Studies on metallothionein and cadmium

Studies on Metallothionein

and Cadmium

A review of recent and earlier data on metallothionein is given together with a description of the present author’s studies and a critical evaluation of theories concerning this protein. The present studies were performed on rabbits and mice. Special interest is focused on the role of metallothionein in the transport of cadmium and in the protection against cadmium toxicity. Purification methods. characterization. toxicity, and distribution of metallothionein with special reference to metallothionein having a high content of cadmium are discussed. Cadmium bound to metallothionein is observed to be five times more toxic than cadmium chloride when administered intravenously or subcutaneously in a single dose. The kidney is the target organ for cadmium bound to metallothionein. whereas the liver is the target organ for cadmium chloride.

INTRODUCTION

Cadmium is a very toxic metal by virtue of its cumulative properties and its ability to cause renal damage after long-term low level exposure. This element occurs as a contaminant in air and in food and enters the body by inhalation and ingestion. In human beings the biological half-life for cadmium is extremely longmore than 20 years. More than 50% of the body burden of cadmium is found in the liver and kidneys together. In human beings exposed excessively to cadmium, accumulation takes place in the renal cortex where tubular damage may be elicited. In a normal Swedish person of 50 years of age, the renal cortex contains about 15 pug of Cd/g, whereas the critical concentration in the kidney has been estimated to be around 200 pg of Cd/g wet weight. Cadmium is also present in tissues other than liver and kidney tissue, but concentrations are generally very low. For details on occurrence, metabolism, and toxicity of cadmium the reader is referred to the review by Friberg et (11. (1974). In 1957 Margoshes and Vallee reported that they had found a low molecular weight protein with high cadmium content in equine renal cortex. Kagi and Vallee (1960, 1961) reported more data on the protein, which they named metallothionein. They purified metallothionein from horse kidney and reported a molecular weight of around 10.000 and a high content of cadmium and zinc, 5.9 and 2.2%. respectively. The sulfur content was high (8.5%). Another unique property of metallothionein was high absorbance of uv light at a wavelength of 250 nm and none at a wavelength of 280 nm, which is in contrast to most other proteins. The absorption at 250 nm was explained as dependent on the cadmium mercaptide bond. When metallothionein was dialyzed at a low pH. cadmium was removed and the metal-free protein thionein was obtained, which had no absorption at 250 nm. The absence of absorbance at 280 nm was due to lack of aromatic amino acids as shown by amino acid analysis. There was a high percentage of cysteine. 30%. in the protein, which explained the observed sulfur content of 8.5%. The affinity 381 0013.9351/78/0153-0381$02.00/O Copynphf 411 rghtq

tit 197X by Academic Press. Inc of reproduction an any form retened

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of thionein for cadmium was much larger than for zinc. Cadmium was found to be bound to metallothionein approximately 3000 times more firmly than zinc (Kagi and Vallee, 1961). Piscator (1964) found large amounts of metallothionein in the livers of cadmiumexposed rabbits and postulated that metallothionein synthesis was induced by cadmium exposure and that metallothionein acted as a detoxification agent for cadmium. Metallothionein was also shown to be present in human beings (Pulido rt ~1.. 1966). Metal analyses of metallothionein isolated from the human kidney (Pulido et n/., 1966) revealed that in addition to cadmium (4.2%), zinc (2.6$%). and copper (0.3%). mercury (0.5%) also bound to this protein. The presence of mercury was explained by therapeutic use of mercury diuretics. In 1970 the above-mentioned five publications were the only ones in which metallothionein was discussed. From the limited amount of data available it seemed likely that metallothionein would be involved in the metabolism of zinc and cadmium. During the period when this work has been going on and after its termination many other groups have started work on metallothionein and a larger number of publications and data are now available. References to such articles will be given in this report. The present studies were undertaken to obtain more information on the role of metallothionein in metabolism, transport, and toxicity of cadmium. The first step was to isolate the protein, and the material used for this purpose consisted of livers from rabbits and mice exposed to cadmium. Different procedures for the purification of metallothionein, such as precipitation, gel chromatography, and isoelectric focusing, were tested. Molecular weight was estimated and amino acid analyses were performed. The studies incmded purification and characterization of metallothionein (Nordberg et al., 1972, 1975b), and studies of the distribution fate, and metabolism of cadmium bound to the protein (Nordberg et nl., 1975a: Nordberg and Nordberg, 1975d). Studies on the toxicity and distribution of metallothionein were performed as well (Nordberg et L/I., 1975a; Nordberg and Nordberg, 1975d). The distribution studies were performed on mice with both rabbit liver and mouse liver metallothionein. Some studies were also made on blood on the cadmium-binding protein found in the red cells and which probably is metallothionein (Nordberg et nl., 1971). On the basis of the results obtained from studies published earlier and additional studies to be described in this summarizing report, the possibility that metallothionein might serve as a part of the transport mechanism for cadmium was examined and that the release of cadmium from metallothionein in the kidney might be the cause of the renal tubular dysfunction. ISOLATION AND CHARACTERIZATION

OF METALLOTHIONEIN

Methds

Methods used to isolate metallothionein included precipitation procedures, ultracentrifugation, chromatography on different Sephadex gels such as G-75 and G-50, and isoelectric focusing. Metal analyses were performed using atomic absorption spectrophotometry (Perkin-Elmer 403 with deuterium correction). For radioactivity measurements

S’l‘UDlES

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383

of rO”Cd, a Packard gamma scintillation system with a NaI crystal was used. For protein separation a column packed with Sephadex gel connected to a fraction collector and a uv cord (254 nm) with a recorder were used. Separation was carried out with buffer applied to the column with a Perpex pump. Spectrophotometry was performed with a double beam spectrophotometer connected to a recorder. The influence of some procedures used in the preparation of metallothionein was studied before the methods used in Nordberg et ~1. (1972, 3975b) were chosen. Since these data have not been published elsewhere, they will be presented in detail here. Livers from rabbits exposed subcutaneously to cadmium chloride were homogenized in Tris buffer (0.01 M Tris in 0.05 M NaCl, pH 8.0). The homogenate was centrifuged on a Spinco L-50 centrifuge at 40.000 rpm (Ih) at +6”C. The supernatant was removed and diluted to 30 ml. Five milliliters was taken for cadmium and zinc analyses. Five milliliters was stored at -24°C for 12 days. Sephadex G-75 chromatography was performed on each of the samples after 0, 1, 7. and 11 days’ storage at +4”C. The distribution of cadmium in the supernatants after 0 and I days of storage was almost identical. Supernatants stored 7 and I I days showed a shift of cadmium binding away from low molecular weight proteins to high molecular weight proteins. This effect of storage was quite clear even in supernatants stored at -24°C. Another study was performed in the same way but with the following time intervals: 0, I. 2. 3. 4, and 7 days’ storage of the supernatant at +4”C. One sample was frozen drop-wise in liquid nitrogen and stored at -65°C for 9 days. Storage at this low temperature allowed the distribution pattern of cadmium to maintain itself regardless of storage time in contrast to storage at +4”C. The addition of mercaptoethanol to the stored supernatants also prevented storage effects. On the basis of these findings metallothionein sofutions were routinely frozen in liquid nitrogen and stored at -65°C. According to Kagi and Vallee (1960. 1961). different fractions of metallothionein were obtained after ion exchange chromatography on DEAE. Isoelectric focusing was chosen as a further separation step for metallothionein. It was found that isoelectric focusing did not work out well at the beginning when the protein sample was applied to the column together with the gradient. Some metal was then detected in all fractions. Precipitation of protein was observed when too high a voltage was applied, thus giving more than I W of effect. When an appropriate voltage was used and the protein sample was applied in the part of a prepared column, where pH was estimated to be 7 to 8. the results were good. TO allow preparation of larger amounts of metallothionein, precipitation procedures in combination with gel filtration were chosen as partly described by Piscator (1964). More detailed information on methods and equipment is given in Nordberg et 171. (1972, 1975b). Rcrbbit Li\,er- Metrrllotl~ionrit~ The study by Nordberg et al. (1972) was performed to purify rabbit liver metallothionein for further studies on metabolism and toxicity of metallothionein. Since the subsequent studies demanded a reasonably large amount of starting material, the precipitation method mentioned above was chosen. Homogenization of livers from rabbits exposed subcutaneously to a total of 10 mg Cd/kg

