Cadmium Binding Components in the Supernatant Fraction of Liver, Kidney and Intestinal Mucosa Homogenates of Cadmium-Administered Rats

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1 Cadmium Binding Components in the Supernatant Fraction of Liver, Kidney and Intestinal Mucosa Homogenates of Cadmium-Administered Rats Keiichi Tanaka and Kaori Sueda Department of Public Health, Faculty of Nutrition, Kobe-Gakuin University, Kobe INTRODUCTION Many studies have recently been performed on the behavior in animals of nonessential heavy metals including cadmium1). Nordberg2) and Lucis3) have shown that cadmium is bound to components in the supernatant fraction of liver and kidney homogenates. Scarcely any reports are available on previous steps or on the absorption of cadmium into the body. Therefore, we studied the intestinal mucosa along with the liver and kidney. In the supernatant fraction of homogenate of rat intestinal mucosa, components were found which combined with cadmium similar to the so-called metallothionein in the liver and kidney. Cadmium administered orally (1mg/100g body weight) was present in the intestinal mucosa in the bound state. The possibility that such binding acts as a barrier against cadmium absorption has been suggested4). In the present study, rats were fed cadmium for 30 days and the binding of cadmium in the supernatant fraction of homogenates of liver, kidney and intestinal mucosa was studied. MATERIALS AND METHODS Male Wistar rats weighing about 90g were fed a solid ration and water containing was injected subcutaneously in a dose of 0.25ml/100g body weight. After 24 hours, animals were sacrificed and liver, kidney and intestinal mucosa were removed. Each of these tissues was homogenized with 5vol of 0.25M sucrose solution. Each homogenate was ultracentrifuged at 105,000g, for 60 minutes to prepare the supernatant fraction. Five ml of the su- equilibrated and eluted with 0.01M Tris buffer, ph 7.4. The flow rate was 25ml/hr, and 5 ml/tube fraction were collected. In the eluate contained in each tube, radioactivity, cadmium a Fuji Electric EA 14 well-type scintillation counter was used. Cadmium concentration was measured with a Hitachi 208 atomic absorption spectrometer, while absorbency was measured in a Hitachi 101 spectrometer, The influence of ph on the binding between cadmium and supernatant components was tested by adjusting the ph of the supernatant fraction to 2.0 with HCl, followed by gel filtration with 0.01N HCl. Pronase digestion was carried out Pronase E (70,000p.u.k./g) purchased from Kaken Chemicals. The reaction products were subjected to gel filtration with Tris buffer. RESULTS AND DISCUSSION Gel filtration pattern of the supernatant fraction of homogenates of liver, kidney and

2 intestinal mucosa in normal and cadmium-treated rats. Fig. 1-a shows the gel filtration pattern of 5ml each of the supernatant fraction of liver, kidney and intestinal mucosa homogenates in normal rats on Sephadex G-75 column. Two kinds of cadmium binding components were found: fr-1 (MW>50,000) eluted at the void volume of G-75 column and fr-2 (MW about 10,000). The elution volume of the binding components in the three tissues showed good agreement. About the same amount of cadmium was detected in each supernatant fraction, and binding to fr-1 and fr-2 seemed to be of similar degree. Fig. 1. Gel filtration of supernatant fraction of intestinal mucosa, kidney and liver homoganates On the other hand, 109Cd injected 24 hours previously was taken up mainly by the liver, followed by the kidney and intestinal mucosa as in the studies on organ levels5). This cadmium was always bound to fr-2. Fig. 1-b shows the gel filtration pattern of the supernatant from liver, kidney and intestinal mucosa homogenates of rats treated with 50ppm cadmium for 30 days. Compared to normal rats, the amount of bound cadmium was increased in all three tissues, especially that bound to fr-2 in liver and kidney supernatants. Along with this, a peak or shoulder. On the other hand, the amount of cadmium bound to fr-1 did not differ much from that in the control group. The pattern of 109Cd radioactivity was also similar to that in the control group. In the group treated with 100ppm (Fig. 1-c), the difference was more pronounced. Cadmium bound to fr-2 in the kidney and liver supernatants showed a mium in the intestinal mucosa was increased in both fr-1 and fr-2. Unlike the control group

