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The interaction of zinc and cadmium in the synthesis of hepatic metallothionein in rats
Toxicology, 36 (1985) 101--108 Elsevier Scientific Publishers Ireland Ltd.
THE INTERACTION OF ZINC AND CADMIUM IN THE SYNTHESIS OF HEPATIC METALLOTHIONEIN IN RATS
A.M. SCHEUHAMMER*, S. ONOSAKA**, K. RODGERS and M.G. CHERIANt
Department of Pathology, University of Western Ontario, London, Ontario N6A 5C1 (Canada)
(Received March 19tb, 1985) (Accepted April 22nd, 1985)
SUMMARY The interaction of injected zinc salts (Zn) and cadmium salts (Cd) with regard to the synthesis of metallothionein (MT) in adult rat liver was investigated. Male rats received an i.p. injection of Zn (20 mg/kg) or Cd (0.6 mg/kg) with or w i t h o u t pretreatment with Zn (20 mg/kg 16 h prior to the second injection). It was found that both metals, when administered singly, induced the synthesis of significant levels of hepatic MT, but that, when the Cd injection followed the Zn injection, synthesis of MT was not additive. When Zn pretreatment was followed by a second Zn injection, MT accumulation was additive (approx. 2-fold of that observed after a single Zn injection). Also, a highly significant positive correlation, (r = 0.97, P < 0.01) was noted between hepatic Zn concentration and hepatic MT concentration, a relationship which was independent of the mode of MT induction. The results of the investigation indicate that: (1) in the presence of pre-existing hepatic Zn--MT, the ability of Cd to induce new MT synthesis is greatly reduced; rather, Cd is sequestered by the pre-existing MT; and (2) Zn may play a major role in the induction of MT synthesis both after Zn administration and after Cd administration.
K e y words: Interaction of zinc and cadmium; Metallothionein synthesis
*Present address: Canadian Wildlife Service, Environment Canada, Ottawa, Ontario KIA
0E7, Canada. **Present address: Department of Nutrition, Kobe-Gakuin University, Tarumi-Ku, Kobe 673, Japan. J'To whom all correspondence should be addressed.
0300-483X/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
101
INTRODUCTION
The induced synthesis of hepatic metallothionein (MT) occurs in a dosedependent manner in response to the administration of excess Zn 2. [1] or Cd 2. [2]. Induction of rat liver MT with Zn produces a Zn--MT with a biological half-life of 1--2 days [3]. Zn released from the protein does not initiate much further MT synthesis, resulting in the depletion of MT within about 1 week following cessation of excess Zn administration [4]. Induction of MT by Cd produces a Cd--MT with a biological half-life of 4--5 days [3,5], and Cd released from the protein is able to induce further MT synthesis resulting in the persistence of Cd--MT through time. Little is known concerning the interaction of Zn and Cd with respect to the induction of MT and the subsequent binding of these metals to the protein. Thus, the following study was undertaken in order to investigate the behaviour of Zn and Cd, both singly and in combination, with regard to the induction of MT in adult rat liver. MATERIALS AND METHODS
Male Sprague--Dawley rats (125--200 g) were given either no injections (Group 1, control), or a single i.p. injection of Zn (20 mg/kg as ZnSO4 in distilled deionized H20; Group 2), or Cd (0.6 mg/kg as CdC12 in H20, Group 3), or were treated with Cd or Zn 16 h after pretreatment with an initial injection of Zn (Groups 4 and 5). Each treatment group contained 3 rats. A schematic representation of the dosing schedule is shown in Fig. 1. Some rats were also given an i.p. injection of [3"~S]cysteine (10 pCi/rat} 0.5 h after metal administration. Upon termination of the experiment, rats were killed by exsanguination from the abdominal aorta while under pentobarbital anesthesia, and livers were removed for metal analysis and MT estimation. To determine hepatic Zn, Cd and Cu concentrations, liver tissue was homogenized in 4 vols. of 0.25 M sucrose, aliquots of the homogenate were digested with concentrated HC1, and metals were estimated by flame atomic absorption spectrophotometry using a Jarrell Ash 810 spectrophotometer as described previously [2]. To determine MT concentrations, aliquots of the
GROUP
I
GROUP 2 GROUP 3.
Control Zn
16 hrs
~ Cd
Sacrificer
i
GROUP 4.
~ Zn
GROUP 5.
~ Zn
GROUP 6.
~
32 hrs =
Zn
~ Cd
I Zn
Sacrifice I ,
Fig. 1. T r e a t m e n t p r o t o c o l for t h e 6 groups of rats used in t h e p r e s e n t s t u d y . Z i n c (20 mg/ kg) a n d Cd (0.6 m g / k g ) were a d m i n i s t e r e d as i n t r a p e r i t o n e a l i n j e c t i o n s in t h e s e q u e n c e s h o w n in t h e figure.