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body weight was followed by precipitation steps with rivanol and potassium bromide, ethanol, and chloroform. After precipitation and centrifugation procedures the supernatant was taken for gel chromatography on Sephadex G-75. The protein solution thus obtained had a protein concentration of 15.8 mgiml (biuret method). The E2.i,,,28,1ratio was 18 and the contents of cadmium and zinc were 583 pg/ml and 218 pgiml, respectively. Metallothionein, 170 mg. was thus obtained from two rabbit livers with mean cadmium concentration of 176 pgig wet weight and a mean zinc concentration of 119 pgig wet weight. Calculations of protein amount were based on results from amino acid analyses and the biuret method. The molecular weight was estimated to 9000 to 10,000 on a well-calibrated Sephadex G-75 column. This protein solution was also used for other metabolism studies (Nordberg et al., 1975a: Nordberg, 1972) as well as for further purification studies. The protein solution, named metallothionein (Nordberg et al.. 1972), was further separated into different forms by isoelectric focusing. One major fraction, which had a low pZ (3.9 at +4”C) and a high content of cadmium, was designated form I. A second fraction, which had a pZ of 4.5 (at +4”C) and contained an equimolar ratio of cadmium and zinc, was called form II. Amino acid analyses of the two forms showed similar content of most amino acids in terms of the percentage of residues. For cysteine this figure was 28%. No aromatic amino acid was present. The molecular weights of the two forms as based on amino acid analyses were calculated at 5600 and 6600, respectively. Addition of approximately 10% cadmium or cadmium and zinc content would increase the molecular weight by 600. Mouse Li\‘er Metallothione~n

In the study by Nordberg et ~1. (1975b), livers from inbred CBA mice were used to prepare metallothionein. The animals were orally exposed to cadmium for 18 weeks at increasing doses from 10 to 400 ppm of Cd and then twice subcutaneously at 0.7 mg Cd/kg body weight and 2.1 mg Cd/kg body weight. For preparation of metallothionein out of these relatively small samples, homogenization was immediately followed by ultracentrifugation at 105,100 g,,. The supernatant was directly applied to a column packed with Sephadex G-75 gel. Storage of homogenates and supernatants was thus avoided. Metallothionein was eluted using this procedure at the same volume as at least one more low molecular weight protein which had an absorbance at 280 nm, meaning that further separation was necessary. For this purpose, the material was rechromatographed on a column packed with Sephadex G-50, which has a somewhat different and more favorable separation characteristic for low molecular weight proteins, 1500 to 30,000. The protein solution thus obtained had an Een,,,.‘ROratio of 8. a CdiZn ratio of 4.5, and a metal content of 2.3%. The fact that the J?~~,,,.‘~~ratio turned out to be about half the earlier reported ratio for rabbit (Nordberg et al., 1972) backs the supposition that this protein solution contained other low molecular weight proteins as well as metallothionein. Recoveries from Sephadex separations were 90 to 1 lo%, calculated from the cadmium content. The material thus obtained (Nordberg et LZ~., 1975b) was subjected to isoelectric focusing. The results differed compared to those obtained for rabbit liver metallo-

STVDIES

ON

MFI-ALL,OTHIONElN

AND

385

CADMIUM

Fract. -

no.

mCd/g KJznlg FII;.

I. Ion

exchange

chromatography

of mouse

hepatic

metallothionein.

thionein. Metallothionein containing almost exclusively cadmium but traces of zinc had a pZ of 4.2 at +8”C. One metal-containing protein peak was also detected in the pH interval of 6 to 7.2 but the gradient in this region was not especially suitable for resolving these different pl’s. Other runs with better resolution were made with the same material, whereupon two main peaks were observed in this pH interval. One of these had a pZ of 5.7. which was earlier reported to be the approximate pZ for hepatic mouse metallothionein Nordberg et NI., 1971). The metallothionein with a pt of 4.2 was used for amino acid analyses. A very high cysteine content, i.e., 34.6% residues, was detected in this form. No aromatic amino acids were present. After this study a new experiment was set up to try to isolate a possible second form of metallothionein in mouse liver. CBA mice were exposed to cadmium (0.5 mg Cd/kg) by subcutaneous injections 3 days/week for 2.5 months. Ion exchange chromatography was performed on the mouse liver metallothionein which was purified on Sephadex G-50. The column was packed with DEAE-Sephadex A-25 and initially 50 ml of 0.02 M Tris and 0.005 M HCl (pH 8.6) were applied; after this 180 ml of linear gradient was obtained by mixing with the limit buffer;

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then 70 ml of limit buffer (0.2 M Tris, 0.05 M HCI, pH 8.6) was passed through the column. The elution rate was 14 mlihr and fractions of 5 ml were collected. Fractions were collected for metal (cadmium and zinc) analysis (Fig. 1) and absorption readings at 250 and 280 nm. One initial minor and two main fractions were obtained. The fraction that eluted last contained hardly any zinc but much cadmium and the second fraction that eluted contained zinc and cadmium. When the same material was subjected to preliminary studies by isoelectric focusing, it was found that a major part of the material that contained cadmium had an isoelectric point of around 4. The results from ion exchange chromatography were in accordance with these preliminary results from isoelectric focusing, i.e., the metallothionein with the highest cadmium/zinc ratio had a low pZ and thus eluted as the last protein fraction on DEAESephadex. Further comparison and identification are warranted for the different forms obtained from isoelectric focusing and ion exchange. The metallothionein taken for this study was not particularly suitable for studies of the form of metallothionein containing mainly zinc, as it contained much cadmium but only a little zinc. The animals used for metallothionein preparation in this study had been exposed to cadmium by subcutaneous injections, while Nordberg et ~1. (1975b) used animals that were orally and subcutaneously exposed to cadmium. It is possible that the variation in combination with the route for exposure and time influences the relative occurrence of the zinc and cadmium forms of metallothionein, respectively. Discussion arld Conclusions on the Zsolation and Characterization of Metallothionein from Rabbit and Mouse Li\>er As mentioned previously, more and more information dealing with research on metallothionein is being published. Different approaches have been applied to subject areas such as characterization of metallothionein (F’ulido et al.. 1966: Btihler and Kagi, 1974; Kagi et al., 1974; Kojima et al., 1976) and biosynthesis and metal binding of metallothionein (Lucis et al., 1970: Squibb and Cousins, 1974: Wisniewska-Knypl et al., 1970; Winge et al., 1975a; Weser et al., 1973). These groups of researchers have used as starting materials the tissues from humans, horses. and rats with either environmentally or artificially accumulated cadmium concentrations. Metallothionein or metallothionein-like protein has also been isolated from several other species, e.g., goldfish (Marafante, 1976). mussels (Noel-Lambot, 1976). marine vertebrates (Olafson and Thompson, 1974), and chickens (Weser et al., 1973). Although different methods for preparation of metallothionein are now available, some difficulties have been observed in the present author’s and other authors’ reports. There are some problems of purification and identification of metallothionein from different species. For rabbit liver, only gel chromatography with Sephadex G-75 yielded a fairly pure metallothionein (Nordberg et al., 1972). For mouse liver, one further separation on Sephadex G-50 was necessary to separate metallothionein from at least one contaminating low molecular weight protein (Nordberg et al., 1975b). This contaminating protein had the same absorbance at 250 nm as at 280 nm and did not contain any metal. It was not further characterized.

