation of MT in relation to the deposition of Zn 2÷ in a number of tissues, following the s.c. administration of different doses of the metal to rats. They concluded that the response of liver, kidney, pancreas and small intestine was nearly uniform, each increase of 1 ~g/g body wt of Zn resulting in the production of 13--16 ~g/g MT. When C d 2÷ w a s used as the inducing metal, however, liver, pancreas and small intestine still responded with 16--17 ~g MT/~g Cd, but the inducibility of MT in the kidney was about 40O7o lower [5]. Sendelbach and Klaassen [6] reported that liver produced 11 ~g M T / ~ g Cd after injection of CdCI2, whereas kidney produced only 3 ~g M T / ~ g Cd. Thus, there is evidence to suggest that the rodent kidney is substantially less responsive than liver with respect to MT induction after exposure to Cd 2÷. However, in none of these studies were the tissue concentrations of other, endogenous MT-inducing metals, namely Zn and Cu, measured after Cd administration. An understanding of the difference in the responses of liver and kidney to Cd exposure is important because these are target organs of Cd toxicity in higher animals. The present study was undertaken in order to investigate the generality of the apparent difference in the tissue response to Cd z÷ by using a non-rodent model, and to examine the role that the endogenous, potent MT inducers, Cu and Zn, might play in the response of avian liver and kidney to dietary Cd. It is likely that the entry of Zn into liver after Cd administration acts additively with Cd to induce hepatic MT in rats [7]. Bremner and Davies [8] suggested that elevated hepatic Zn could contribute to MT induction in rat liver following Cu 2÷ injection, although it has also been shown that Cu initiates MT m R N A transcription prior to increases in hepatic Zn concentration [9]. Here we report the effects of dietary Cd 2÷ on the concentrations of Cd, Zn, Cu and MT in the livers and kidneys of ring doves (Streptopelia risoria). Our results demonstrate that, in these birds, most of the apparent difference in hepatic and renal MT induction in response to dietary Cd can be accounted for by changes in the tissue levels of Zn and Cu. Materials and methods
Animals and treatment Adult, aviary-bred ring doves (Streptopelia r&oria) were housed as malefemale pairs. Pairs were assigned to one of 3 experimental groups, 15 pairs/ group, as part of a study to investigate the reproductive effects of chronic dietary metal exposure. Controls (Group 1) received a semipurified chicken diet (ICN Nutritional Biochemicals, Cleveland, OH) containing, on a dry weight basis, 0.2 Cg/g Cd, and supplemented with 7.4 ~g/g Zn (as ZnCO3) and 4 ~g/g Cu (as C u S O 4 . 5 H 2 0 ). To generate a range of tissue-Cd and -MT concentrations, Groups 2 and 3 received the same diet supplemented with 2 and 20 ~g/g Cd (as CdClz) respectively. Food and deionized water were provided ad lib. All birds were sacrificed by decapitation 5 months after the start of the experiment.
152
Tissue preparation and analyses Livers and kidneys were removed at sacrifice and stored at - 8 5 ° C until required. For metal analysis, approximately 0.5 g wet tissue was lyophylized, weighed, and digested in H N O 3 (Merck G R grade, low in mercury). Cd, Zn, and Cu were measured in tissue digests by flame atomic absorption spectrophotometry using a Perkin-Elmer 3030 spectrophotometer. MT was estimated in fresh tissue by the method of Scheuhammer and Cherian [10] as modified by Scheuhammer [11]. M T results were converted to dry weight concentrations after measuring the water content of each tissue sample. Although MT from S. risoria has been only partially characterized (Scheuhammer, unpubl, data), M T from other avian species has been thoroughly studied and found to be structurally and biochemically similar to m a m m a l i a n MT [12,13]. We are confident that the wellstandardized Ag-saturation assay can be used to accurately measure induced levels of MT in ring dove tissues, and those of other avian species [11]. Regressions and other statistical analyses were performed using the S P S S / P C (SPSS Inc., Chicago, IL), SAS (SAS Institute) and Sigmaplot (Jandel Scientific, Sausalito, CA) computer programs. Results and discussion