102
non-acidified h o m o g e n a t e s were c e n t r i f u g e d at 18 0 0 0 g f o r 20 min at 4°C and MT was m e a s u r e d in t h e resulting p o s t - m i t o c h o n d r i a l s u p e r n a t a n t s b y the Cd s a t u r a t i o n (Cd-hem) m e t h o d o f O n o s a k a and Cherian [2,6] w i t h o u t modification. In those animals which had also been injected with [3sS] c y s t e i n e , aliquots o f the p o s t - m i t o c h o n d r i a l s u p e r n a t a n t were f u r t h e r c e n t r i f u g e d f o r 1 h at 105 0 0 0 g and t h e resulting s u p e r n a t a n t s were f r a c t i o n a t e d o n S e p h a d e x G-75 c o l u m n s (0.9 × 60 cm) e l u t e d with 10 mM Tris--HC1 (pH 8.6). F r a c t i o n s (1 ml) were c o l l e c t e d and a n a l y z e d for 35S o n a Packard b e t a liquid scintillation c o u n t e r following a p p r o p r i a t e sample p r e p a r a t i o n [7]. T h e p e r c e n t a g e o f the t o t a l r a d i o a c t i v i t y r e c o v e r e d in t h e MT f r a c t i o n was calculated. In o r d e r t o c o m p a r e t h e distribution o f Cd and Zn in the h e p a t i c c y t o s o l s o f rats injected with Cd alone (group 3) and t h o s e injected with b o t h Zn and Cd ( g r o u p 5), the c y t o s o l s ( 1 0 5 0 0 0 g f o r 1 h) were p r e p a r e d f r o m a 30% liver h o m o g e n a t e . A l i q u o t s (0.25 ml) o f t h e c y t o s o l s were applied o n calibrated S e p h a d e x G-75 c o l u m n s (0.9 × 60 cm), and 1 ml fractions were collected. T h e c o n c e n t r a t i o n s o f Cd and Zn in each f r a c t i o n were e s t i m a t e d by flame atomic absorption spectrophotometry. RESULTS A single injection o f Zn (20 mg/kg) or Cd (0.6 mg/kg) resulted in the significant a c c u m u l a t i o n o f metals and i n d u c t i o n o f M T synthesis in rat liver b y 16 h (Table I). No direct c o m p a r i s o n o f t h e hepatic MT levels a f t e r Zn and Cd injections can be m a d e in these e x p e r i m e n t s because o f t h e p o t e n t i a l d i f f e r e n c e s in t h e t u r n o v e r rates o f Zn and Cd used in this study. T h e hepatic Zn levels o f rats injected with b o t h Cd and Zn were increased significantly as c o m p a r e d t o t h e c o n t r o l values. C a d m i u m was d e t e c t e d o n l y in livers o f rats which were injected with c a d m i u m chloride. T h e r e were n o changes in hepatic
TABLE I HEPATIC METAL AND METALLOTHIONEIN CONCENTRATIONS (ug/g WET WT) Group
Zn
Cd
Cu
MT b
4.7 ± 1.2 4.3 *- 0.6 4.2 *_0.2
40 756 417 489 780 1590
1 (control)
28.1 ± 3.4 a
n.d.
2 (Zn, 16 h)
69.9 ± 20.1"
n.d.
3 (Cd, 16 h)
46.0 ± 5.5*
5.3 ± 0.5
4 (Zn, 3 2 h)
62.4 ± 13.1"
n.d.
4.4 ± 0.4
5 (Zn---Cd)
66.2 ± 13.2"
6.5 ± 2.5
4.5 ± 0.4
n.d.
4.3 ± 0.6
6 (Zn--Zn)
119 ± 38*
± 25 ± 110" ~ 57* ± 240* ~ 156" ± 572*
aValues are means +_S.D., n = 3. bMT was measured by Cd-hem method. n.d., not detected (<0.1 ug/g). *Significantly different from control, P < 0.01.