STLlDIES

OS

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AND

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387

Another problem is interlaboratory comparison of different forms of metallothionein. Several investigators have used various methods for the preparation of the different forms of metallothionein. Fulido ef ~1. (1966) originally used column electrophoresis and obtained an incomplete separation of their metallothionein into two or three forms. Ion exchange chromatography has been used by a number of authors (Shaikh and Lucis. 1971; Winge and Rajagopalan. 1972: Winge et nl.. 1975a; Kagi et LI/. , 1974). Btihler and Kagi (1974), who used ion exchange chromatography on DEAE-A25. separated two distinct forms of metallothionein from human liver. Isoelectric focusing has been used for the preparation of different forms, mainly by this author. Cherian (1974) also used this method for separation of two forms of metallothionein from rat liver. No detailed characterization of the two forms was attempted by Cherian. The isoelectric points of his two forms were 4.2 and 4.7 and did agree well with this author’s results from isoelectric focusing of rabbit and mouse liver (Nordberg er (11.. 1972, 1975b). Results based on ion exchange chromatography are difficult to compare with results based on isoelectric focusing (Nordberg et (11.~1972: Nordberg et crl., 1975b). due to the differences in the basic principles of these two techniques. Results based on analysis of amino acid composition are more appropriate for comparison and will be the subject of the following discussion. Descriptions of the amino acid composition of liver metallothionein in various species have not revealed any major differences. There are some clear minor differences. however. Valine seems to be absent from liver metallothioneins from rabbit (Nordberg et ~1.. 1972) but to be present in those from horse (Kagi et t/l.. 1974: Kojima et (II., 1976), rat (Winge rt L/I., 1975a: Bremner and Davies, 1975: Weser et (I/., 1973), chicken (Weser et al.. 1973), mouse (Nordberg, rt crl., 1975b). and human beings (Btihler and Kagi. 1974). Arginine is. on the contrary, only present in liver metallothionein from horse. Estimation of the molecular weight of metallothionein also poses a problem. Methods used include gel filtration, sedimentation equilibrium, and calculation from amino acid analyses. Theoretically gel filtration is related not to molecular weight but to molecular size (Stokes radius). However. for practical reasons, it is convenient to express molecular size in terms of molecular weight. This practice is only useful when marker proteins are used which have the same shape as the tested protein. Because different markers have been chosen for determining molecular weights, inconsistency has arisen. In addition to the data on molecular weight of metallothionein which were mentioned in the introduction. Kagi (1970) reported a molecular weight of 6600 from amino acid analyses. Kagi et ~1. (1974) verified by sedimentation equilibrium the molecular weight for metallothionein to be 6600. On the basis of studies on a calibrated Sephadex G-50 column. Weser et (11. (1973) calculated a molecular weight of approximately 19.000 for hepatic metallothionein from chicken and rat. Winge ct trl. (1975b) determined a molecular weight of 10.000 by means of sedimentation equilibruim for rat liver metallothionein. This value was in accordance with calculations derived from amino acid analyses by the same authors. On the other hand, amino acid analyses on mouse and rabbit hepatic metallothioneins gave a much lower molecular weight than did estimations based on gel filtration (Sephadex

388

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G-75) (Nordberg et al., 1972, Nordberg et al., 1975b). This would be expected for metallothionein, since it has probably a shape different from that of the marker proteins. Storage and oxidizing processes may increase the molecular weight of metallothionein, which may cause erroneous results if gel chromatography is performed. Calculation of molecular weight by amino acid analyses with correction for peptide bonds and addition of estimated metal content is considered by this author to be a reliable and precise method. Kojima et rd. (1976) determined the amino acid sequence of equine renal cortex metallothionein-IB. They then calculated the chain weight at a little more than 6000 and estimated the molecular weight with the metal added to be approximately 6700. These estimations were in good accordance with minimum molecular weights found for rabbit liver metallothionein forms I and II (Nordberg et al., 1972) and for mouse liver metallothionein form I (Nordberg et cd.. 1975b). Mention should also be made of studies on the binding of metals other than cadmium to metallothionein or metallothionein-like proteins. Metallothionein with zinc as the main metal. zinc-thionein, has been isolated and characterized by amino acid analyses from different species such as humans (Buhler and Kagi. 1974), horses (Kagi et al., 1974), and rats (Bremner and Davies. 1975). Other reports indicate that zinc exposure as well as starvation would induce metallothionein synthesis in the liver (Webb. 1972; Chen et al., 1974: Bremner et 01.. 1973). Evans rr al. (1975) studied copper binding proteins from neonatal and adult rat liver. By amino acid analyses the neonatal protein was shown to be very similar to metallothionein. which was in accordance with data taken from Porter (1974). The adult hepatic rat protein with a high content of copper was similar to copper chelatin, described by Winge et 01. (1975b). There are publications indicating that metallothionein in the kidney but not in the liver would be induced by mercury (Wisniewska-Knypl et N/., 1970; Nordberg et rrl., 1974: Piotrowski et ~1.. 1974: Cherian and Clarkson, 1976). On the other hand Winge et ~11.(1975a) reported that exposure to mercury induced metallothionein in the liver with approximately the same amino acid composition as zincthionein and cadmium-zinc-thionein. It is evident that metallothionein might play an important role in the metabolism of some essential and nonessential metals.

Espr~imental

TOXICITY Sidirs

OF RABBIT

LIVER METALLOTHIONEIN

The comparative toxicity of cadmium injected bound to rabbit liver metallothionein versus cadmium injected as cadmium chloride was studied in mice (Nordberg et ~1.. 1975a). The effects of varying the dose and time of observation were also studied. Injections were either intravenous or subcutaneous. No differences in toxicity between intravenous and subcutaneous injections of cadmium bound to metallothionein were evident with the doses and survival times employed. For the subcutaneous injections two solutions were used. one containing cadmium- and zinc -metallothionein (Nordberg ef ~1.. 1972) and the other containing cadmium chloride and zinc chloride with the same molar ratio of cadmium and zinc as in the metallothionein (Nordberg PT crl., 1972). The doses were 6. 2.5. 1.5,

STUDIES

ON

~ME.T.~l,I.OTHIONEIS

ANI)

(:.I\DMItIhf

389

1.3, and I. 1 mg of Cd/kg body weight and the animals were observed for various times up to 30 days. In these dosage groups. none of the animals injected with cadmium chloride died during the time interval studied. All animals injected with the two higher doses of cadmium bound to metallothionein (6 and 2.5 mg of Cd/kg) died within 3.5 days. Two of the five animals in the 1.5 mg of Cd/kg dose group died within 4 days from uremia as verified by measurements of urinary creatinine. This was true as well for animals given I .3 and I. 1 mg of Cd/kg body weight in which death occurred within 7 days after injection. No metal analyses were performed on the liver and kidneys. On the basis of the results obtained from the mortality experiment, a further study was designed in which lower doses of metallothionein were used. Different combinations of the solutions were injected subcutaneously at different places on the backs of the animals. Injected animals were observed for I2 to 14 days and thereafter killed by cervical dislocation under slight ether anesthesia. Kidney and liver tissues were then taken for cadmium and zinc analyses and for histological examination. The animals given the highest subcutaneous dose of cadmium bound to metallothionein. i.e.. 0.6 mg of MT-Cd as cadmium + 0.5 mg of Cd, had 8.3 pg of Cd/g wet weight (mean) in the kidney. The corresponding mean figure for animals given 0.3 mg of MT-Cd as cadmium + 1.O mg of Cd was 1 I .5 pg. Animals exposed to combinations of cadmium and cadmium-zinc solutions had kidney values of about 4 pg of Cdlg wet weight. Histological examination showed dose-dependent renal damage in the animals injected with cadmium bound to metallothionein. Discusion

