effects of interaction between65zn, cadmium, and copper in rats

�9 1988 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/88/1713-0285502.00

Effects of Interaction Between 65Zn, Cadmium, and Copper in Rats

JADWIGA CHMIELNICKA,* ELZBIETA KOMSTA-,SZUMSKA, AND GRAZYNA ZARI~BA

Department of Toxicological Chemistry, Institute of Environmental Research and Bioanalysis, Medical Academy, Narutowicza 120 a,

90-145 L6d~, Poland

Received November 6, 1986; Accepted April 24, 1987

ABSTRACT

Distribution and retention of zinc in the presence of cadmium and copper was studied in rats exposed repeatedly to these metals.

The experiment was performed on white rats of the Wistar strain. The animals were divided into four groups/five rats each: 1) 65ZnCI2; 2) 65ZNC12 + CdCI2; 3) 65ZnC12 + CuCI2; and 4) control ~sroup. Rats were administered sc every other day for two weeks:

ZnCI2-5 mg Zn/kg; CdCI2-0,3 Cd/kg; and CUC12-2 mg Cu/kg. The zinc content was measured in rat tissues by ~-counting. Effect of Cd and Cu on subcellular distribution of zinc in the kidney and liver and on the level of metallothionein were also examined.

Whole body retention of zinc under the influence of cadmium was lower than that observed in animals treated with zinc alone. However, copper increased twofold the whole body retention of zinc. Cadmium elevated the accumulation of zinc only in the kidneys nuclear fraction and liver soluble fraction.

In the kidneys and liver, copper elevated the accumulation of zinc, in the nuclear, mitochondrial, and soluble fractions. The level of metallothionein-like proteins (MT) in the kidneys after a combined supply of zinc and copper was significantly increased with respect to the group of animals treated with zinc alone.

These results indicated complex interactions between cadmium, copper, and zinc that can affect the metabolism of each of the metals.

*Author to whom all correspondence and reprint requesL~ should be addressed.

Biological Trace Element Research 2 8 5 Vol. 17, 1988

286 Chmielnicka, Komsta-Szumska, and Zarr

Index Entries: Zinc, retention of; copper, cadmium; metallothionein; fractions, nuclear, soluble, and mitochondria].

INTRODUCTION

Zinc status or the tissue levels of zinc may be important factors in the regulation of induced metallothionein synthesis by other metals (1). Cad- mium, in many respects an antimetabolite to zinc, thus could influence zinc metabolism.

The antagonism between cadmium and essential cations in the body may be confounded by interactions between the essential ions them- selves. Variations in copper and zinc intake, for example, could modify the accumulation of cadmium in the body (2,3,4). The effects of zinc on cadmium toxicity thus should depend, to some extent, on whether zinc interacts directly with cadmium, or whether it is acting as an antagonist of copper. In the first case, zinc could exert a pro-active effect, in the second, copper would exacerbate the toxic effects of cadmium.

Interactions between zinc, cadmium, and copper may involve var- ious processes, such as competition for membrane sites, diversion of metal from low mol wt proteins to high ones, or alteration in organ distribution.

At present, the data on the interaction of these metals at the cellular level are still scarce.

This paper aims to elucidate the effects of cadmium or copper on the body retention and organ distribution of 65Zn administered to rats subcutaneously and simultaneously with these metals. In addition, it deals with the effects of these elements on subcellular distribution of zinc in the kidney and liver and on the level of metallothionein.

MATERIALS AND METHODS

The experiments were performed on white rats of the Wistar strain, average body wt 200 g. The animals were divided into four groups of five rats each: 1) 65ZNC12; 2) 65ZNC12 + CdC12; 3) 65ZnC12 + CuCl2; 4) control group. The animals were administered subcutaneously every other day for 2 wk: 65ZnC12, at the dose of 5 mg Zn/kg; CdC12, at the dose 0.3 mg Cd/kg, and CuC12 at the dose 2 mg Cu/kg. The specific activity of 65Zn was 5 ~Ci/dose. The animals were killed by heart puncture under light narcosis 24 h after administration of the last dose. The zinc content was estimated in the following tissues: blood, liver, kidneys, lungs, heart, spleen, skin, tail, bowels, muscle, and bones. The tissues were dissolved in a mixture of hot H2SO4 and HNO3 and measured in a USB-2 scintilla- tion counter (with a NaI/T1 crystal). Liver and kidneys were homoge- nized and fractionated by the method of Shibko et al. (5). The content of

Biological Trace Element Research Vol. 17, 1988

65Zn, Cadmium, and Copper Interactions in Rats 287

metallothionein-like proteins was estimated in the liver and kidneys by a radio-chemical method according to Zelazowski and Piotrowski (6).