103
TABLE II % OF [3sS]CYSTEINE INCORPORATION INTO MT [35S]cysteine was administered as an i.p. injection (10 uCi/rat) 0.5 h after the last dose o f metal and rats were sacrificed 15.5 h later. The liver cytosol fractions (105 000 g, 1 h) were prepared and fractionated on Sephadex G-75 columns. The ~sS-radioactivity associated with the 10 000 mol. wt fractions ( V e / V o 1.9 to 2 . t ) are expressed as percent of the total radioactivity in the cytosol. The results are mean values of 2 separate experiments. Group Group Group Group
1 (control)
3 (Cd) 5 (Zn--Cd) 6 (Zn--Zn)
6 30 15
26
copper levels after Zn and/or Cd treatments. When a Zn injection was followed by a second Zn injection (group 6), the level of MT was approximately 2-fold that observed after a single Zn injection. The hepatic Zn level also increased after the second Zn injection (69.9 in group 2 vs. 119 in group 6). However, when a Zn injection was followed by a Cd injection, the resulting level of MT was less than additive. In order to compare the new MT synthesis induced by a second injection of Zn or Cd, rats were injected with [3SS]cysteine at about 15.5 h before sacrifice. The fractionation of liver cytosols (105 000 g for 1 h) from control rats showed only a 6% of 3sS-incorporation to MT fraction. A significant increase in MT synthesis after Cd in iection was indicated by the 30% incorporation of [3SS]cysteine into MT fractions. The injection of Cd or Zn to Zn pretreated rats (groups 5 and 6) also resulted in increased incorporation of [3sS] cysteine to MT fractions. However, the new MT synthesis initiated by an injection of Cd in Zn-pretreated rats (group 5), as measured by [35S] cysteine incorporation, was lower than both injection of Cd alone (group 3) and a second injection of Zn in Zn pretreated rats (group 6). When the hepatic levels of Zn and MT in rats of various groups were compared, a direct correlation was observed (Fig. 2). These results demonstrate that regardless of whether Zn, Cd or both were used to induce hepatic MT synthesis, the resulting MT levels were highly correlated with the hepatic Zn concentration. The distribution of Cd in the hepatic cytosols of rats injected with Cd salts alone and those injected with both Zn and Cd salts showed identical patterns (Fig. 3). In both these groups, the hepatic Cd was exclusively bound to a 10 000 molecular weight fraction ( V e / V o ~ 2), identical to the position where a standard sample of MT was eluted out. The pattern of distribution of Zn among protein fractions was also similar in the 2 groups. A significant a m o u n t of Zn was associated with MT fraction in both of the groups.
104
2000
,
r
I000
~
150
Zn (pg/g liver)
Fig. 2. R e l a t i o n s h i p b e t w e e n Z n a n d M T c o n c e n t r a t i o n in t h e liver. P o i n t s are values f r o m all individual rats o f all 6 e x p e r i m e n t a l g r o u p s (as d e p i c t e d in Fig. 1). T h e c o r r e l a t i o n is h i g h l y significant; r = 0 . 9 7 , P < 0.01. Also s b o w n is t h e best-fit linear regression line, e q u a t i o n Y = 1 5 . 9 x - 366.
DISCUSSION
A major finding of the present study is that, in the presence of preexisting hepatic Zn--MT, the effectiveness of Cd to initiate new MT synthesis is much reduced, whereas the ability of Zn to induce further synthesis is unimpaired (Table I). A single injection of either Cd or Zn salt resulted in the synthesis of hepatic MT, but the induction of MT by these metals was not additive when an injection of Zn was followed by an injection of Cd. We suggest that this is probably due to the in vivo displacement of Zn from preexisting Zn-MT by Cd in Zn-pretreated rat liver. Additional support for this interpretation is the finding that [35S] cysteine incorporation into the MT fraction 16 h after Cd injection was reduced by approximately one-half by a pre-injection of Zn, again indicating that, although there is no reduction in Cd uptake by the liver after Zn-pretreatment (Table I), the accumulated Cd does n o t initiate much new MT synthesis. These results in adult rats confirm the data of Panemangalore and Cherian [7] who reported that, in 5-day-old rat pups, the administration of Cd (1 mg/kg s.c.} did not produce any alteration in the hepatic MT or Zn levels, but an injection of Zn (20 mg/kg s.c.) caused a 45% increase in the hepatic MT level. Neonatal rats have naturally high levels of hepatic Zn--MT [7--9], and MT may thus serve as a Zn-donor during rapid postnatal growth and development when the metabolic demands for Zn are high [ 1 0 - 1 2 ]. The presence of high endogenous levels of Zn--MT in neonates is thus analagous to Zn-injected adult rats with respect to MT concentrations and the behaviour of subsequently administered Cd. The second major finding of the present study is that, within 16--32 h of metal administration, hepatic MT concentrations are highly correlated with hepatic Zn concentrations regardless of whether MT is induced by Zn, or Cd,
105
I0
C d - injected
5~;~=~.m~_o" -o-,~o--o-o- ';
,
2
-
-o-o.o,.o.o.~ Zn and Cd injected
-16
2
i
I
.
_
_
~ _
>_~o-o-o-o-o-o( I
15
, 'o. . . . . ¢ . . . . o-~-~-~ 2
25
3
Ve/Vo Fig. 3. Gel filtration of liver cytosols. The cytosol (105 000 g, 1 h) samples were prepared f r o m livers of rats injected witb Cd alone (upper panel) or those injected with Zn and Cd (lower panel) and fractionated on calibrated Sephadex G-75 columns (0.9 x 60 cm). 1 ml fractions were collected and metals (Zn and Cd) were analyzed by a t o m i c absorption spectropbotometry.
or both. The suggestion has previously been made [1,13] that, under a variety of conditions, Zn 2÷ may be the primary inducer of MT at least in certain organs, and that other metals such as Cd 2+, Cu 2÷ and Hg 2÷ may induce the synthesis of MT by virtue of their ability to somehow alter the intracellular Zn pool. Subsequent to MT induction by Zn, these other metals would be transferred to MT from their initial macromolecular binding sites because of their higher affinity for MT. Zn would be displaced from MT in the process.