~~tul Conc~lrrsions

on tllc To.ri1,it.v c!f’ Rrlhhit Lil-cJr M~Jtrrllc)tlliot~l~in (Nordberg et rrl., 1972) was shown to have a protective

Rabbit metallothionein role against testicular damage caused by cadmium (Nordberg, 1971). In the same experiment. he as well pointed out that part of injected metallothionein was distributed to the kidney and could cause renal damage. No other data were available in the literature on the toxicity of cadmium bound to metallothionein at the time these studies (Nordberg ct NI., 1975a) were published. Observations by Nordberg rt al. (1975a) indicated that cadmium bound to metallothionein would be about five times more toxic than cadmium chloride. Cadmium, 1.1 mgikg body weight. bound to metallothionein killed mice within 7 days, while cadmium chloride could be given in larger doses (2.5 and 6 mgikg) without killing any mice within 30 and 5 days, respectively. Animals exposed to cadmium bound to metallothionein had prominent kidney damage while animals exposed to cadmium chloride showed no damage. Smaller doses did not kill the animals within 2 weeks. Renal damage was observed at renal cadmium concentrations around 10 pgig wet weight. Webb and Etienne (1977) reported LD,,, values of 0.28 and 0.32 mg of cadmium. respectively, bound to metallothionein/kg body weight in rats given intravenous injections of hepatic metallothionein which had been induced by cadmium in rats and rabbits. The injected rats were not pretreated with cadmium or zinc and therefore could not have synthesized their own metallothionein to any significant degree. Kidney damage was shown by histological examination. Cadmium concentrations in kidney were reported to be 10 to 15 pg of Cd/g wet weight, which seems

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to be the critical concentration after a single injection of metallothionein in rats and mice, as well as in rabbits (Nordberg and Nordberg, 1975~; Fowler and Nordberg, 1978). Their results support data obtained by Nordberg et al. (1975a). Whether cadmium or thionein constitutes the toxic part of metallothionein has been further discussed by Webb and Etienne (1977). These authors showed that zinc-induced thionein was nontoxic at a level of 2.4 mg of zinc bound to metallothionein. This backs the suggestions by Nordberg et al. (1975a) that the renal damage which takes place after injection of a toxic dose of cadmium metallothionein is due to release of cadmium and not to the thionein part of metallothionein. Data by Cherian and Shaikh (1975) and Nordberg and Nordberg ( 1975~) demonstrated that only part of the cadmium in the kidney was bound to metallothionein a short time (minutes to some hours) after injection of cadmium metallothionein. This further supported the theory that released cadmium is responsible for the kidney damage. Since the animals in our studies and those by Webb and Etienne (1977) synthesized practically no metallothionein of their own. a time lag probably existed before metallothionein became available to bind the “free” cadmium in the kidney. Such an initial deficiency in binding renal cadmium to metallothionein was further indicated by the studies to be described later in this report. As stated in the introduction, a normal Swedish person exposed to cadmium and not having any kidney damage has a renal concentration of 15 pg of Cd/g wet weight. This is about the same concentration that caused renal damage in mice and rats when cadmium bound to metallothionein was injected. In man, a continuous exposure to small amounts of cadmium mainly takes place via food, which causes a continuous synthesis of metallothionein. Thus, there is normally no release of “free” cadmium. When exposure to cadmium is excessive it might well be that the capacity of metallothionein in the kidney is not sufficient to bind all cadmium, whereupon cadmium is free to cause renal damage.

Repeated

PROTEIN BINDING Exposure to Cadmium

OF CADMIUM

IN BLOOD

The binding pattern of cadmium in the blood was studied under conditions of repeated exposure in mice. The animals were given subcutaneous injections of cadmium (0.25 or 0.50 mg of Cd/kg body weight) as radioactive cadmium chloride for 5 days/week for half a year (Nordberg et N/.. 1971). Whole blood was withdrawn by heart puncture. Samples of plasma and blood cell hemolysate were separated on Sephadex G-75. The distribution pattern of cadmium in a hemolysate from a mouse in the 0.5 mg of Cd/kg body weight group showed that 64% of the total cadmium was recovered in fractions corresponding to the elution volume of low molecular weight proteins, which could well be metallothionein. Only 17% of the total cadmium was recovered in hemoglobin-containing fractions. Figures for mice given 0.25 mg/kg body weight were 30% in “metallothionein” fractions and 40% in hemoglobincontaining fractions. A considerable part of erythrocyte cadmium was therefore suggested to be bound to metallothionein. Plasma from the mice described above was also subjected to protein separation

SI‘“D,F,S

ON

ME?‘i\LLOTHIONEIN

AND

CrZDMIUM

391

on Sephadex G-75 under the same conditions as above. The small amount of cadmium in plasma was noted to be bound mostly to proteins with a molecular weight corresponding to albumin. A minor portion of the cadmium was bound to a substance having the same molecular weight as metallothionein. The amount of protein suspected to be metallothionein on the basis of Sephadex G-75 chromatography was considered to be too small for amino acid analyses. The substance to which cadmium was bound in plasma thus could not be identified for certain. Single Exposwe

to Crrdmilrm

A new experiment was designed for further study of cadmium-binding proteins in the blood. Twenty-five mice were divided into five groups and given a single dose of radioactive (rO”Cd) cadmium chloride (1.0 mg of Cd/kg body weight. 100 $X/mouse). They were killed after different time intervals: 20 min (n = 4), 4 (II = 4). 24 (n = 4). 48 (n = 4). 96 (n = 4), and 192 (II = 5) hr. Whole blood was withdrawn from the eye vessels with heparinized capillaries, was weighed, counted for radioactivity. and separated into plasma and erythrocytes by centrifugation. A few plasma samples (one at each of 4. 24. and 96 hr) with hemolysis were totally excluded from the study. Radioactivity was counted in plasma and erythrocytes. Blood cells were washed by adding 0.9% saline, the radioactivity was counted. and the cells were hemolyzed by adding deionized water and frozen at -60°C. The cadmium concentrations of erythrocytes and plasma are seen in Fig. 2. A rapid clearance of cadmium from the plasma along with an increase of cadmium in the erythrocytes is observed between 4 and 48 hr. To find out the distribution among proteins in erythrocytes and plasma. gel chromatography on Sephadex G75 was performed. Radioactivity was detected in the major plasma protein fractions 20 min and 4 hr after injection. At 24, 48, 96, and 192 hr part of the plasma cadmium was found in the major plasma protein fractions and part in fractions with the same elution volume as metallothionein. Figure 3 shows distribution patterns from plasma obtained 20 min, 96. and 192 hr after injection. Figure 4 shows the distribution pattern of cadmium after gel chromatography of hemolysates from animals killed 96 and 192 hr after injection. Both high and low molecular weight fractions contained cadmium. However, cadmium was not found to be bound particularly to fractions containing hemoglobin. Low molecular weight fractions (22-25) contained a major part of the cadmium and the metal was thus likely to be bound to metallothionein. These fractions (metallothionein) were pooled and concentrated on an Amicon cell with a UM-2 filter. The protein solution obtained was taken for another study which is described in the section dealing with distribution of metallothionein. The protein solution from blood cell hemolysate was further characterized by isoelectric focusing as described in principle by Nordberg et rrl. ( 1975b), whereupon cadmium was discovered to be bound to the protein appearing at a pH slightly below 6. This indicated that cadmium in the blood might be bound to a form of metallothionein that contained zinc as well. Zinc analyses performed by atomic absorption spectrophotometry showed that the fractions obtained by isoelectric focusing of cadmium binding protein from hemolysates did indeed contain zinc.