None of the birds died over the course of the experiment, nor did they exhibit overt signs of Cd toxicity. Based on the average amount of food consumed, the estimated doses of Cd were: < 0 . 2 ~ m o l / k g / d a y (Group 1), 2 ~ m o l / k g / d a y (Group 2), and 20 g m o l / k g / d a y (Group 3). All groups had an average daily dietary intake of 6 gmol C u / k g and 113 ~mol Z n / k g . For all three dietary groups, kidneys contained higher average Cd concentrations than livers (Table I). This preferential accumulation of Cd by kidney in response to low, oral dosing protocols has been observed in other species [11,14,15]. Renal Cd concentrations did not exceed 230 ~g/g (2.1 ~mol/g) dry wt (,'o50 ~g/g wet wt, based on average water content of ring dove kidney tissue) in any individual bird, and averaged < 180 ~g/g (< 1.6 gmol/g) dry wt in Group 3. These levels are insufficient to cause significant renal toxicity after chronic, oral exposure in adult birds [16] or m a m m a l s [17]. Whereas kidney tended to accumulate Cd at a rate 2--3-times that of liver (Fig. 1; Cdk~d = 2.652 Cd~ v + 0.082), the rate of MT accumulation by each tissue was roughly equivalent (Fig. 2; MTk~d = 0.975 MTI~v + 0.096). (For ease of comparison, regression equations referred to in the text are collected in Table II.) These results are consistent with those reported by others [17,6] and might be interpreted as demonstrating that the kidney is, on average, only about 35°7o as responsive as the liver in synthesizing MT after Cd incorporation. However, when tissue-Zn and -Cu concentrations are also considered, a different conclusion is reached. One-way analysis of variance revealed that hepatic and renal Zn, and renal Cu, were significantly affected by changes in dietary Cd concentration (P < 0.001; Table I). In addition, in the liver, Cd and Zn concentrations were positively correlated (r = 0.61, P < 0.01). Cd and Cu concentrations were also
153
U,
Liver Kidney
Liver Kidney
Group 2
Group 3
0.501 +_ 0 . 2 2 6 1.590 _ 0 . 4 2 4
0.063 _ 0.021 0.143 ± 0 . 0 4 2
0.012 _ 0.004 a 0.043 ± 0 . 0 1 4
Zn
2.5 _ 0.9 3.6 + 0.6
1.8 __. 0.7 2.9 ± 0.6
1.6 _ 0.6 2.8 ± 0.7
Cu
0.26 _+ 0.08 0.69 + 0 . 2 0
0.23 _ 0.05 0 . 4 0 ± 0.12
0.23 ± 0 . 0 6 0.38 ___ 0.09
MT
0 a m o l / g d r y wt) A F T E R D I E T A R Y C d E X P O S U R E
0.285 ± 0 . 1 3 4 0.441 + 0 . 1 2 8
0.084 _ 0.094 0.156 _ 0.074
0.048 ± 0.056 0 . 0 7 7 ± 0.041
as d e t e r m i n e d b y a n a l y s i s o f v a r i a n c e .
Note: C h a n g e s in d i e t a r y C d ( G r o u p ) s i g n i f i c a n t l y a f f e c t e d c o n c e n t r a t i o n s o f all 3 metals a n d M T in b o t h tissues ( P < 0.001), except liver C u ( P = 0.093),
"Values a r e m e a n s ± S . D .
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Group 1
Cd
TISSUE METAL AND MT CONCENTRATIONS
TABLE I
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.
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Fig. 1. The relationship between hepatic and renal Cd concentrations after chronic dietary Cd exposure. The linear regression equation is Cdk~d = 0.082 + 2.652Cd,~v; r = 0.904, P < 0.01. Data are plotted in log/log form for illustrative purposes. Male ( • ) ; female (O).