106
Consistent with this theory is the observation that, when tissue Zn levels are reduced, such as in the pancreas during Zn deficiency, Cd is unable to induce the synthesis of MT [ 1 ]. However, this proposed mechanism cannot be extrapolated to certain metals such as Ag, which will readily displace Zn from MT, but cause only a minor increase in tissue MT content [14]. Similarly, the failure of Cd to induce MT synthesis in the Zn-deficient pancreas [15] may be due to a general failure in Zn-dependent gene expression [12] as a consequence of Zn deficiency. The results of the present study can be interpreted as lending support for a major role of Zn 2÷ in the induction of MT after the administration of Zn and Cd salts. We observed that control rat livers contained a basal level of Zn of about 25--30 pg/g wet wt and a basal level of MT, as measured by the Cdhem method, of about 40 ug/g (Table I). Sixteen hours after a single injection of Zn, Zn levels had increased by approximately 40 ~g/g and were accompanied by a corresponding increase in MT levels of approximately 700 pg/g. Thus, the accumulation of 1 ~g/g of excess Zn in the liver resulted in the de novo synthesis of about 17--18 pg MT/g liver. Similarly, after the administration of a single dose of Cd, the hepatic Zn concentration increased by about 20 pg/g above control levels, corresponding to an increase in MT of about 400 pg/g. Thus, an accumulation of 1 pg/g of excess Zn was accompanied by an increase of about 20 pg MT/g, an almost identical value to that observed after Zn injection. Hepatic Cd concentrations, on the other hand, were only modestly increased, the molar increase in hepatic Zn (after Cd injection) being 8-fold that of Cd itself. It should also be noted that, after Cd injection, the a m o u n t of hepatic MT which is induced, is 5--10-fold that required to completely complex the entire hepatic Cd load (assuming a minimum of 3 and a maximum of 7 g atom Cd/mol MT). If only Cd is involved in the direct induction of MT after Cd injection, it may be questioned how and why such a large excess of MT is synthesized. If, however, the involvement of Zn is also hypothesized, such seeming excesses are better accounted for. Figure 2 further illustrates the highly significant correlation between hepatic MT concentration and hepatic Zn concentration, and demonstrates that, regardless of the mode of induction of MT -- whether by 1 or 2 Zn injections, 1 Cd injection, or a combination of a Zn injection followed by a Cd injection -- an increase of 1 pg Zn/g above basal values results in the synthesis of about 15--20 pg MT/g in the liver. It should also be mentioned that a significant a m o u n t of Zn is also detected in MT, isolated from various organs after injection of Cd [2]. Further studies designed to elucidate the possible role of Zn in the initial induction of MT synthesis in response to the administration of various metals are warranted. ACKNOWLEDGEMENT
This work was supported by grants from Medical Research Council of Canada. The authors wish to thank Mrs. Colleen Alford and Miss Paula Fenner for their help in typing this manuscript.
107
REFERENCES 1 S. Onosaka and M.G. Cherian, The induced synthesis of metallothionein in various tissues of rats in response to metals. II. Influence of zinc and specific effect on pancreatic metallothionein. Toxicology, 23 ( 1982 ) 11. 2 S. Onosaka and M.G. Cherian, The induced synthesis of metallothionein in various tissues of rat in response to metals. I. Effect of repeated injection of cadmium salts. Toxicology, 22 (1981) 91. 3 S.H. Ob, J.T. Deagen, P.D. Whanger and P.H. Weswig, Biological functions of metallothionein. IV. Biosynthesis and degradation of liver and kidney metallothionein in rats fed diets containing zinc or cadmium. Bioinorg. Chem., 8 (1978) 245. 4 S.H. Oh, N. Nakaue, J.T. Deagen, P.D. Whanger and G.H. Arscott, Accumulation and depletion of zinc in chick tissue metallothionein. J. Nutri., 109 {1979) 1720. 5 R.W. Chen, P.D. Whanger and P.H. Werwig, Biological functions of metallothionein. I. Syntbesis and degradation of rat liver metallothionein. Biochem. Med., 12 {1975) 95. 6 S. Onosaka and M.G. Cherian, Comparison of metallotbionein determintion by polarographic and cadmium saturation methods. Toxicol. Appl. Pharmacol., 63 (1982) 270. 7 M. Panemangalore and M.G. Cherian, Metabolism of parenterally administered zinc and cadmium in livers of newborn rats. Chem.-Biol. Interact., 45 (1983) 327. 8 J.U. Bell, Native metallothionein levels in rat hepatic cytosol, during perinatal development. Toxicol. Appl. Pharmacol., 50 (1979) 101. 9 K.-L. Wong and C.D. Klaassen, Isolation and characterization of metallothionein which is highly concentrated in newborn rat liver. J. Biol. Chem., 254 (1979) 12,399. 10 M. Webb and K. Cain, Functions of metallothionein. Biochem. Pharmacol., 31 (1982) 137. 