Protection by metallothionein against cadmium toxicity

Comp. Biochem. Physiol. Vol. 73C, No. 1, pp. 135 to 139, 1982 Printed in Great Britain

0306-4492/82/050135-05503.00/0 © 1982 Pergamon Press Ltd

PROTECTION BY METALLOTHIONEIN AGAINST CADMIUM TOXICITY HIDEAKI KITO*, TE1JIRO TAZAWAt, YOUKI OSE*, TAKAHIKO SATO* and TETSUYA ISHIKAWA* *Department of Environmental Hygiene, Gifu College of Pharmacy, 6-1, Mitahora-higashi 5 chome, Gifu 502, Japan tHokkaido Institute of Public Health, 12 chome Nishi, 19 Jyo Kita, Kita-ku, Sapporo 060, Japan (Received 16 November 1981) Abstract 1. The protective effect against Cd toxicity of prior exposure to Cd or Zn solutions at low concentration was studied. 2. Carp were bred in tap water (A), 1 ppm Cd solution (B) and 5 ppm Zn solution (C) for 14 days and then transferred into 15 ppm Cd solution. The survival ratio of carp decreased in the order: (C):(B):(A). 3. Binding capacity of Cd to high molecular and metallothionein fractions in the cytoplasmic solutions of the hepato-pancreas was studied and the binding capacity to the metallothionein fraction was stronger than that to the high molecular fraction. The authors recognized that Zn in the metallothionein fraction is substituted by Cd.

INTRODUCTION

It has been reported that m a n y m a m m a l s produced a tolerance against heavy metals by prior administration of small a m o u n t s of these metals and that metallothionein is concerned with this effect (Yoshikawa et al., 1970). T h o u g h it was considered that fish are usually exposed to heavy metals repeatedly through water pollution, there is little information o n the relationship between heavy metals a n d metallothionein (MT) in fish. In a previous paper (Kito et al., 1982), the authors suggested that M T is induced in the carp by breeding in Cd and Z n solutions. In this paper, the protective effect against Cd in fish by prior exposure to low concentrations of Cd or Zn, and the role of M T in this protective effect were investigated.

heavy metals in eluted fractions were analysed with an atomic absorption spectrophotometer. The binding capacity of Cd to H M F and M T F in the hepato-pancreas

Carp were kept in tap water, 1 ppm Cd and 5 ppm Zn solutions for 14 days before being killed; the cytoplasmic solutions were prepared from the dissected hepato-pancreas. One milliliter of Cd solution (in 0.01 M Tris-HC1 buffer, pH 7.4) was added to 3 ml of cytoplasmic solution to provide Cd in amounts of 3.75, 7.5, 18, 75 and 75/~g. Each of these samples was applied to a column of Sephadex G-75, and the metal contents in the eluted fractions were anlysed with an atomic absorption spectrophotometer. RESULTS Effect o f prior exposure on Cd toxicity

METHODS

Results of survival ratio in 15 p p m Cd solution following prior exposure to low concentrations of metal are shown in Fig. 1. This experiment was repeated three times and similar results were obtained. All carp in group (A) died after 15 hr and those in group (B) died after 20 hr. O n e carp in group (C) lived for 26 hr. A mucus-like white substance was observed on the gills of the dead fish. It was obvious that Cd tolerance against high concentration of Cd was improved by prior exposure to low concentrations of metal. Cd a n d Z n content in the ultracentrifuged s u p e r n a t a n t in each organ after pre-exposure followed by 15 ppm Cd exposure are shown in Figs 2 and 3. The Cd concentration in the supernatant of each organ increased after exposure to 15 ppm Cd. In kidney and gill, the Cd concentration in the precipitate in group (B) was less than in groups (A) and (C). Z n concentration in the kidney was higher than in other organs. Changes of the elution pattern of hepato-pancreas, kidney and gill before and after 15 p p m Cd exposure are shown in Figs 4, 5 and 6, respectively.

Carp were obtained from a breeding pond and kept in tap water for more than a week and then used for the experiments. A hundred litre of solution was put in polypropylene tanks and aerated. Effect of prior exposure on Cd toxicity Groups of fifteen carp were maintained in tap water (A), 1 ppm Cd solution (B) and 5 ppm Zn solution (C), respectively, for 14 days and then transferred into 15ppmCd solution for 18 hr. The solutions were changed at 24hr intervals during pre-exposure and 4 h r intervals during 15 ppm Cd exposure. Water temperature was kept at 2OC. After pre-exposure and 15 ppm Cd exposure, three carp from each group were killed. The cytoplasmic solution of each dissected organ was prepared by ultracentrifugation as described in the previous paper (Kito et al., 1982). Aliquots of the cytoplasmic solution were digested by HNO3 H202 and extracted by the DDTC MIBK method (Pharm. Soc. Japan, 1980). This sample was analysed with a Shimadzu AA-610S atomic absorption spectrophotometer. Other aliquots were applied to a column of Sephadex G-75 (1.8 × 46.5 cm), and 135

136

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138

Table 1. Increase of cadmium concentrations in metallothionein (MTF) and high molecular fractions (HMF) after exposure to 15 ppm cadmium solution for 18 hr following prior exposure to tap water, 1 ppm cadmium or 5 ppm zinc solutions for 14 days.