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X’lONI(:A

NORDBERG

ngCdlg

1

I

4 24 -Concentrattan 0---Oconcentratlon

96

48 ,n blaod cells I” pLwna

I

Standard

error

192 tb”S

FIG. 2. Concentrations of cadmium in plasma and blood cells. respectively in mice given a single subcutaneous injection of “‘TdCI, (I mg of Cd/kg body weight) and killed at various times after injection. Vertical bars indicate the standard error and the circles indicate mean values.

Discussiotl ntld Conclusions otl Pwtcin Birlditlg oj‘ Cndmium in Blood The distribution of cadmium in blood after repeated exposure to cadmium has previously been studied by Carlson and Friberg (1957). They reported that cadmium was mainly in the erythrocytes. at least partly bound to hemoglobin. Their results were based on column electrophoresis studies of the hemolysate in starch. The data presented by Nordberg rt (11. (1971) showed that cadmium was chiefly bound to a low molecular weight protein in the erythrocytes after repeated exposure to the metal. This protein was clearly distinguishable from hemoglobin where less cadmium was found. Only a minor portion of the total blood cadmium was detectable in plasma. where part of it was bound to low molecular weight proteins, probably metallothionein. The content and distribution of cadmium in blood after a single exposure to

STLIDIES

ON

ME’I‘AI.LOT~~IONEIN

AND

393

(:ADMICM

0.D

A

B 250

150 100

1

L \

p. 500 i, 200

,\; 5

10

‘I

,J, 15

20

i

‘,,L

r

25

30

Fraction “p

ID.

C 300

0,4

250

0,3

200

o-2

150 100

41 I r 5

10

15

20

25

30 Fraction

0 np

Frc.. 3. Gel chromatography on Sephadex G-75 of plasma from mice given a single subcutaneous injection of 109CdCI, (I mg of Cd/kg). Column dimensions were 355 x 26 mm. Elution with 0.01 M Tris buffer in 0.05 M NaCl (pH 8.0) at a flow rate of 14 mlihr. Volume of fractions: 5 ml. OD at 254 nm was continuously monitored. (A) 20 min after injection: 20.000 cpm = 45.4 ng of Cd; (B) 96 hr after injection: 100 cpm = 0.028 ng of Cd: (C) 192 hr after injection: 100 cpm = 0.028 ng of Cd.

394

MONICA

NORDBERG

cadmium have been studied by Nordberg (1972), who reported details about the cadmium distribution in blood cells but not in plasma. The results summarized here were in agreement with those results. An increase of cadmium in the erythrocytes was thus observed 24 hr after injection. As in the previous studies by Nordberg (1972), cadmium in erythrocytes was found to be bound to a low molecular weight protein at 24 hr or more after injection. Webb and Verschoyle (1976) found that the amount of cadmium bound in plasma increased with increasing doses of cadmium. Plasma levels of cadmium were shown to be 0.27 to 1.21 rig/g 48 hr after a single intravenous injection of 0.5 to 2 mg of Cd/kg body weight. These authors did not study the distribution of cadmium among plasma proteins. By using cadmium with a high specific radioactivity, the distribution of cadmium in plasma was followed by gel chromatography in the present studies. At 24 hr or longer after the single injection, a considerable proportion of plasma cadmium was bound to low molecular weight protein. The amounts of protein involved in cadmium binding in erythrocytes and plasma of mice were too small to allow further identification by amino acid analyses. Even though it has not been possible to identify the low molecular weight cadmium-binding protein in blood. it is likely that it is synonymous with metallothionein. The observed increase of cadmium in erythrocytes 24 hr after injection and the appearance of cadmium in a low molecular weight protein are probably the result of the increase in synthesis of metallothionein in the liver. It is reasonable to assume that cadmium is transported in the blood partly bound to metallothionein. The low molecular weight protein to which cadmium is bound is easily filtered through the glomerular membrane and subsequently reabsorbed in the tubules, which could be the explanation for cadmium accumulation in the kidney. The amount of cadmium bound to low molecular weight protein in plasma is directly available for such transport. The possibility that cadmium bound to other proteins can contribute to the renal accumulation has not yet been studied. DISTRIBUTION OF CADMIUM 1~ vitro Lnbeting of M~t~tllothionein

BOUND

TO METALLOTHIONEIN

Nonradioactive and radioactive protein solutions were employed along with atomic absorption spectrophotometry, whole body measurements, and whole body autoradiography to study the distribution and fate of cadmium bound to metallothionein after injection into mice. As whole body autoradiography was to be performed. the labeled metallothionein had to have a high radioactivity. It was considered of importance to use wellcharacterized and purified metallothionein, which was available as nonradioactive material. Since it might be difficult to maintain this high purity in an in Go-labeled substance, labeling in vitro seemed to be preferable. logCadmium, the only radioactive Cd-isotope available in carrier-free form, was used for the in LWl labeling. The use of carrier-free cadmium meant that mainly radioactivity and only minor amounts of cadmium were added to the metallothionein in the labeling process. During the in vitro labeling, it was noted that an excess of cadmium (i.e.. more cadmium was added to the metallothionein solution

STUDIES

ON

MMETALLO?‘HIONEIN

AND

CADMIUM

395

than could be bound to the protein) brought about changes in some of the characteristics of metallothionein, e.g., on Sephadex (G-75) chromatography it was found that a peak appeared with a higher VJV,, ratio than is typical for metallothionein. Since SH-groups of cysteine in metallothionein are bound to cadmium and this bond is known to have a specific high absorption shoulder at 250 nm, the spectrum of the labeled metallothionein was used to provide information as to the saturation level. By carefully adding cadmium until no further increase in absorbance at 250 nm was observed, the above mentioned changes could be avoided. Distribution

c?fInjected Rabbit Lh-rr Mrtallothionein

in Mice

In connection with the toxicity study (Nordberg et (11.. 1975a), which was described earlier in this report, data on the distribution of hepatic rabbit metallothionein were obtained. As a rule, mice that had received cadmium and zinc along with rabbit liver metallothionein (Nordberg ct NI., 1972) showed higher cadmium concentrations in the kidney than in the liver. In contrast, mice given cadmium and zinc in the absence of metallothionein had the highest concentrations of cadmium in the liver. It must be kept in mind that these studies were performed with heterologous metallothionein, i.e., rabbit liver metallothionein injected into mice. In an attempt to gain support for the postulations that the accumulation of cadmium in the kidney and the kidney damage seen histologically in the cadmium and metallothionein-exposed mice were truly effects of metallothionein, studies with speciesspecific protein were also performed.