11 M. Webb, Functions of hepatic and renal metallotbionein in the control of the metabolism of certain bivalent cations, in J.H.R. Kagi and M. Nordberg (Eds.), Metallotbionein, Birkhauser Verlag, Basel/Boston/Stuttgart, 1979, p. 313. 12 B.L. Vallee and K.H. Falchuk, Zinc and gene expression. Phil. Trans. R. Soc. Lond., 294 (1981) 185. l g F.O. Brady, M. Panemangalore, F.A. Day, A.M. Fiskin and G. Peterson, In vivo and ex vivo induction of rat liver metallothionein, in J.H.R. Kagi and M. Nordberg (Eds.), Metallothionein, Birkhauser Verlag, Basel/Boston/Stuttgart, 1979, p. 261. 14 D.R. Winge, R. Premakumar and K.V. Rajagopalan, Metal-induced formation of metallothionein in rat liver. Arch. Biochem. Biophys., 170 (1975) 242. 15 D.M. Templeton and M.G. Cherian, Effects of zinc deficiency on preexisting cadmiummetallothionein in the pancreas. Toxicology, 29 (1984) 251.
108
THE INTERACTION OF ZINC AND CADMIUM IN THE SYNTHESIS OF HEPATIC METALLOTHIONEIN IN RATS
A.M. SCHEUHAMMER*, S. ONOSAKA**, K. RODGERS and M.G. CHERIANt
Department of Pathology, University of Western Ontario, London, Ontario N6A 5C1 (Canada)
(Received March 19tb, 1985) (Accepted April 22nd, 1985)
SUMMARY The interaction of injected zinc salts (Zn) and cadmium salts (Cd) with regard to the synthesis of metallothionein (MT) in adult rat liver was investigated. Male rats received an i.p. injection of Zn (20 mg/kg) or Cd (0.6 mg/kg) with or w i t h o u t pretreatment with Zn (20 mg/kg 16 h prior to the second injection). It was found that both metals, when administered singly, induced the synthesis of significant levels of hepatic MT, but that, when the Cd injection followed the Zn injection, synthesis of MT was not additive. When Zn pretreatment was followed by a second Zn injection, MT accumulation was additive (approx. 2-fold of that observed after a single Zn injection). Also, a highly significant positive correlation, (r = 0.97, P < 0.01) was noted between hepatic Zn concentration and hepatic MT concentration, a relationship which was independent of the mode of MT induction. The results of the investigation indicate that: (1) in the presence of pre-existing hepatic Zn--MT, the ability of Cd to induce new MT synthesis is greatly reduced; rather, Cd is sequestered by the pre-existing MT; and (2) Zn may play a major role in the induction of MT synthesis both after Zn administration and after Cd administration.
K e y words: Interaction of zinc and cadmium; Metallothionein synthesis
*Present address: Canadian Wildlife Service, Environment Canada, Ottawa, Ontario KIA
0E7, Canada. **Present address: Department of Nutrition, Kobe-Gakuin University, Tarumi-Ku, Kobe 673, Japan. J'To whom all correspondence should be addressed.
0300-483X/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
101
INTRODUCTION
The induced synthesis of hepatic metallothionein (MT) occurs in a dosedependent manner in response to the administration of excess Zn 2. [1] or Cd 2. [2]. Induction of rat liver MT with Zn produces a Zn--MT with a biological half-life of 1--2 days [3]. Zn released from the protein does not initiate much further MT synthesis, resulting in the depletion of MT within about 1 week following cessation of excess Zn administration [4]. Induction of MT by Cd produces a Cd--MT with a biological half-life of 4--5 days [3,5], and Cd released from the protein is able to induce further MT synthesis resulting in the persistence of Cd--MT through time. Little is known concerning the interaction of Zn and Cd with respect to the induction of MT and the subsequent binding of these metals to the protein. Thus, the following study was undertaken in order to investigate the behaviour of Zn and Cd, both singly and in combination, with regard to the induction of MT in adult rat liver. MATERIALS AND METHODS
Male Sprague--Dawley rats (125--200 g) were given either no injections (Group 1, control), or a single i.p. injection of Zn (20 mg/kg as ZnSO4 in distilled deionized H20; Group 2), or Cd (0.6 mg/kg as CdC12 in H20, Group 3), or were treated with Cd or Zn 16 h after pretreatment with an initial injection of Zn (Groups 4 and 5). Each treatment group contained 3 rats. A schematic representation of the dosing schedule is shown in Fig. 1. Some rats were also given an i.p. injection of [3"~S]cysteine (10 pCi/rat} 0.5 h after metal administration. Upon termination of the experiment, rats were killed by exsanguination from the abdominal aorta while under pentobarbital anesthesia, and livers were removed for metal analysis and MT estimation. To determine hepatic Zn, Cd and Cu concentrations, liver tissue was homogenized in 4 vols. of 0.25 M sucrose, aliquots of the homogenate were digested with concentrated HC1, and metals were estimated by flame atomic absorption spectrophotometry using a Jarrell Ash 810 spectrophotometer as described previously [2]. To determine MT concentrations, aliquots of the
GROUP
I
GROUP 2 GROUP 3.