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Protective effect of metallothionein

139

Hepato-pancreas. In group (A), a low Zn peak in M T F was observed after prior exposure, but this peak disappeared after 15 ppm Cd exposure. The low Cd peak observed in M T F after pre-exposure increased after 15 ppm Cd exposure. The Cd content in H M F and M T F were nearly equal after 1 5 p p m C d exposure. In group (B), Zn and Cd peaks were found in M T F after prior exposure. After 15 ppm Cd exposure, the Cd peak in M T F increased, as shown in group (A), but the Zn peak did not disappear unlike group (A). In group (C), a high Zn peak was found in both the M T F and H M F after prior exposure. After 15 ppm Cd exposure, the Cd peak in M T F and the Zn peaks in M T F and H M F all increased. Kidney. In group (A), the Cd peak was low in M T F and H M F after prior exposure and increased in H M F as well as M T F after 15 ppm Cd exposure. In group (B) the Cd peak was found in M T F after prior exposure~ while after 15 ppm Cd exposure the peak increased and the Cd peak in H M F appeared. In group (C) the Zn peak was found in M T F after prior exposure but after the 15 ppm Cd exposure the Zn peak decreased while the Cd peak in M T F increased greatly. Gill. In group (A) a large Cd peak was observed in H M F after 15 ppm Cd exposure. In group (B) the Cd peak was found in M T F after prior exposure and the Cd peak in H M F appeared after 15 ppm Cd exposure. In group (C), the Zn peak was found in M T F after prior exposure. After the 15 p p m C d exposure this peak disappeared and the Cd peak appeared in M T F and HMF. These results suggested that M T was induced by the presence of Zn and thus restrained the Cd toxicity by binding to the Cd. The increase of Cd content in H M F and M T F from prior exposure to 1 5 p p m C d solution is listed in Table 1. In the hepato-pancreas the increase of Cd content in M T F was in the order (C), (B) and (A). In the H M F group (A) showed a higher value than that in the MTF. In the kidney the same tendency was observed. It appears that thionein was induced by prior exposure to a low concentration of Cd or Zn and that Cd was bound to the induced thionein during exposure to the high concentration of Cd. In the gills, increase of Cd in M T F was low in each group, but in group (A) it was high in HMF.

group after prior exposure and the contents of Cd and Zn in M T F were determined. The results are shown in Fig. 8. In all groups, the content of Cd in M T F increased and that of Zn correspondingly decreased. The total contents of Cd and Zn increased only slightly, with the increase of added Cd. Presumably Zn in M T F was replaced by added Cd.

Binding capacity o f Cd to H M F and M T F in the hepatopancreas Cd was added in vitro to the supernatants of the hepato-pancreas after prior exposure. Cd bound to H M F and M T F is shown in Fig. 7. In each group, Cd binding to M T F increased at first, but it saturated easily, and Cd binding to H M F increased. This shows that the affinity of M T F to Cd is stronger than that of H M F . In group (C) the saturation content of Cd bound to M T F was larger than that of the other groups. Presumably the content of M T in group (C) was larger than that in the others and the larger amount of Cd was captured by MT.

REFERENCES

Substitution o f Z n to Cd in M T F in vitro

Cd was added in vitro to the supernatants of each

DISCUSSION Cd has a high affinity for thiol group and its target appears to be thiol rich proteins. MT has many thiol groups and easily binds Cd; it is to be expected that MT binds Cd and plays an important part in detoxication in animals. Leber et al. (1976) reported that tolerance against Cd is produced by prior exposure to Zn in mammals and that this is concerned with MT. In the present experiments, the survival ratio of fish was the lowest in group (A) and the Cd content in H M F was high after prior exposure to only tap water. But following prior exposure to a low concentration of Cd or Zn survival increased and the Cd content in M T F increased. This suggests that M T is concerned with this detoxication effect. Tanaka et al. (1977) suggested that Cd has a higher affinity to M T than to H M F in vitro and MT-bound Zn was replaced by Cd in vivo and in vitro in rats. Leber et al. (1976) reported that the mechanism of the tolerance effect is due to the substitution of Zn by Cd in Zn-thionein in the case of prior exposure to Zn. The present experiment suggested that Cd has a higher affinity to M T F than to H M F in carp and recognized the substitution of Zn to Cd in M T F in vitro. Also, the Zn peak disappeared and the Cd peak increased in M T F due to the 15 p p m C d exposure after prior exposure to tap water, again suggesting that Zn was substituted by Cd in M T F #1 t:ivo. On the basis of these results it is suggested that the detoxication effect of prior exposure is due to the induction of MT and the capture of Cd by MT. Acknowled,qements This work was supported by the Grant in Aid of Scientific Research from the Ministry of Education, Culture and Science.

K1TO H., OSE Y., TAZAWA T., SATO T. & ISHIKAWAT. (1982) Comp. Biochem. Physiol. 73C, 129-134. LEBER A. P. • MIYA T. S. (1976) A mechanism for cadmium- and zinc-induced tolerance to cadmium toxicity: Involvement of metallothionein. Toxicol. appl. Pharmacol. 37, 403~414. Pharmaceutical Society of Japan (1980) Standard Methods of Analysis for Hy,qienie Chemists, p. 7. Kinbara Publication, Tokyo. TANAKA K., ONOSAKA S., DOl M. & OKAHARA K. (1977) Substitution of zinc bound to metallothionein for cadmium in vitro and in vivo. J. hy,q. chem. Japan 23~ 229-234. YOSm~:AWA H. (1970) Preventive effect of pretreatment with low dose of metals on the acute toxicity of metals in mice. Ind. Hlth 8, 184-191.