The work on species-specific protein consisted of two studies on in \,itr-o-labeled protein presented by Nordberg and Nordberg (1975b). One study employed partially purified metallothionein-like cadmium binding protein (CdBP) and the other study used only one highly purified and well-characterized form of metallothionein (CdMTl). Studies on ‘09C’dBP. In the first study CdBP from mouse liver (Nordberg et ~1.. 1975b) only purified on Sephadex G-50 was labeled in \vitro with ‘09Cd and injected intravenously into the tails of mice. The dose of cadmium amounted to 0.075 mgi kg body weight. Animals injected with ‘09CdCl., served as controls. Whole body measurements were performed immediately after injection and before killing 2, 5, and 20 min. and 4, 24. and 96 hr after injection. The whole body measurements showed no differences in body burden between the lo9CdBP and ‘09CdCl, groups as long as animals were killed within 2 to 20 min. Animals injected with *09CdBP showed differences compared to animals injected with lo9CdCl, after 4 to 96 hr. Whole body counting showed that only about 50% of the body burden just after injection remained in the body. Autoradiography was performed according to a method described by Ullberg ( 1954). Sections of the frozen mouse were prepared at 20 pm on a tape and apposed to Structurix or Kodirex X-ray film. In animals injected with lo9CdBP 2 min to 96 hr after injection, uptake of cadmium in the kidney cortex was prominent. For animals killed 20 min after injection a blackening of the urinary bladder was

396

MONICA

NORDBERG

also observed, thus indicating that cadmium was present as well in the urine. This was only observed in animals in the 20-min group. For animals injected with ‘WdCl,, the liver contained most of the cadmium. The mice were then thawed and whatever remained of the organs was removed. Organ weights were recorded and y-counting was performed. Animals exposed to cadmium chloride were found to have much higher concentrations of cadmium in liver than in kidney. For animals exposed to cadmiumbinding protein, kidney values tended to increase up to 20 min after injection of ‘09CdBP but then remained constant and much higher than corresponding liver values. Kidneys were subjected to gel chromatography on Sephadex G-75. The results are given in Fig. 5. Only part of the cadmium content in kidney in animals injected with 109CdCl, was found to be bound to low molecular weight proteins [fractions 25-30). even after96 hr. In animals injected with lo9CdBP most of the radioactivity was in the low molecular weight fractions S min after injection. At 24 hr relatively less cadmium in the low molecular weight fractions was detected. At 96 hr the distribution pattern was more similar to the one of 5 min. Sr~rdic).sotr ‘09CdMT/. The second part of this study was performed at National Institute of Environmental Health Sciences on C57BL16JH(J67) mice with metallothionein prepared from CBA mice. This metallothionein had been purified and characterized by isoelectric focusing, whereupon it was found to contain one form with 1055 (w/w) cadmium and approximately 0.4% (w/w) zinc. This form. designated CdMTI. was radiolabeled i/r ~~?t.o with royCdCI, and intravenously injected into the tails of the mice. That only one form of metallothionein was used constituted the key difference between this and the first part of this study and that conducted by Nordberg and Nordberg ( 197%). The animals were divided into four groups of from three to four individuals and received 0.03 mg or 0.08 mg of Cd/kg body weight. calculated from the cadmium content of metallothionein. Controls received only ‘09CdC1,. No whole body counter was available. The animals were put into metabolism cages and urine and feces were collected. Five minutes or four hours after injection the animals were killed. Blood. liver, kidneys. testes, pancreas, urinary bladder, hair. tail. heart. tibia. and muscle were taken for measurements of radioactivity in order to come as closely as possible to whole body counting. Almost no radioactivity was found in the feces, urine, or urinary bladder. meaning that no appreciable cadmium excretion took place. More than 80% of the radioactivity in animals injected with radiolabeled CdMTl appeared in the kidney 4 hr after injection. Animals injected with ro9CdMT1 have about 10 times more cadmium in kidneys than animals given ‘09CdCl., . Electronmicroscopy was performed and did not show any kidney damage in mice representing both groups of animals. Distribrrrion of Ittjectrcf Hrmolysute in Micr

CLtdttlittttt-Bitldittl:

Ptwtvitt

Isolcrtrd

.fiottt Mousr

Solutions prepared by gel filtration (Fig. 4) of hemolysates of mice given a single injection of radiolabeled cadmium (1 mg of Cd/kg body weight) and killed 96 and 192 hr later were taken for further study of the distribution of cadmium bound to

SI‘LlD1E.S

ON

~lE’~.-ZLLO?‘HION~I~

AND

397

C:.-\DMIL’M A

ng Cd 7.0s

OD 05- A 1.7. LO-

6.0. 5.0-

O,4 o-

0.5. 0.4. 0.3.

3.0.

0%

20.

0.1.

I o-

Cd in Iroction~ OD 254 nm =

ngcd 0.D

B 60. so4.0. 3 o20. 1.0.

0 Cdin5

5 mlhclion

15

10 -.-.

H

c

20

d5

30

Fracl. no

.

0.D 254 nm Y---‘-

FIG. 4. Gel chromatography on Sephadex G-75 of hemolysates of hlood cells from mice given a single subcutaneous injection of “YdCI. (1 mg of Cd/kg body weight). Column dimensions were 355 x 26 mm. Elution with 0.01 M Tris buffer in 0.05 M NaCl tpH 8.0) at a flow rate of 14 ml/hr. Volume of fractions: 5 ml. OD at 254 nm was continuously monitored. The indicated fractions were pooled, concentrated and taken for further studies. (A) 96 hr after injection: (B) 192 hr after injection.

the protein with some features similar to metallothionein. Mice were intravenously injected with these solutions (approximately 2 ng of Cd/mouse). The animals were divided into groups of 2 to 5 animals (Table 1). These animals were killed after 4 and 96 hr. Animals intravenously injected with lo9CdCl, served as controls. The animals were subjected to whole body counting. which was performed immediately after injection and just before killing. Animals were put into metabolism cages and urine was collected 4 hr after injection and 24 hr before killing. Blood, liver, kidney, pancreas, testes, spleen, and urinary bladder were removed, weighed. and counted for radioactivity. Animals injected with cadmium bound to

398

MONICA

NORDBERG

TABLE AMMOUNT

OF CADMIUM”

IN ‘I’HL KI~SFY NITH HFMOLYSATL

Cadmium compound and time after injection A” 4 hr Mean 96 hr Mean B’ 4 hr Mean 96 hr Mean CM 4 hr Mean 96 hr Mean

1 OF MICE INJM-T‘ED OF MI(X

INTRAVLN~I~SI.~

Kidney 6.1 6.9 10.1 6.5 4.5 6.8 6.2 6.6 6.4 55.9 74.9 68.2 67.4 67.4 so.3 58.9 75.9 66.6 63.0 68.5 71.3 73.1 72.2

” Percentage of amxrnt injected. b A. r’VdC1,. c B. ro9Cd-binding protein from 96-hr hemolysate d C, “Wd-binding protein from 192-hr hemolysate

protein isolated from mice killed after a 96-hr observation time had kidney values of 66.3 and 58.9% of the dose, depending upon their own observation time, 4 or 96 hr (Table 1). Corresponding figures for animals injected with protein isolated from animals killed after 192 hr were 68.5 and 72.2% of the dose (Table 1). These values are about 10 times higher than those seen in animals injected with lo9CdCl,. Autoradiography performed on mice killed 4 hr after injection showed accumulation of cadmium in the kidney cortex in animals that had received cadmium bound to metallothionein and in the liver in those given cadmium chloride. The autoradiographic patterns were similar to the ones observed in mice injected with hepatic metallothionein. Discussion and Conclusions on the Distribution of Cadmium Bound to Metallothionein

In mice injected with rabbit liver metallothionein, the target organ was shown to be the kidney both with regard to uptake of cadmium and the appearance of dam-