Control Zn
16 hrs
~ Cd
Sacrificer
i
GROUP 4.
~ Zn
GROUP 5.
~ Zn
GROUP 6.
~
32 hrs =
Zn
~ Cd
I Zn
Sacrifice I ,
Fig. 1. T r e a t m e n t p r o t o c o l for t h e 6 groups of rats used in t h e p r e s e n t s t u d y . Z i n c (20 mg/ kg) a n d Cd (0.6 m g / k g ) were a d m i n i s t e r e d as i n t r a p e r i t o n e a l i n j e c t i o n s in t h e s e q u e n c e s h o w n in t h e figure.
102
non-acidified h o m o g e n a t e s were c e n t r i f u g e d at 18 0 0 0 g f o r 20 min at 4°C and MT was m e a s u r e d in t h e resulting p o s t - m i t o c h o n d r i a l s u p e r n a t a n t s b y the Cd s a t u r a t i o n (Cd-hem) m e t h o d o f O n o s a k a and Cherian [2,6] w i t h o u t modification. In those animals which had also been injected with [3sS] c y s t e i n e , aliquots o f the p o s t - m i t o c h o n d r i a l s u p e r n a t a n t were f u r t h e r c e n t r i f u g e d f o r 1 h at 105 0 0 0 g and t h e resulting s u p e r n a t a n t s were f r a c t i o n a t e d o n S e p h a d e x G-75 c o l u m n s (0.9 × 60 cm) e l u t e d with 10 mM Tris--HC1 (pH 8.6). F r a c t i o n s (1 ml) were c o l l e c t e d and a n a l y z e d for 35S o n a Packard b e t a liquid scintillation c o u n t e r following a p p r o p r i a t e sample p r e p a r a t i o n [7]. T h e p e r c e n t a g e o f the t o t a l r a d i o a c t i v i t y r e c o v e r e d in t h e MT f r a c t i o n was calculated. In o r d e r t o c o m p a r e t h e distribution o f Cd and Zn in the h e p a t i c c y t o s o l s o f rats injected with Cd alone (group 3) and t h o s e injected with b o t h Zn and Cd ( g r o u p 5), the c y t o s o l s ( 1 0 5 0 0 0 g f o r 1 h) were p r e p a r e d f r o m a 30% liver h o m o g e n a t e . A l i q u o t s (0.25 ml) o f t h e c y t o s o l s were applied o n calibrated S e p h a d e x G-75 c o l u m n s (0.9 × 60 cm), and 1 ml fractions were collected. T h e c o n c e n t r a t i o n s o f Cd and Zn in each f r a c t i o n were e s t i m a t e d by flame atomic absorption spectrophotometry. RESULTS A single injection o f Zn (20 mg/kg) or Cd (0.6 mg/kg) resulted in the significant a c c u m u l a t i o n o f metals and i n d u c t i o n o f M T synthesis in rat liver b y 16 h (Table I). No direct c o m p a r i s o n o f t h e hepatic MT levels a f t e r Zn and Cd injections can be m a d e in these e x p e r i m e n t s because o f t h e p o t e n t i a l d i f f e r e n c e s in t h e t u r n o v e r rates o f Zn and Cd used in this study. T h e hepatic Zn levels o f rats injected with b o t h Cd and Zn were increased significantly as c o m p a r e d t o t h e c o n t r o l values. C a d m i u m was d e t e c t e d o n l y in livers o f rats which were injected with c a d m i u m chloride. T h e r e were n o changes in hepatic
TABLE I HEPATIC METAL AND METALLOTHIONEIN CONCENTRATIONS (ug/g WET WT) Group
Zn
Cd
Cu
MT b
4.7 ± 1.2 4.3 *- 0.6 4.2 *_0.2
40 756 417 489 780 1590
1 (control)
28.1 ± 3.4 a
n.d.
2 (Zn, 16 h)
69.9 ± 20.1"
n.d.