RESULTS

As shown in Table 1, the retention of 65Zn in the rat after subcutane- ous injections of zinc chloride did not exceed 20%. The highest accumulation of zinc was found in the spleen, heart, and skin. In the other organs, the levels of zinc ranged from 4.6-10 lag Zn/g tissue. The lowest amounts of 65Zn were found in the blood. Zinc was retained in all the examined subcellular fractions of rat kidneys (Fig. 1) at approximately the same level (ca. 0.5 lag Zn/g tissue); only in the microsomal and soluble fractions did the concentration of zinc reach 1.4 lag Zn/g.

In the liver (Fig. 1), the highest levels of 65Zn were bound with the nuclear (3.3 ~g Zn/g) and soluble fractions (2.9 lag Zn/g). In the other fractions of the liver, the level of zinc did not exceed 0.6 lag Zn/g tissue.

The content of metallothionein (Table 2) in the kidneys of rats exposed to zinc did not increase significantly as compared to control animals, whereas in the liver it increased fourfold.

Simultaneous supply of zinc with cadmium contributed to decrease in the retention of 65Zn (Table 1).

In the kidneys, cadmium elevated the accumulation of 65Zn only in the nuclear and mitochondrial fractions (Fig. 1), but increased it consid- erably in the microsomal fraction. Cadmium did not induce significant alterations in the concentration of zinc in subcellular fractions of the liver, except for an increase in the soluble fraction.

After combined administration of zinc and cadmium, the level of metallothionein (Table 2) in rat kidneys and liver was only slightly elevated with respect to the experimental group given only zinc.

Zinc, administered in combination with copper, brought about an over twofold increase of the whole body retention of 65Zn (Table 1). In- creased content of zinc was found in the kidneys, liver, spleen, lungs, blood, skin, and bowels.

In the kidneys, copper elevated the accumulation of 65Zn, particu- larly in the soluble (fivefold) nuclear, mitochondrial, and lysosomal fractions (Fig. 1).

On the other hand, in the liver the highest increase of the zinc concentration was found in the nuclear, mitochondrial, heavy lysosomal, and soluble fractions.

The level of metallothionein (Table 2) in the kidneys and liver after a combined supply of zinc and copper was found to be significantly different from the control group.

In the group given zinc and copper, a 15-fold increase of the metallothionein concentration was found in the kidneys, which is


288 Chmielnicka, Komsta-Szumska, and Zar~ba

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Biological Trace Element Research VoL 17, 1988

65Zn, Cadmium, and Copper Interactions in Rats 289

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Fig. 1. The distribution of zinc in the homogenate and subcellular fractions of rat kidneys and liver. Rats Exposed to: 1) 65Zn; 2) 65Zn + Cd; 3) 65Zn + Cu. Fraction symbols: N = nuclear; M = mitochondrial; LH = heavy lysosomal; L L = light lysosomal; P = microsomal; and S = soluble.

Biological Trace Element Research VoL 17, 1988

290 Chmielnicka, Komsta-Szumska, and Zar~ba

Table 2 The Level of Metallothionein ~ in Kidneys and Liver

of Control Rats, Exposed Repeatedly to Zinc Chloride ~', Cadmium Chloride", or Copper Chloride ~

Kind of exposure" Kidneys Liver

Control 0.22 _+ 0.05 0.10 + 0.05 Zn 0.27 -+ 0.06 0.42 _+ 0.07 I Zn + Cd 0.29 + 0.04 0.45 _+ 0.07 t Zn + Cu 0.38 -+ 0.06 r 0.40 _+ 0.10 r

"Expressed as metallothionein in mg/g. ~'7 x 5 mg Zrdkg. '7 x 0.3 mg Cd/kg. ~7 x 2 mg Cu/kg. ~Means and range values from five rats. ~Significantly different from control (p < 0.05).

confirmed by a considerable elevation of the concentration metal in the soluble fraction of the kidneys (Fig. 1).

DISCUSSION

The decrease in the retention of 65Zn observed in response to re- peated administrations of cadmium in this study (Table 1) in rats is con- sistent with the result of Magos and Webb (7), although these authors applied zinc and cadmium in single doses. There are no changes in liver and kidney zinc levels after zinc and cadmium treatments. It was con- cluded that the metabolism of endogenous and exogenous zinc in rat liver and kidneys is different (8). However, a decrease in the retention of 65Zn in the muscle and bones in the presence of cadmium, as observed in this study, is in general agreement with our previous work (8) in which the levels of endogenous zinc were followed.

The induction of zinc-thionein in the liver of rats exposed either parenterally or perorally to zinc is well documented (1). Zinc-thionein is induced also in the rat kidneys by injection of zinc; the induced amounts of zinc-thionein in both the liver and kidneys are dependent upon the injected amount (9).