STUDIES

ON

METALLOTMlONElN

AND

CrZDMICTM

399

age. As this was observed with heterologous metallothionein, studies with homologous protein were performed. When mice were injected intravenously with crude hepatic mouse metallothionein (0.075 mg of Cd/kg body weight) consisting of at least two forms of metallothionein and minor amounts of some other proteins, the protein was taken up in the kidney. The homologous protein thus behaved similarly to the heterologous one. A high urinary excretion of cadmium was noted shortly after injection. In contrast, in animals injected intravenously with 0.08 or 0.03 mg of Cd/kg body weight of pure metallothionein with a high ratio of cadmium to protein, almost all radioactivity accumulated in the kidney. No appreciable urinary cadmium excretion was observed. The same was true when mice were exposed (intravenous injections) to a metallothionein-like compound from hemolysate at a dose of 2 ng of Cd/mouse (=0.08 pg of Cd/kg body weight), i.e., a cadmium dose 1000 times lower than in the other experiments. Distribution of radioactive cadmium bound to metallothionein has been studied by other authors as well (Cherian and Shaikh, 1975; Tanaka et (11.. 1975: Cherian et nl., 1976). Cherian and Shaikh (1975) reported results from experiments in which they gave intravenous injections of radioactive cadmium-binding protein to rats in doses of 10. 30, and 200 pg of Cd (0.05, 0.15. and 1 mg of Cd/kg body weight). The protein used by Cherian and Shaikh (1975) was labeled in r’ii~ with lo9Cd and isolated by Sephadex G-75 only. It consisted of at least two forms of cadmium-binding protein (metallothionein) as shown by isoelectric focusing (Cherian. 1974). Reported amounts of cadmium in kidney were 5.8, 13.0, and 71.4 pg. respectively. 3 hr after injection. Cherian and Shaikh (1975) stated that cadmium was excreted in the urine. It is seen from their data that only 1% of the lowest dose of cadmium was excreted while 23 and 32% of the higher doses were detected in the urine. Their data thus indicate a threshold for renal handling of metallothionein. Cherian and Shaikh (1975) also performed gel chromatography on urine. whereupon they identified the radioactive peak as corresponding to the metallothionein peak. The rapid clearance of metallothionein from the blood and the tinding of a high urinary excretion at higher doses made them express doubts as to the role of metallothionein in the transport of cadmium to the kidneys. However, their own data showed that when small amounts of cadmium are given, practically no excretion takes place. Hepatic rat metallothionein labeled in \ri\w with lo9Cd and purified only by gel chromatography was injected intravenously into rats by Tanaka et czl. (1975). They used a dose of 0.23 mg of Cd/kg body weight and killed the animals 0.5 to 168 hr after injection. One hour after injection, about 27% of the injected dose. or around 7 pg of Cd/g, was detected in the kidney. About 60% of the administered dose was recovered in the urine. It is reasonable to assume that both dose and purity of injected metallothionein are of importance for the renal handling of the protein. When pure homologous metallothionein was injected in relatively small doses (Nordberg and Nordberg. 1975d), the protein went to the kidney and no excretion of cadmium was observed in the urine. However. when crude metallothionein with the same amount of cadmium was injected. cadmium excretion in urine was evident. The reason for the

400

MONICA

NORDBERG

excretion of cadmium may be that the total protein load has become too great and results in an incomplete reabsorption. The possibility cannot be excluded, however, that different forms of metallothionein may be handled differently by the kidney or that tubular dysfunction has contributed. Absorbed metallothionein will be catabolized in the kidney. Cherian and Shaikh (1975) used an in L~~IYIdoubly labeled cadmium-binding protein obtained by gel filtration. A major part of this “metallothionein” was taken up in the kidney, where it was reported to be partly degraded within 3 hr. Webb and Etienne (1977) studied in \G~Y)and iw i,itro degradation of the protein moiety of metallothionein labeled with 35S. The in rqitro study with sliced kidney cortex gave less than 10% degradation. When the protein was incubated with kidney homogenate, a timedependent hydrolysis was reported. After 4.5 hr 41.4% was reported to be degraded. This may be compared to the half-life for rat hepatic metallothionein. which was reported to be 4.2 days (Chen rt rrl.. 1975). Synthesis of metallothionein in the kidney has been reported to be slow, in contrast to that in liver. Webb and Daniel (1975) cultured pig kidney cells itz \,itro in the presence of cadmium. After 8 days they found a small amount of metallothionein. which was identified by gel chromatography. In vi\v experiments also show a much longer time lag in the kidney than in the liver for the synthesis of metallothionein (Webb and Daniel, 1975; see also Fig. 5). Available data thus suggest that cadmium bound to metallothionein is filtered through the glomeruli and reabsorbed almost completely. Cadmium will be released by the catabolism of metallothionein, whereupon it stimulates a new synthesis of metallothionein that will bind cadmium. A continuous turnover is thus suggested. There is also the possibility that released cadmium will bind to hepatic metallothionein which has been transported to the kidney. Normally, the result is that cadmium is not excreted but remains in the kidney. A continuous rise in cadmium concentration will occur. which may somewhat explain the long biological half-life of cadmium in the kidney. When exposure is excessive. the concentrations in the kidney may be so high that metallothionein no longer has the capacity to bind all released cadmium. The excess cadmium may then interfere with reabsorption systems in the kidney and renal tubular dysfunction can occur. GENERAL

SUMMARY

Metallothionein was isolated from livers of cadmium exposed rabbits and mice. Using isoelectric focusing, metallothionein was separated into different forms; rabbit hepatic metallothionein form I had a pl of 3.9 and form II had a p1 of 4.5. Mouse hepatic metallothionein form I had a pl of 4.2. The minimum molecular weights of thionein, which is the metal-free part of metallothionein, were calculated by amino acid analyses to be around 5600, 6600, and 6300 for the three respective forms. A low molecular weight cadmium-binding protein was detected in blood, both in erythrocytes and plasma of mice exposed to cadmium. This protein was detected by radioactive measurements of ioyCd at an elution volume identical to the one that had been obtained for metallothionein by gel chromatography. This protein is probably metallothionein and acts as a transport protein for cadmium. Cad-

4ND

401

CADMIUM

A

. 0.5

l.

*

01

r-,

‘

,

,.*. *.“I,

‘. ,

,

9..

;

,

.

t ,

,o-

B

25io5,oI-

FIN.. 5. Gel chromatography on Sephadex G-75 of kidneys from mice exposed to ‘YdCI, (left) and ‘O”CdBP (right). The column dimensions were 371 8 26 mm. Elution was carried out with 0.01 M Tris buffer in 0.05 M NaCl (pH 8.0) at a flow rate of 14 mlihr. Volume of fractions: 5 ml. (A) 5 min after injection: (B) 4 hr after injection: (C) 24 hr after injection: (D) 96 hr after injection.

mium-binding protein in hemolysates was further shown to have a pl of around 6 and contained cadmium as well as zinc. Due to the small amounts of the protein in blood, it was not possible to make a final identification. Cadmium bound to metallothionein was found to be much more toxic than cadmium given as cadmium chloride in experiments using mice. Using autoradiographic technique and metal analyses, it was also found that the cadmium bound

402

MONICA

NORDBERG

to metallothionein was taken up by the kidney to a much greater extent than cadmium received as cadmium chloride. Histological examination showed kidney damage in animals exposed to large doses of metallothionein. Excretion of cadmium in urine was noted when mice were exposed to relatively large doses of crude metallothionein from mouse liver. However, smaller amounts of pure homologous metallothionein were almost completely reabsorbed in the kidney. These findings together with other published data make it possible to propose the following model for cadmium transport and accumulation in the kidney. Exposure to cadmium induces synthesis of metallothionein in the liver and this protein appears to be involved in the transport of cadmium in the blood. Cadmium circulates in the blood bound to metallothionein in both erythrocytes and plasma. Metallothionein is cleared from the plasma and is taken up in the kidney. In the kidney, there will be a continuous release of cadmium from catabolized metallothionein. Released cadmium will probably stimulate the synthesis of metallothionein. There is also the possibility that cadmium may bind to reabsorbed metallothionein, which in turn will be catabolized, this procedure being repeated time and again and preventing the secretion and excretion of cadmium. Cadmium in kidney will thus have binding sites available all the time. The result is a continuous accumulation of cadmium in the kidney. When exposure to cadmium is excessive, the renal cadmium will eventually reach a level, the critical concentration, where the binding capacity of renal metallothionein for cadmium is exceeded and tubular dysfunction will occur. ACKNOWLEDGMENTS Grants for this research were obtained from the Swedish 13X-775. 26X-775) and the Folksam Insurance Company. National Institute of Environmental Health Sciences, North ing the author to use laboratory facilities.