c o r r e l a t e d (r = 0.43, P < 0.01), t h o u g h n o t as well as C d a n d Z n . M u l t i p l e stepwise r e g r e s s i o n d e m o n s t r a t e d t h a t , w i t h r e g a r d t o t h e i r utility as p r e d i c t o r s o f h e p a t i c M T c o n c e n t r a t i o n , t h e r a n k o r d e r o f m e t a l s was Z n > C d > C u . Z n c o n c e n t r a t i o n was b e t t e r c o r r e l a t e d w i t h M T t h a n was C d , in a c c o r d w i t h t h e results o f S c h e u h a m m e r et al. [7] w h o o b s e r v e d t h a t h e p a t i c M T in rats was h i g h l y c o r r e l a t e d w i t h h e p a t i c Z n r e g a r d l e s s o f w h e t h e r M T was i n d u c e d b y Z n 2÷, C d 2+, o r b o t h . T h e m o l a r i n c r e a s e in h e p a t i c Z n c o n c e n t r a t i o n a f t e r C d 2÷
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Metallothionein production: similar responsiveness of avian liver and kidney to chronic cadmium administration A . M . S c h e u h a m m e r a,* a n d D . M . T e m p l e t o n b ~Environment Canada, Canadian Wildlife Service, 100 Gamelin Boulevard, Ottawa, Ontario KIA OH3 and ~University of Toronto, Department of Clinical Biochemistry, Banting Institute, 100 College Street, Toronto, Ontario M5G 1L5 (Canada) (Received May 22nd, 1989; accepted September 6th, 1989)
Summary The accumulation of hepatic and renal Cd, Zn, Cu, and metallothionein (MT) was investigated in ringed turtle doves (Streptopelia risoria) chronically exposed to 3 different concentrations of dietary Cd. When only tissue-Cd was considered as an inducer of MT, kidney was found to be 35070 as responsive as liver in producing MT. However, when all potentially relevant inducing metals (Cd + Zn + Cu) were taken into account, kidney was found to be 85°7o as responsive as liver. The greater production of MT/mol Cd in liver was accounted for mainly by a greater co-accumulation of Zn/mol Cd in liver than in kidney. We conclude that the apparent tissue specificity in expression of MT may be overestimated by failure to consider fluctuations in multiple inducers. Variability in tissue-MT concentrations after chronic dietary Cd administration is best accounted for by a consideration of tissueCd, -Zn, and -Cu, rather than tissue-Cd alone. Key words: Birds; Cadmium; Copper; Liver; Kidney; Metallothionein synthesis; Zinc
Introduction T h e s y n t h e s i s o f m e t a l l o t h i o n e i n ( M T ) c a n b e i n d u c e d b y several m e t a l s . T h e most effective of these are the essential elements Zn and Cu, and the non-essential m e t a l s C d a n d H g [1,2]. A l t h o u g h s e v e r a l o t h e r m e t a l i o n s c a n c a u s e m e a s u r a b l e i n c r e a s e s in tissue M T , t h e s e i n c r e a s e s a r e g e n e r a l l y l o w in c o m p a r i s o n to those produced by the more effective inducers. Various organs have been comp a r e d w i t h r e s p e c t to t h e i r M T " i n d u c i b i l i t y " o r " i n d u c t i o n p o t e n t i a l " , t h a t is, t h e a m o u n t o f M T t h e y p r o d u c e in r e s p o n s e to a d m i n i s t r a t i o n o f a g i v e n i n d u c ing m e t a l . O l a f s o n [3] f o u n d t h e r e s p o n s e o f liver to b e a p p r o x i m a t e l y t w i c e t h a t o f k i d n e y a f t e r s.c. a d m i n i s t r a t i o n o f e i t h e r C d 2÷ o r Z n 2÷ to m i c e . I n d u c i b i l i t y was *To whom correspondence should be sent. 0300-483x/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
151