Administration of either 65Zn or 65Zn and cadmium to the rat re- sulted in the synthesis of hepatic metallothionein, but the induction of metallothionein by these metals was not additive when these two metals were injected together (Table 2). Scheuhammer et al. (10) demonstrated also that in the presence of pre-existing hepatic Zn-MT, the ability of cadmium to induce new MT synthesis was greatly reduced. These authors suggested zinc may play a major role in the induction of metallothionein synthesis both after zinc administration and cadmium administration.

At liver zinc concentrations greater than 30 p~g/g tissue hepatic metallothioneins are induced, thus serving as a second line of homeo- static control by sequestering zinc in the liver (11). With time, zinc was


~SZn, Cadmium, and Copper Interactions in Rats 291

depleted from the soluble fraction of the liver, whereas cadmium re- mained fixed. Cadmium does not disappear from metallothionein as it is degraded (12), whereas Zn disappears (13,14). Petering et al. (3) suggest that binding of cadmium to metallothionein occurs with the loss of zinc and copper from the protein, thus apparently reducing the number of binding sites on metallothionein for copper and zinc that are used for its normal unspecified functions in kidneys.

There are clearly complex interactions between the chemically simi- lar metals cadmium, copper, and zinc, and these interactions can significantly affect the metabolism of each of the metals.

Copper in high concentrations induces synthesis Cu-thionein in liver and kidneys (15,16). Interaction between copper and zinc could be partially explained by competition for binding sites on a protein carrier such as albumin in blood plasma. Copper and zinc in blood are mainly associated with proteins, often as cofactors of enzymes.

The suggestion has previously been made (10,17,18) that, under a variety of conditions, Zn -'§ may be the primary inducer of MT and that other metals, such as Cd 2+, Cu 2+, and Hg 2+, may induce the synthesis of MT by virtue of their ability somehow after the intracellular Zn.

Variations in copper and zinc intake may modify the accumulation of cadmium in the body and ultimately the development of cadmium- induced renal dysfunction.

These results, as well as those of our previous studies (19), confirm our hypothesis that interaction processes between cadmium, zinc, and copper are dependent on the extent of interaction between endogenous metals at the subcellular level.

ACKNOWLEDGMENT

This work was performed within the framework of Project CPBR 11.11.81.1. Polish People's Republic "Occupational Medicine."

REFERENCES

1. J. H. R. K~gi and M. Nordberg, MetalIothfonein, Experientia Supplementum 34, Birh~iuser Verlag. Boston. pp. 41-124 (1979).

2. K. Nakamura, E. Suzuki, Y. Sugiura, and T. Takata, Ind. Health 1, 17 (1979). 3. H. Petering, J. Loftsgaavden, J. Schneider, and B. Fowler, Environ. Hlth.

Perspect., 54, 73 (1984). 4. M. P. Waalkes and L. A. Poirier, Toxicol. Appl. Pharmacol. 7, 539 (1984). 5. S. Shibko, P. Koivistoinen, C. A. Tratnyck, A. R. Newhall, and L.

Friedman, Anal. Chem. 19, 514 (1967). 6. A. Zelazowski and J. K. Piotrowski, Acta Biochem. Pol. 24, 97 (1977). 7. L. Magos and M. Webb, Arch. Toxicol. 36, 53 (1976). 8. J. Chmielnicka, E. Komsta-Szumska and G. Zar~ba, Arch. Toxicol. 53, 165

(1983). 9. K. T. Suzuki and M. Yamarnura, Biochem. PharmacoI. 28, 2852 (1979).


292 Chmielnicka, Komsta-Szumska, and Zarr

10. A. M. Scheuhamer, S. Onosaka, K. Rodgers, and M. G. Cherian, Toxicol. 36, 101 (1985).

11. M. Webb and K. Cain, Biochem. Pharmacol. 31, 137 (1982). i2. R. W. Chen, P. D. Whanger, and P. H. Weswig, Biochem. Med. 12, 95 (1975). 13. S. L. Feldman and R. J. Cousins, Biochem. J. 160, 583 (1976). 14. S. H. Oh, J. T. Deagen, P. D. Whanger, and P. H. Weswig, Bioinorg. Chem.

8, 245 (1978). 15. D. R. Winge, R. Premakumar, and K. V. Rajagopalan, Arch. Biochem.

Biophys. 170, 242 (1975). 16. I. Bremner and N. T. Davies, Biochem. J. 149, 733 (1975). 17. F. O. Brady, M. Panemangalore, F. A. Day, A. M. Fiskin, and G. Peterson,

Metallothionein, J. H. R. Kagi and M. Nordberg, eds., Birkh/iuser Verlag. Boston, p. 261 (1979).

18. S. Onosaka and M. G. Cherian, Toxicology 23, 11 (1982). 19. J. Chmielnicka, E. Brze;tnicka, E. Bem, and M. Kasperek. Environ. Res. 37,

419 (1985).

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effects of interaction between65zn, cadmium, and copper in rats

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