3 and the group treated with 50ppm Cd, free, unbound cadmium appeared. This tendency was also noted in the group treated with 200ppm Cd (Fig. 1-d). The amount of cadmium in fr-2 of the kidney and liver was increased further, and free cadmium was noted in the intestinal mucosa. The elution pattern of 109Cd was similar in each dose group. Thus, cadmium entering the body appears to be bound mostly to fr-2 of the liver, followed by fr-2 of the kidney. Amount of bound cadmium in the supernatant fraction of liver, kidney and intestinal mucosa homogenates of normal and cadmium-treated rats Fig. 2. summarizes the amount of bound cadmium in 1ml of supernatant fractions of the three tissues in groups of 4 rats each, maintained on drinking water containing 0, 50, 100 or 200ppm cadmium. In the control group, cadmium bound similarly to fr-1 and fr-2 in each tissue, without significant organ difference. In the treated groups, no marked difference was noted between fr-1 and fr-2 in the intestinal mucosa. In the liver and kidney, however, almost all cadmium was bound to fr-2. The amount of bound cadmium per one ml of supernatant fraction of the kidney was similar to that of the liver, but when more than 100ppm was used, the amount exceeds that of the liver. At 24 hours after injection, as shown in Fig. 1, 109Cd was distributed concentration of Cd in drinking water (ppm) **p<0.01 Fig. 2. Bound cadmium in supernatant fraction of intestinal mucosa, kidney and liver homoganates Table. 1. Effect of Cadmium administration on body and tissue weight in overwheling abundance in the liver. These results are due to the long half-life of cadmium in the kidney6) or transfer of cadmium from other organs to the kidney5). On the other hand, the amount of bound cadmium in the intestinal mucosa failed to increase as markedly as in the liver and kidney even if the ingestion of cadmium increased. In the groups treated with 100 or 200ppm cadmium, free cadmium was detected in the supernatant of the intestinal mucosa (fig. 1-c and d). The cadmium binding components in the liver and kidney have been reported to be induced by cadmium administration7,8). Cadmium binding components in the intestinal mucosa are probably not revealed by such a procedure. When the amount of cadmium administered exceeds 100ppm, free cadmium appears in the intestinal mucosa and body weight falls, while the weight of the intestinal mucosa increases markedly (Table 1). Above 100ppm, the ability of the intestinal mucosa to block cadmium seems to be exceeded.

4 This speculation needs further investigation. Effect of ph and pronase on the binding of cadmium with the supernatant components in liver, kidney and intestinal mucosa homogenates. In a study of the chemical property of cadmium binding components, the effect of nonphysiological ph and incubation with pronase was investigated. The findings in the supernatant fraction of liver homogenate are shown in Fig. 3. At ph 2.0, cadmium bound to fr-1 and fr-2 was all freed (Fig. 3-a). Incubation with pronase for 2 or 6 hours resulted in disappearance of radioactivity in fr-1, and transfer of radioactivity in fr-2 to a low molecular side (Fig. 3-b). After longer incubation, inorganic cadmium was released (Fig. 3-c). Besides the liver, supernatants of kidney and intestinal mucosa homogenates were studied and similar results were obtained. Therefore, these cadmium binding components in the three tissues all appear to be protein. CONCLUSION Fig. 3. Effect of ph and pronase on binding of Cadmium to liver supernatant fraction (LSF) 30 days, cadmium was retained especially in fr-2 of the liver and kidney. 1. In the supernatant fraction of homogenates of liver, kidney and intestinal mucosa, a cadmium binding component (fr-1) with a molecular weight above 50,000 and another one (fr-2) with a molecular weight of about 10,000 were found. 2. In rats maintained on water containing 50,100 or 200ppm of cadmium for 3. The ability of the supernatant fraction of intestinal mucosa homogenate to bind cadmium failed to increase markedly even when the dose of cadmium was increased. In groups treated with 100 and 200ppm of cadmium, free cadmium was detected.. 4. Regardless of the amount of cadmium retained in the body, cadmium which entered the body anew was mostly bound to fr-2 in the liver. 5. The cadmium binding components in the supernatant fraction of liver, kidney and intestinal mucosa homogenates are proteins. ACKNOWLEDGMENT We are indebted to Professor Itsuhiko Mori, Kobe Women's Pharmaceutical College, for

5 radioisotope experiments, and also to Mr. Hideki Uejima for cooperation in part of this experiment. REFERENCES 1) Kimura, M.: Metallothionein, Igaku no Ayumi, 83, 1-10 (1972). 2) Nordberg, G. F., Piscator, M. and Lind, B.: Distribution of cadmium among protein fractions of mouse liver, Acta Pharmacol. Toxicol., 29, (1971). 3) Shaikh, Z. A. and Lucis, O. J.: Cadmium and zinc binding in mammalian liver and kidneys, Arch. Environ. Health, 24, (1972). 4) Tanaka, K., Nishiguchi, K. and Okahara, K.: Fate of heavy metal in animals: Binding of cadmium to the supernatant components in rat intestinal mucosa, J. Hyg. Chem., 19, (1973). 5) Sayato, Y., Hasegawa, A, and Ando, M.: Hygienic chemical studies on poisonous metals. I. Researches on the body retention, excretion and distribution pattern of cadmium, J. Hyg. Chem., 17, (1971). 6) Shaikh, Z. A. and Lucis, O. J.,: Biological differences in cadmium and zinc turnover, Arch. Environ. Health, 24, (1972). 7) Shaikh, Z. A. and Lucis, O. J.: Induction of cadmium-binding protein, Fed. Proc., 29, 298 Abs (1970). 8) Shaikh, Z. A. and Lucis, O. J.: The nature and biosynthesis of cadmium binding proteins, Fed. Proc., 30, 238 Abs (1971).