3 (Cd, 16 h)
46.0 ± 5.5*
5.3 ± 0.5
4 (Zn, 3 2 h)
62.4 ± 13.1"
n.d.
4.4 ± 0.4
5 (Zn---Cd)
66.2 ± 13.2"
6.5 ± 2.5
4.5 ± 0.4
n.d.
4.3 ± 0.6
6 (Zn--Zn)
119 ± 38*
± 25 ± 110" ~ 57* ± 240* ~ 156" ± 572*
aValues are means +_S.D., n = 3. bMT was measured by Cd-hem method. n.d., not detected (<0.1 ug/g). *Significantly different from control, P < 0.01.
103
TABLE II % OF [3sS]CYSTEINE INCORPORATION INTO MT [35S]cysteine was administered as an i.p. injection (10 uCi/rat) 0.5 h after the last dose o f metal and rats were sacrificed 15.5 h later. The liver cytosol fractions (105 000 g, 1 h) were prepared and fractionated on Sephadex G-75 columns. The ~sS-radioactivity associated with the 10 000 mol. wt fractions ( V e / V o 1.9 to 2 . t ) are expressed as percent of the total radioactivity in the cytosol. The results are mean values of 2 separate experiments. Group Group Group Group
1 (control)
3 (Cd) 5 (Zn--Cd) 6 (Zn--Zn)
6 30 15
26
copper levels after Zn and/or Cd treatments. When a Zn injection was followed by a second Zn injection (group 6), the level of MT was approximately 2-fold that observed after a single Zn injection. The hepatic Zn level also increased after the second Zn injection (69.9 in group 2 vs. 119 in group 6). However, when a Zn injection was followed by a Cd injection, the resulting level of MT was less than additive. In order to compare the new MT synthesis induced by a second injection of Zn or Cd, rats were injected with [3SS]cysteine at about 15.5 h before sacrifice. The fractionation of liver cytosols (105 000 g for 1 h) from control rats showed only a 6% of 3sS-incorporation to MT fraction. A significant increase in MT synthesis after Cd in iection was indicated by the 30% incorporation of [3SS]cysteine into MT fractions. The injection of Cd or Zn to Zn pretreated rats (groups 5 and 6) also resulted in increased incorporation of [3sS] cysteine to MT fractions. However, the new MT synthesis initiated by an injection of Cd in Zn-pretreated rats (group 5), as measured by [35S] cysteine incorporation, was lower than both injection of Cd alone (group 3) and a second injection of Zn in Zn pretreated rats (group 6). When the hepatic levels of Zn and MT in rats of various groups were compared, a direct correlation was observed (Fig. 2). These results demonstrate that regardless of whether Zn, Cd or both were used to induce hepatic MT synthesis, the resulting MT levels were highly correlated with the hepatic Zn concentration. The distribution of Cd in the hepatic cytosols of rats injected with Cd salts alone and those injected with both Zn and Cd salts showed identical patterns (Fig. 3). In both these groups, the hepatic Cd was exclusively bound to a 10 000 molecular weight fraction ( V e / V o ~ 2), identical to the position where a standard sample of MT was eluted out. The pattern of distribution of Zn among protein fractions was also similar in the 2 groups. A significant a m o u n t of Zn was associated with MT fraction in both of the groups.
104
2000
,
r
I000
~
150
Zn (pg/g liver)
Fig. 2. R e l a t i o n s h i p b e t w e e n Z n a n d M T c o n c e n t r a t i o n in t h e liver. P o i n t s are values f r o m all individual rats o f all 6 e x p e r i m e n t a l g r o u p s (as d e p i c t e d in Fig. 1). T h e c o r r e l a t i o n is h i g h l y significant; r = 0 . 9 7 , P < 0.01. Also s b o w n is t h e best-fit linear regression line, e q u a t i o n Y = 1 5 . 9 x - 366.
DISCUSSION
A major finding of the present study is that, in the presence of preexisting hepatic Zn--MT, the effectiveness of Cd to initiate new MT synthesis is much reduced, whereas the ability of Zn to induce further synthesis is unimpaired (Table I). A single injection of either Cd or Zn salt resulted in the synthesis of hepatic MT, but the induction of MT by these metals was not additive when an injection of Zn was followed by an injection of Cd. We suggest that this is probably due to the in vivo displacement of Zn from preexisting Zn-MT by Cd in Zn-pretreated rat liver. Additional support for this interpretation is the finding that [35S] cysteine incorporation into the MT fraction 16 h after Cd injection was reduced by approximately one-half by a pre-injection of Zn, again indicating that, although there is no reduction in Cd uptake by the liver after Zn-pretreatment (Table I), the accumulated Cd does n o t initiate much new MT synthesis. These results in adult rats confirm the data of Panemangalore and Cherian [7] who reported that, in 5-day-old rat pups, the administration of Cd (1 mg/kg s.c.} did not produce any alteration in the hepatic MT or Zn levels, but an injection of Zn (20 mg/kg s.c.) caused a 45% increase in the hepatic MT level. Neonatal rats have naturally high levels of hepatic Zn--MT [7--9], and MT may thus serve as a Zn-donor during rapid postnatal growth and development when the metabolic demands for Zn are high [ 1 0 - 1 2 ]. The presence of high endogenous levels of Zn--MT in neonates is thus analagous to Zn-injected adult rats with respect to MT concentrations and the behaviour of subsequently administered Cd. The second major finding of the present study is that, within 16--32 h of metal administration, hepatic MT concentrations are highly correlated with hepatic Zn concentrations regardless of whether MT is induced by Zn, or Cd,
105
I0
C d - injected
5~;~=~.m~_o" -o-,~o--o-o- ';
,
2
-
-o-o.o,.o.o.~ Zn and Cd injected
-16
2
i
I
.