determined in relation to the dose administered, however, and not the concentration of metal accumulated by the respective tissues. Onosaka and Cherian [4] studied the accumulation of MT in relation to the deposition of Zn 2÷ in a number of tissues, following the s.c. administration of different doses of the metal to rats. They concluded that the response of liver, kidney, pancreas and small intestine was nearly uniform, each increase of 1 ~g/g body wt of Zn resulting in the production of 13--16 ~g/g MT. When C d 2÷ w a s used as the inducing metal, however, liver, pancreas and small intestine still responded with 16--17 ~g MT/~g Cd, but the inducibility of MT in the kidney was about 40O7o lower [5]. Sendelbach and Klaassen [6] reported that liver produced 11 ~g M T / ~ g Cd after injection of CdCI2, whereas kidney produced only 3 ~g M T / ~ g Cd. Thus, there is evidence to suggest that the rodent kidney is substantially less responsive than liver with respect to MT induction after exposure to Cd 2÷. However, in none of these studies were the tissue concentrations of other, endogenous MT-inducing metals, namely Zn and Cu, measured after Cd administration. An understanding of the difference in the responses of liver and kidney to Cd exposure is important because these are target organs of Cd toxicity in higher animals. The present study was undertaken in order to investigate the generality of the apparent difference in the tissue response to Cd z÷ by using a non-rodent model, and to examine the role that the endogenous, potent MT inducers, Cu and Zn, might play in the response of avian liver and kidney to dietary Cd. It is likely that the entry of Zn into liver after Cd administration acts additively with Cd to induce hepatic MT in rats [7]. Bremner and Davies [8] suggested that elevated hepatic Zn could contribute to MT induction in rat liver following Cu 2÷ injection, although it has also been shown that Cu initiates MT m R N A transcription prior to increases in hepatic Zn concentration [9]. Here we report the effects of dietary Cd 2÷ on the concentrations of Cd, Zn, Cu and MT in the livers and kidneys of ring doves (Streptopelia risoria). Our results demonstrate that, in these birds, most of the apparent difference in hepatic and renal MT induction in response to dietary Cd can be accounted for by changes in the tissue levels of Zn and Cu. Materials and methods
Animals and treatment Adult, aviary-bred ring doves (Streptopelia r&oria) were housed as malefemale pairs. Pairs were assigned to one of 3 experimental groups, 15 pairs/ group, as part of a study to investigate the reproductive effects of chronic dietary metal exposure. Controls (Group 1) received a semipurified chicken diet (ICN Nutritional Biochemicals, Cleveland, OH) containing, on a dry weight basis, 0.2 Cg/g Cd, and supplemented with 7.4 ~g/g Zn (as ZnCO3) and 4 ~g/g Cu (as C u S O 4 . 5 H 2 0 ). To generate a range of tissue-Cd and -MT concentrations, Groups 2 and 3 received the same diet supplemented with 2 and 20 ~g/g Cd (as CdClz) respectively. Food and deionized water were provided ad lib. All birds were sacrificed by decapitation 5 months after the start of the experiment.
152
Tissue preparation and analyses Livers and kidneys were removed at sacrifice and stored at - 8 5 ° C until required. For metal analysis, approximately 0.5 g wet tissue was lyophylized, weighed, and digested in H N O 3 (Merck G R grade, low in mercury). Cd, Zn, and Cu were measured in tissue digests by flame atomic absorption spectrophotometry using a Perkin-Elmer 3030 spectrophotometer. MT was estimated in fresh tissue by the method of Scheuhammer and Cherian [10] as modified by Scheuhammer [11]. M T results were converted to dry weight concentrations after measuring the water content of each tissue sample. Although MT from S. risoria has been only partially characterized (Scheuhammer, unpubl, data), M T from other avian species has been thoroughly studied and found to be structurally and biochemically similar to m a m m a l i a n MT [12,13]. We are confident that the wellstandardized Ag-saturation assay can be used to accurately measure induced levels of MT in ring dove tissues, and those of other avian species [11]. Regressions and other statistical analyses were performed using the S P S S / P C (SPSS Inc., Chicago, IL), SAS (SAS Institute) and Sigmaplot (Jandel Scientific, Sausalito, CA) computer programs. Results and discussion