Medical Carolina

Research is gratefully

Council thanked

(Project

No.

for allow-

REFERENCES Bremner. I., and Davies. N. T. (1975). The induction of metallothionein in rat liver by zinc injection and restriction in food intake. Biochrm. .I. 149, 733-738. Bremner. .I.. Davies, N. T.. and Mills. C. F. (1973). The effect of zinc deficiency and food restriction on hepatic zinc proteins in the rat. Biocltem. Sot. Trans. 1, 982-985. Buhler. R. H. 0.. and Kagi. .I. H. R. (1974). Human hepatic metallothioneins. FEBS Let. 39,229-234. Carlson. L. A.. and Friberg. L. (1957). The distribution of cadmium in blood after repeated exposure. Sc~and. J. C/in. Lab. Invcjst. 9, I-4. Chen, R. W.. Eakin. D. J.. and Whanger, P. D. (1974). Biological function of metallothionein: II, Its role in zinc metabolism in the rat. Nlrtr. Rep. Inr. 10, 195-200. Chen. R. W.. Whanger, P. D.. and Weswig. P. H. (1975). Biological function of metallothionein: 1. Synthesis and degradation of rat liver metallothionein. Biochen~. Med. 12, 95- 105. Cherian. M. G. (1974). Isolation and purification of cadmium binding proteins from rat liver. Bioclwm. Biophys. Res. Commr/n. 61. 920-926. Cherian. M. G.. and Clarkson. T. W. (1976). Biochemical changes in rat kidney on exposure to elemental mercury vapor: Effect on biosynthesis of metallothionein. Chem. Biol. Infemct. 12, 109- 120. Cherian. M. G., Goyer. R. A.. and Delaquerriere-Richardson, L. (1976). Cadmium-metallothioneininduced nephropathy. T~~.rico/. Appl. Phavmacol. 38. 399-408. Cherian. M. G.. and Shaikh, Z. A. (1975). Metabolism of intravenously injected cadmium-binding protein. BI’D(.IIcuI. Biophy.s. Rrs. Commun. 65, 863 -869. Evans. G. W., Wolenetz, M. L.. and Grace, C. J. (1975). Copper-binding proteins in the neonatal and adult rat liver soluble fraction. Nlrfr-. Rep. Inr. 12, 261-269.

STUDIES

ON

METALLOTHIONEIN

AND

CADMIUM

403

Fowler, B. A. and Nordberg, G. F. (1978). Rena1 toxicity of cadmium metallothionein: morphometric and X-ray microanalytical studies. Torico/. Appl. Phur-rtitrcol. in press. Friberg. L.. Piscator. M.. Nordberg. G. F., and Kjellstrom. T. (1974). “Cadmium in the Environment.” CRC Press Division, Chemical Rubber. Co.. Cleveland, Ohio. Kagi. J. H. R. (1970). Hepatic metallothionein. Ei,e/rtlt Ittt. Corrgr. Rioc~llcrr~.. III/. Ut~ion Bioc~hc,tn. 130 (Abstract). Kagi. J. H. R., Himmelhoch. S. R.. Whanger. P. D.. Bethune, J. L., and Vallee. B. L. ( 1974). Equine hepatic and renal metallothioneins purification. molecular weight. amino acid composition and metal content. ./. Bid. Churn. 249. 3537-3541. Kagi. J. H. R.. and Vallee. B. L. (1960). Metallothionein: A cadmium and zinc containing protein from equine renal cortex. J. Bic~l. Chem. 235. 3460-3465. Kagi. J. H. R.. and Vallee. B. L. ( 1961). Metallothionein: A cadmium and zinc containing protein from equine renal cortex: II. Physicochemical properties. .I. B/o/. Cltcm. 236, 2435-1442. Kojima. Y.. Berger. C.. Vallee. B. L., and Kagi. J. H. R. (1976). Amino-acid sequence of equine renal metallothionein~lB. Pn,c,. Not. Acc~tl. SC,;. USA 37, 3413-3417. Lucis. 0. J.. Shaikh. Z. A.. and Embil. J. A. (1970). Cadmium as a trace element and cadmium binding components in human cells. E.t-pc,ric~,iti
404

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Shaikh. 2. A.. and Lucis. 0. J. (1971). Induction of cadmium binding protein. Fd. Pwc. Ahrr. 29. 298. Squibb. K. S.. and Cousins. R. J. ( 1974). Control of cadmium-binding protein synthesis in rat liver. E/fviro,l. Ph.YAiO/. Bi~~~~/lc/?l. 4. 24-30. Tanaka. K.. Sueda. K.. Onosaka. S.. and Okahara. K. (1975). Fate of r”‘Cd-labeled metallothionein in rats. To.\-ic,o/. A&. Ph~~rm~d. 33. 258-266. Ullberg. S. (1954). Studies on the distribution and fate of S” labeled benzyl penicillin in the body. Ac,frr Radio/. Suppl. 118, I - I 10. Webb, M. ( 1972). Binding of cadmium ions by rat liver and kidney. Bioc~/z~,/~. P/rtrr/,~c~~~/. 21, 2751-2765. Webb. M.. and Daniel. M. (197.5). Induced synthesis of metallothionein by pig kidney cells i/r r,itr’o in response to cadmium. C/rc,rr. Biol. Ifrte~~~ct. 10, 169-276. Webb. M.. and Etienne. A. T. (1977). Studies on toxicity and metabolism of cadmium-thionein. Bioc~lic,rn. f’hrrrmtrc~c~l. 26. 25 -30. Webb, M.. and Verschoyle. R. D. ( 1976). An investigation of the role of metallothioneins in protection against the acute toxicity of the cadmium ion. Bioc h(,m. Phtrrnrtrc~~l. 25, 673-679. Weser. U.. Rupp. H.. Donay. F.. Linneman. F.. Voelter. W.. Voetsch. W.. and Jung, G. (1973). Characterization of Cdd. Znthionein (metallothionein) isolated from rat and chicken liver. El/r. .I. BI’O~~IIPIII. 39. 127- 140. Winge. D. R., Premakumar, R.. and Rajagopalan, K. V. t 1975a). Metal-induced formation of metallothionein in rat liver. AK/I. Biwhcm. Biophy.s. 170, 242-252. Winge. D. R.. Premakumar. R., Wiley. R. D.. and Rajagopalan. K. V. (1975b). Copper-chelatin: Purification and properties of a copper-binding protein from rat liver. AK/I. Bicwhc,~. Bioplfys. 170. 253-266. Winge. D. R.. and Rajagopalan, K. V. (1972). Purification and some properties of Cd-binding protein from rat liver. Awir. Bkw/w~~~. Biophys. 153. 755-761. Wisniewska-Knypl. J. M.. Trojanowska. B.. Piotrowski. J.. and Jakubowski, M. (1970). Binding of mercury in the rat kidney by metallothionein. T~~.ric~~~/. Appl. Plrrrrrhclc~~~l. 16, 7544763.