_
_
~ _
>_~o-o-o-o-o-o( I
15
, 'o. . . . . ¢ . . . . o-~-~-~ 2
25
3
Ve/Vo Fig. 3. Gel filtration of liver cytosols. The cytosol (105 000 g, 1 h) samples were prepared f r o m livers of rats injected witb Cd alone (upper panel) or those injected with Zn and Cd (lower panel) and fractionated on calibrated Sephadex G-75 columns (0.9 x 60 cm). 1 ml fractions were collected and metals (Zn and Cd) were analyzed by a t o m i c absorption spectropbotometry.
or both. The suggestion has previously been made [1,13] that, under a variety of conditions, Zn 2÷ may be the primary inducer of MT at least in certain organs, and that other metals such as Cd 2+, Cu 2÷ and Hg 2÷ may induce the synthesis of MT by virtue of their ability to somehow alter the intracellular Zn pool. Subsequent to MT induction by Zn, these other metals would be transferred to MT from their initial macromolecular binding sites because of their higher affinity for MT. Zn would be displaced from MT in the process.
106
Consistent with this theory is the observation that, when tissue Zn levels are reduced, such as in the pancreas during Zn deficiency, Cd is unable to induce the synthesis of MT [ 1 ]. However, this proposed mechanism cannot be extrapolated to certain metals such as Ag, which will readily displace Zn from MT, but cause only a minor increase in tissue MT content [14]. Similarly, the failure of Cd to induce MT synthesis in the Zn-deficient pancreas [15] may be due to a general failure in Zn-dependent gene expression [12] as a consequence of Zn deficiency. The results of the present study can be interpreted as lending support for a major role of Zn 2÷ in the induction of MT after the administration of Zn and Cd salts. We observed that control rat livers contained a basal level of Zn of about 25--30 pg/g wet wt and a basal level of MT, as measured by the Cdhem method, of about 40 ug/g (Table I). Sixteen hours after a single injection of Zn, Zn levels had increased by approximately 40 ~g/g and were accompanied by a corresponding increase in MT levels of approximately 700 pg/g. Thus, the accumulation of 1 ~g/g of excess Zn in the liver resulted in the de novo synthesis of about 17--18 pg MT/g liver. Similarly, after the administration of a single dose of Cd, the hepatic Zn concentration increased by about 20 pg/g above control levels, corresponding to an increase in MT of about 400 pg/g. Thus, an accumulation of 1 pg/g of excess Zn was accompanied by an increase of about 20 pg MT/g, an almost identical value to that observed after Zn injection. Hepatic Cd concentrations, on the other hand, were only modestly increased, the molar increase in hepatic Zn (after Cd injection) being 8-fold that of Cd itself. It should also be noted that, after Cd injection, the a m o u n t of hepatic MT which is induced, is 5--10-fold that required to completely complex the entire hepatic Cd load (assuming a minimum of 3 and a maximum of 7 g atom Cd/mol MT). If only Cd is involved in the direct induction of MT after Cd injection, it may be questioned how and why such a large excess of MT is synthesized. If, however, the involvement of Zn is also hypothesized, such seeming excesses are better accounted for. Figure 2 further illustrates the highly significant correlation between hepatic MT concentration and hepatic Zn concentration, and demonstrates that, regardless of the mode of induction of MT -- whether by 1 or 2 Zn injections, 1 Cd injection, or a combination of a Zn injection followed by a Cd injection -- an increase of 1 pg Zn/g above basal values results in the synthesis of about 15--20 pg MT/g in the liver. It should also be mentioned that a significant a m o u n t of Zn is also detected in MT, isolated from various organs after injection of Cd [2]. Further studies designed to elucidate the possible role of Zn in the initial induction of MT synthesis in response to the administration of various metals are warranted. ACKNOWLEDGEMENT
This work was supported by grants from Medical Research Council of Canada. The authors wish to thank Mrs. Colleen Alford and Miss Paula Fenner for their help in typing this manuscript.
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