None of the birds died over the course of the experiment, nor did they exhibit overt signs of Cd toxicity. Based on the average amount of food consumed, the estimated doses of Cd were: < 0 . 2 ~ m o l / k g / d a y (Group 1), 2 ~ m o l / k g / d a y (Group 2), and 20 g m o l / k g / d a y (Group 3). All groups had an average daily dietary intake of 6 gmol C u / k g and 113 ~mol Z n / k g . For all three dietary groups, kidneys contained higher average Cd concentrations than livers (Table I). This preferential accumulation of Cd by kidney in response to low, oral dosing protocols has been observed in other species [11,14,15]. Renal Cd concentrations did not exceed 230 ~g/g (2.1 ~mol/g) dry wt (,'o50 ~g/g wet wt, based on average water content of ring dove kidney tissue) in any individual bird, and averaged < 180 ~g/g (< 1.6 gmol/g) dry wt in Group 3. These levels are insufficient to cause significant renal toxicity after chronic, oral exposure in adult birds [16] or m a m m a l s [17]. Whereas kidney tended to accumulate Cd at a rate 2--3-times that of liver (Fig. 1; Cdk~d = 2.652 Cd~ v + 0.082), the rate of MT accumulation by each tissue was roughly equivalent (Fig. 2; MTk~d = 0.975 MTI~v + 0.096). (For ease of comparison, regression equations referred to in the text are collected in Table II.) These results are consistent with those reported by others [17,6] and might be interpreted as demonstrating that the kidney is, on average, only about 35°7o as responsive as the liver in synthesizing MT after Cd incorporation. However, when tissue-Zn and -Cu concentrations are also considered, a different conclusion is reached. One-way analysis of variance revealed that hepatic and renal Zn, and renal Cu, were significantly affected by changes in dietary Cd concentration (P < 0.001; Table I). In addition, in the liver, Cd and Zn concentrations were positively correlated (r = 0.61, P < 0.01). Cd and Cu concentrations were also
153
U,
Liver Kidney
Liver Kidney
Group 2
Group 3
0.501 +_ 0 . 2 2 6 1.590 _ 0 . 4 2 4
0.063 _ 0.021 0.143 ± 0 . 0 4 2
0.012 _ 0.004 a 0.043 ± 0 . 0 1 4
Zn
2.5 _ 0.9 3.6 + 0.6
1.8 __. 0.7 2.9 ± 0.6
1.6 _ 0.6 2.8 ± 0.7
Cu
0.26 _+ 0.08 0.69 + 0 . 2 0
0.23 _ 0.05 0 . 4 0 ± 0.12
0.23 ± 0 . 0 6 0.38 ___ 0.09
MT
0 a m o l / g d r y wt) A F T E R D I E T A R Y C d E X P O S U R E
0.285 ± 0 . 1 3 4 0.441 + 0 . 1 2 8
0.084 _ 0.094 0.156 _ 0.074
0.048 ± 0.056 0 . 0 7 7 ± 0.041
as d e t e r m i n e d b y a n a l y s i s o f v a r i a n c e .
Note: C h a n g e s in d i e t a r y C d ( G r o u p ) s i g n i f i c a n t l y a f f e c t e d c o n c e n t r a t i o n s o f all 3 metals a n d M T in b o t h tissues ( P < 0.001), except liver C u ( P = 0.093),
"Values a r e m e a n s ± S . D .
Liver Kidney
Group 1
Cd
TISSUE METAL AND MT CONCENTRATIONS
TABLE I
0
i-
1.00
"13 E~
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0
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0.10
13 £3 >I,I Z {:3 Y
0.01
•
•
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0.01
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•
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0.10
LIVER Cd (/~mol/g
.
.
.
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1.00 dry wt.)
Fig. 1. The relationship between hepatic and renal Cd concentrations after chronic dietary Cd exposure. The linear regression equation is Cdk~d = 0.082 + 2.652Cd,~v; r = 0.904, P < 0.01. Data are plotted in log/log form for illustrative purposes. Male ( • ) ; female (O).
c o r r e l a t e d (r = 0.43, P < 0.01), t h o u g h n o t as well as C d a n d Z n . M u l t i p l e stepwise r e g r e s s i o n d e m o n s t r a t e d t h a t , w i t h r e g a r d t o t h e i r utility as p r e d i c t o r s o f h e p a t i c M T c o n c e n t r a t i o n , t h e r a n k o r d e r o f m e t a l s was Z n > C d > C u . Z n c o n c e n t r a t i o n was b e t t e r c o r r e l a t e d w i t h M T t h a n was C d , in a c c o r d w i t h t h e results o f S c h e u h a m m e r et al. [7] w h o o b s e r v e d t h a t h e p a t i c M T in rats was h i g h l y c o r r e l a t e d w i t h h e p a t i c Z n r e g a r d l e s s o f w h e t h e r M T was i n d u c e d b y Z n 2÷, C d 2+, o r b o t h . T h e m o l a r i n c r e a s e in h e p a t i c Z n c o n c e n t r a t i o n a f t e r C d 2÷
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